Review

pubs.acs.org/CR

Single Particle Tracking: From Theory to Biophysical Applications

Hao Shen,† Lawrence J. Tauzin,† Rashad Baiyasi,‡ Wenxiao Wang,‡ Nicholas Moringo,† Bo Shuang,†
and Christy F. Landes*,†,‡,§
†
Department of Chemistry and ‡Department of Electrical and Computer Engineering, §Smalley-Curl Institute, Rice University,
Houston, Texas 77251, United States

ABSTRACT: After three decades of developments, single particle tracking (SPT) has
become a powerful tool to interrogate dynamics in a range of materials including live cells
and novel catalytic supports because of its ability to reveal dynamics in the structure−
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function relationships underlying the heterogeneous nature of such systems. In this review,
we summarize the algorithms behind, and practical applications of, SPT. We first cover the
theoretical background including particle identification, localization, and trajectory
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reconstruction. General instrumentation and recent developments to achieve two- and

three-dimensional subdiffraction localization and SPT are discussed. We then highlight
some applications of SPT to study various biological and synthetic materials systems.
Finally, we provide our perspective regarding several directions for future advancements in
the theory and application of SPT.

CONTENTS 5.3. Nonconfocal Based Active Tracking 7365

6. Conclusion and Perspective 7366
1. Introduction 7331 Author Information 7367
2. Theoretical Foundations for Superlocalization Corresponding Author 7367
and SPT 7334 ORCID 7367
2.1. Optical Diffraction Limit 7334 Notes 7367
2.2. Fourier Optics Provide a Tool To Predict Biographies 7367
Point Spread Functions 7335 Acknowledgments 7367
2.3. Fisher Information and the Cramér−Rao References 7367
Lower Bound 7336
2.4. Localization Methods 7337
2.5. Simulating Experimental Data 7339
1. INTRODUCTION
2.6. SPT Methods 7339
2.7. Mean Square Displacement (MSD) Analysis 7342 Single-molecule microscopy1−9 and super-resolution micros-
2.8. Ergodic Hypothesis in SPT 7343 copy10−14 have revolutionized our ability to study fundamental
3. Revealing Dynamic Processes Using 2D SPT 7343 biology15−17 and general surface sciences.18−24 Single-molecule
3.1. Two-Dimensional Interfacial Dynamics 7343 techniques have enabled us to understand details about, for
3.2. Imaging 2D Structures with SPT 7345 example, the movement of molecular motors inside cells,25,26
3.3. Enhanced Spatial Resolution as a Stepping and mechanisms of biomolecular separation efficiency at
Stone to Improved 2D SPT 7346 chromatographic interfaces.27−33 Such advancements in the
3.4. Correlation Analysis 7348 fundamental understanding of these areas could dramatically
3.5. Differential Interference Contrast (DIC) Mi- influence our daily lives in the future.
croscopy 7350 Single particle tracking (SPT), as illustrated in Figure 1, has
4. Revealing Dynamic Processes Using 3D SPT 7350 played a central role in many of the advances in single-molecule
4.1. Multifocal Plane Microscopy (MPM) 7351 microscopy.34−37 In a typical SPT measurement, the molecule
4.2. Astigmatic 3D Detection Using a Cylindrical of interest is imaged through an optical microscope by a photon
Lens 7352 detector and its motion is recorded in a continuous fashion to
4.3. Engineered 3D Point Spread Functions generate a trajectory. Like other single-molecule phenomena,
Using Phase Masks for 3D Imaging and SPT 7355 while the behavior of a single trajectory might be stochastic, the
4.4. High Temporal Resolution in Single Particle statistical behavior from many trajectories may reveal additional
Tracking 7359
5. Active Feedback Tracking 7360 Special Issue: Super-Resolution and Single-Molecule Imaging
5.1. ABEL Trap 7360
5.2. Confocal Based Active Tracking 7362 Received: December 14, 2016
Published: May 18, 2017

© 2017 American Chemical Society 7331 DOI: 10.1021/acs.chemrev.6b00815

Chem. Rev. 2017, 117, 7331−7376
Chemical Reviews
Figure 1. Schematic representation of SPT. Particles are localized by
finding the central locations of their point spread functions (A),
localizations belonging to the same particle at different times are
connected using algorithms such as nearest neighbor (B), and their
chronological series of linked localizations form a time series trajectory
(C). Dynamic information is extracted based on a statistical analysis of
the trajectories (D), through, for example, mean square displacement
(MSD) analysis.
chemical or physical properties combining diffusion, adsorp-
tion−desorption dynamics, folding−unfolding dynamics, trap-
ping, or other processes.35,36,38,39
There are other techniques that can provide dynamic
information about two-dimensional (2D) and three-dimen-
sional (3D) motion, namely fluorescence recovery after
photobleaching (FRAP)40−42 and fluorescence correlation
spectroscopy (FCS),43,44 both of which have been extensively
reviewed previously. Briefly, in FRAP, an intense laser beam is
focused onto a small region to photobleach fluorescently
labeled molecules of interest. After the laser illumination stops,
the fluorescence intensity is recorded as fluorescent species in
the surroundings diffuse back into the bleached region through
Brownian motion. The rate of diffusion can then be readily
calculated from the time-varying fluorescence signal.45,46 FCS is
an autocorrelation based technique that measures the
fluorescence intensity fluctuation as a function of time due to
the diffusion of fluorescently labeled molecules. Autocorrelation
analysis of the temporal fluctuations in fluorescence intensity
provides information such as the diffusion coefficient, the
analyte concentration, and the hydrodynamic radius, via the
Stokes−Einstein equation.47−49
Although FRAP and FCS offer dynamics information based
on real-time observations, they can be surpassed by SPT in two
ways. First, FRAP and FCS only provide average information,
and function-dependent heterogeneity is lost for analytes such
as nanoparticles or biomolecules. Second, although FRAP and
FCS provide millisecond temporal resolution, their spatial
resolutions are still diffraction limited (see section 2.1 for
details), which limits their application for nanoscale processes.
However, it is also worth noting that some recent studies have
combined super-resolution approaches with FCS, such as
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fluorescence correlation spectroscopy super-resolution optical
fluctuation imaging (fcsSOFI; see section 3.4 for details).50
Aside from the case in which the analyte of interest is itself
emissive, in SPT a fluorescent tag is attached to the analyte
molecule and it is the emission of this tag that is tracked.
Indeed, the development of SPT techniques is closely tied to
the continually growing library of fluorescent labels available for
experimental use.51,52 Ideally, the tagging species needs to be
small enough to not interfere with the mobility and
functionality of the analyte; on the other hand, it must be
bright enough, i.e. exhibit high quantum yield, and have a
robust photobleaching lifetime to be measured over reasonable
periods of time. Unfortunately, few fluorescent tags can fully
meet these two requirements, as there is a trade-off between
size and robust photophysical properties.
While Robert Brown’s use of an optical microscope to
observe the motion of pollen suspended in water led to our
earliest understanding of Brownian motion,53 the modern era’s
advancements in SPT began with the ability to video track
motion. For instance, De Brabander et al. microinjected 40 nm
AuNPs stabilized with polyethylene glycol and bovine serum
albumin (BSA) into the cytoplasm of PTK-2 cells, where the
motion of the AuNPs was manually tracked via video-enhanced
contrast microscopy.54 The AuNPs, invisible to the naked eye,
strongly scattered light and were thus clearly seen as black dots
after background subtraction and contrast enhancement.
Polystyrene beads,55 latex beads,56 and silica particles57 were
other early analytes of choice during the inception of SPT. For
example, Gratton et al. tracked the 3D motion of 500 nm
polystyrene spheres using a two-photon light microscope.58
In 1993, one important milestone in SPT was developed by
Betzig and Chichester, in which a single fluorescent molecule
was detected at room temperature.59 Using single fluorescent
molecules as tags for analytes was soon adapted to SPT. Due to
the small physical sizes of organic dyes (1−2 nm), they have
been attached to a variety of molecules including proteins,60
antisense DNA strands,61 mRNA,62 polymer chains,63 and
antibodies.64 However, because some organic dye molecules are
highly toxic to living cells, the successful synthesis of fluorescent
proteins in the mid-1990s was another important advance.65,66
The significance of fluorescent proteins is that they can be
genetically encoded, making live cell imaging possible. Since
then, there has been an explosion of SPT studies of various
systems in living cells, including gene expression,67,68 protein
DNA interaction,69,70 and dynamic processes on cell mem-
branes.39,71 An alternative choice for tagging is quantum dots
(QDs),72−75 which have high quantum yields and do not suffer
from photobleaching. QDs used for fluorescent tags are
generally larger than either organic dyes or fluorescent proteins,
but are smaller than AuNPs or polymeric spheres: a typical QD
tag has an overall diameter of 10−30 nm including an inorganic
core and necessary stabilization ligands. It is important to note
that, despite many advances in single fluorescent probes, the
current library of SPT tags leaves considerable room for
improvement through optimization of sizes, quantum yields,
and photobleaching lifetimes.
Single fluorescent molecules are often the tagging species of
choice; thus the development of highly sensitive detectors has
been crucial for advances in SPT. The core components of
state-of-the-art detectors are semiconductor materials that can
convert photons into electrical signals based on the creation of
electron−hole pairs in low light environments.76,77 Depending
on the excitation geometry and sample conditions, two types of
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Figure 2. Scheme of biophysical time scales relevant to single-molecule studies. “Cy” stands for cytoplasm and “Mb” stands for membrane. Reprinted
with permission from ref 77. Copyright 2007 Taylor & Francis.
detectors are most commonly used: point detectors and wide-
field detectors.78,79 Point detectors such as photomultiplier
tubes (PMT) and avalanche photodiodes (APD) have little to
negligible readout noise and dark counts, and can operate at
picosecond temporal resolution; therefore they are commonly
used in confocal based experiments.77,80 However, raster-
scanning is needed for imaging purposes; thus the applications
of point detectors are limited in large data output scenarios.
High sensitivity wide-field detectors are ideal for single-
molecule imaging. The most commonly seen wide-field
detectors are cameras based on charge-coupled devices
(CCD). To achieve single-molecule sensitivity, an image
intensifier is placed in front of the CCD chip. These so-called
“intensified CCD (ICCD) cameras” have quantum efficiencies
ranging from 20 to 50%.77 Another popular choice is the
electron multiplying CCD (EMCCD) camera, which utilizes a
shift register and an output amplifier to achieve large data
output and high sensitivity simultaneously. The quantum
efficiency of an EMCCD camera can be >90%. The drawback
of EMCCD cameras is that the stochastic electron multi-
plication mechanism also amplifies the shot noise in the signal,
resulting in lower photodetection efficiency. Another option for
wide-field detection is the scientific complementary metal oxide
semiconductor (sCMOS) camera. Unlike CCD based cameras
that utilize the entire pixel area for photon collection, sCMOS
cameras have supporting electronic devices integrated into each
pixel, meaning that only part of the entire pixel is photon
sensitive. Also, sCMOS cameras usually have intense dark noise
and readout noise, so single photon detection cannot be
currently achieved on a sCMOS camera.81 The quantum
efficiency of sCMOS cameras is ∼70%, lower than that of
EMCCD cameras. However, simulation results in previous
work suggest that sCMOS cameras can outperform EMCCD
cameras in spot localization, because the sCMOS does not
amplify shot noise.81 The temporal resolution of single-
molecule experiments is usually dictated by the frame rate of
the detector. CCD based cameras have native frame rates of
∼100 fps, while sCMOS cameras can achieve several hundred
frames per second. Although many advances have been made in
wide-field detectors, the temporal resolution of these cameras
cannot cover the entire time scale for single-molecule studies
(Figure 2). To overcome these limitations, novel photon
detectors have been designed in the past few years, such as
single-photon avalanche photodiode detector (SPAD) arrays to
simultaneously achieve large data throughput and fast frame
rates up to several thousand frames per second.77,79,81,82 Recent
reviews of the latest detector technologies discuss their
applications, advantages, and disadvantages in detail.79,81
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Moreover, not only are engineers making scientific cameras
more sensitive and with faster frame rates, scientists are also
improving the excitation and detection geometries to achieve
an effectively faster temporal resolution. We will discuss some
of the recent progress in pushing the temporal resolution of
single-molecule techniques in section 4.4.
Although advancements in SPT techniques have evolved in
the past three decades with the development of tagging species,
improved photon detectors, and more complicated excitation-
detection pathways, the goal of SPT has remained unchanged:
to understand the motion of single particles or molecules in
various environments and to extract mechanistic descriptions of
their dynamics. With modern advances, SPT can achieve tens of
microseconds temporal resolution with down to 1 nm spatial
resolution.83 In comparison to imaging macroscale objects,
imaging a nanoscale tagged analyte under an optical microscope
presents unique challenges. Because of the diffraction of light,
one cannot resolve any structures smaller than ∼250 nm with
light from the visible spectrum using traditional microscopy
methods. In the past decade, several methods have been
developed to overcome this diffraction limit. Although they are
all termed as “super-resolution localization” or “sub-diffraction
imaging”, they fall into two different categories. The first type,
represented by techniques such as stimulated emission
depletion (STED) microscopy,84,85 ground state depletion
(GSD) microscopy,11,86 and reversible saturable optical linear
fluorescence transitions (RESOLFT),87,88 are deterministic
methods where the emission signals from point-like objects
are directly modulated. In STED, for example, a red-shifted
donut-shaped depletion laser pulse forces excited fluorophores
into the ground state; therefore, spontaneous fluorescence
emission cannot occur in the periphery of the excitation spot.
Implementation of this depletion laser beam allows the residual
fluorescent region to be much smaller as compared to the
original diffraction limited size, and ∼30 nm lateral resolutions
are achieved. The other category, known as stochastic
functional techniques and represented by stochastic optical
reconstruction microscopy (STORM)12 and photoactivated
localization microscopy (PALM),10,52,89 use mathematical
analyses to push the resolution limit. The diffraction-limited
image of a single fluorophore or any point-like emitter can be
analyzed to determine the emitter position if a sufficient
number of photons are collected. 2D STORM and PALM
usually provide lateral resolution between 20 and 40 nm. These
2D super-resolution techniques were later modified for 3D
imaging as well.90−95 Welsher and Yang summarize the spatial
and temporal resolutions for the commonly used super-
resolution techniques (Figure 3) in their recent publication.83
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Chemical Reviews
We will discuss some of these methods as they relate to 2D and
3D SPT in later sections.
Figure 3. Spatiotemporal resolutions of various imaging methods. The
left bound of each rectangle stands for the best spatial resolution for
that particular method, while the right bound stands for the largest
applicable scale. Reprinted with permission from ref 83. Copyright
2015 Royal Society of Chemistry.
There are a few previous reviews on the subject of
SPT.35,36,39,71,96−98 Most of these reviews focus on the optical
implementation to achieve single particle detection, and
applications in biophysics. In this review, we aim at providing
the readers a broad view of SPT, from the theoretical
foundations to the recent developments in optical instrumen-
tations and analytical methodologies, and finally discuss the
application of SPT in a variety of different systems including
living cells, as well as organic and inorganic surfaces. We start
our discussion by presenting an overview of the theoretical
analysis and modeling for superlocalization and the various
ways to achieve SPT in section 2. This section also introduces
some of the commonly used data analyses in SPT, such as
extracting mean square displacement (MSD) and calculating
the corresponding diffusion coefficients. The important ergodic
assumption and some nonergodic examples are also discussed
in this section. In section 3, we focus on 2D SPT techniques
and their applications. We introduce a range of excitation
Figure 4. Comparison of the Airy disk (A) and Gaussian (B) point spread function models. (C) Comparison of cross section of a pixelated image,
along with best fit Gaussian and Airy models. (D) Gaussian and Airy curves as in (C), but with a logarithmic scale. While the Gaussian
approximation is valid near the peak, it fails to capture the ring structure of the Airy disk. (C) and (D) reprinted with permission from ref 106.
Copyright 2014 Nature Publishing Group.
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geometries for SPT, and the representative methods to achieve
2D superlocalization. We also present SPT studies at inorganic
and synthetic polymeric surfaces in this section to demonstrate
this technique being applicable to a wide range of systems.
Sections 4 and 5 further extend SPT to 3D superlocalization. In
section 4, we cover 3D techniques including multifocal plane
microscopy, astigmatism based microscopy, and phase modu-
lation-based techniques. Finally, in section 5 we introduce
active feedback tracking and highlight the technique with
highest spatiotemporal resolution to date.
2. THEORETICAL FOUNDATIONS FOR
SUPERLOCALIZATION AND SPT
2.1. Optical Diffraction Limit
The diffraction of light limits the resolution of a conventional
microscope such that a point-like emitter is imaged instead as a
blur known as the point spread function (PSF). For an optical
microscope, the diffraction limit is around 250 nm, defined by
λ
d = 2NA , where λ is the wavelength of light and NA is the
numerical aperture. It is the overlapping of PSFs from emitters
spaced closer together than the diffraction limit that defines the
resolution of traditional fluorescence microscopy. One of the
primary goals of modern SPT is to localize individual emitters
with subdiffraction limited resolution. While techniques exist to
localize emitters with overlapping PSFs,99,100 most SPT
experiments are based on imaging and subsequently localizing
distinctly separated PSFs.
Localization techniques exist that do not require a PSF
model, but for fitting-based techniques prior knowledge of the
PSF is necessary. The PSF is the impulse response of the
optical system, and can be experimentally determined by
imaging a single point source of light. Determining the PSF
theoretically requires the derivation of equations to describe the
shape of the PSF. High precision models such as the Richards−
Wolf model101 and the Gibson−Lanni102 model are rigorous
methods to calculate highly accurate PSFs, but they involve
evaluating integrals. The Airy disk PSF (eq 1, Figure 4A)
describes the intensity at point (x,y) for the paraxial wide-field
fluorescence microscopy PSF, where J1(x) is the first-order
2π
Bessel function of the first kind and kem = λ for emission
em
wavelength λem and is a valid representation in many
cases.103−105
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⎡ ⎤2 example, an isotropic point emitter at the front focal plane is

⎢ J1(kem NA x 2 + y 2 ) ⎥ described by a delta function. Its Fourier transform is constant
PSFAiry = 2
⎢ k NA x 2 + y 2 ⎥ valued at all points in space,109 equivalent to an infinite plane
⎣ em ⎦ (1)
wave. When simulating experimental situations, the aperture
In general, the equations that most precisely describe the functions as a low-pass filter, yielding the circle function shown
PSF can be prohibitively complex, but in most fitting-based as a discrete representation in Figure 5A. The Fourier transform
single-molecule localization algorithms the approximate
Gaussian PSF model (eq 2, Figure 4B) suffices. Small and
Stahlheber describe the reasoning behind the use of the
Gaussian approximation in their review “Fluorophore local-
ization algorithms for super-resolution microscopy”.106 Fitting
the Airy PSF shown in Figure 4A can be computationally
demanding, and the improved speed of the Gaussian PSF
(shown in eq 2 and Figure 4B), with Gaussian standard
deviation σxy and amplitude A, is appropriate in most
experimental situations,104,106,107 particularly for freely rotating
emitters.108
⎛ x2 + y2 ⎞
PSFGaussian = A exp⎜⎜ − ⎟
⎝ 2σxy2 ⎟⎠ (2)
Parts C and D in Figure 4 compare the fits for the Gaussian
and Airy PSF models for a cross section of pixelated data.106
While the Airy and Gaussian PSFs appear very close, the log-
scale plot in Figure 4D shows that the Gaussian PSF fails to
capture the effect of the ring structure of the Airy PSF. Since
most of the intensity of the PSF for an isotropic emitter is
contained in the bright central lobe, the Gaussian PSF is an
appropriate approximation for the 2D PSF. In fact, for high NA,
a Gaussian model with background is a better approximation
than the Airy disk PSF.108
2.2. Fourier Optics Provide a Tool To Predict Point Spread
Functions Figure 5. (A) Discrete representation of a plane wave truncated by the
Fourier optics techniques provide a method by which aperture. (B) Fourier transform of (A), resulting in a diffraction-
researchers can understand the propagation of light as it passes limited jinc function, rather than a delta function. The magnitude
squared of the jinc function is the Airy disk PSF.
through an optical system, important for purposes such as
simulating the optical response of a specific system and
engineering PSFs for 3D or supertemporal resolution
microscopy (see section 4.4). Fourier optics is based on the of the truncated plane wave is the function shown in Figure 5B,
paraxial limit of the angular spectrum method, and uses a spatial called by names such as besinc, jinc, or sombrero function. The
Fourier transform to represent an optical wave as a super- squared magnitude of this function represents the PSFthe
position of plane waves with different wave vectors.109,110 The classic Airy diskdisplaying a finite width due to the diffraction
continuous spatial Fourier transform in 2D, F( f x,f y), of a of light about the aperture. When observing an input located at
function f(x,y) is given by a distance d in front of a thin lens, the optical wave at the back
∞
focal plane can be written as
F(fx , f y ) = ∫ ∫−∞ f (x , y)e−2π i(f x+f y) dx dy
x y
(3)
A ⎛ (f − d)(x 2 + y 2 ) ⎞ ⎛ x y ⎞
where x and y are the traditional spatial Cartesian coordinates, g (x , y ) = exp⎜iπ ⎟F ⎜ , ⎟
iλf ⎝ λf 2 ⎠ ⎝ λf λf ⎠ (5)
x′ and y′ are coordinates of spatial frequency, and i = −1 ,
while the analogous discrete transformation is given by where f is the focal length, and
∞ ∞
⎛x y⎞ ∞
F(x′, y′) = ∑ ∑ f (x , y)e−2π i(x ′ x + y ′ y) F ⎜ , ⎟ = -{f (x′, y′)} = ∫ ∫−∞ f (x′, y′)
−∞ −∞ (4) ⎝ λf λf ⎠
for the discrete valued function f(x,y). Conceptually, a 2D ⎛ xx′ + yy′ ⎞
spatial Fourier transform switches between spatial coordinates exp⎜ −2π i ⎟ dx′ dy′
⎝ λf ⎠ (6)
and spatial frequency for an optical wave.111 With the angular
spectrum representation, an optical system is reduced to a is the Fourier transform of the optical wave at the input plane.
series of phase shifts, convolutions, and Fourier transforms.109 When d = f such that the input is at the front focal plane, as is
Interestingly, a thin lens performs an analog 2D spatial the case for a 4f system used for engineering 3D PSFs as
Fourier transform on incident light. Specifically, the optical described in section 4.3, the relationship between g(x,y) and
wave at the image plane (back focal plane) is the Fourier f(x′,y′) is an exact Fourier transform.109 When the input source
transform of the optical wave at the front focal plane.109 As an is not located in the focal plane, the quadratic factor in eq 6
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Chemical Reviews
Figure 6. Simulated diffraction limited PSF recorded in images with different magnifications. (A) Optical plane wave with uniform phase pattern,
truncated by the lens aperture, approximated with a 200 × 200 pixel image. Using a discrete 2D Fourier transform and specifying different N, where
N is the number of pixels across one side of the final image, will generate images with different pixel sizes (B−E). Shared color map represents
normalized intensity.
results in more complicated defocused PSFs that can be used to
engineer PSFs as discussed in section 4.3.110
Discrete Fourier transforms such as the fast Fourier
transform (FFT) can be used to generate the pixelated images
that will be formed in a diffraction-limited system, useful for
testing localization methods with ground-truth simulated data.
Relating the formulas for discrete and continuous Fourier
mλ
transformation reveals the relationship p2 = 2m 1NA , where p2 is
2 fundamental limit on precision for localization with an Airy
the size of square pixels capturing the image g(x,y) and m1 (m2)
is the number of pixels in the image f(x′,y′) (g(x,y)). Figure 6
shows an aperture-restricted plane wave (A) and the resultant
FFT with different numbers of pixels in the transform (B−E).
2.3. Fisher Information and the Cramér−Rao Lower Bound
Before discussing the estimators that are used in the localization
step of SPT algorithms, it is important to understand the
concepts of estimator accuracy and precision. When estimating
a parameter based on collected data, accuracy and precision
describe the bias and standard deviation of the estimator,
respectively. The bias of an estimator is b(θ̂) = E[θ̂] − θ, the
difference between the expected value of the estimator and the
actual value of the parameter being estimated. In practice, the
expected value of the estimator is approximated by averaging
the estimated values obtained from a large number of trials. An
unbiased estimator is preferable to a biased estimator. The
standard deviation of an estimator is the square root of the
variance, and describes how close multiple estimations of the
same parameter will be to each other. A high precision
estimator will have low standard deviation, which means that
the results will be closely clustered together.
The lower limit on the variance of an estimator is given by
the Cramér−Rao lower bound (CRLB).105,112,113 Determining
the variance and bias of an estimator requires simulated data
with known ground truth. Most commonly used estimators are
unbiased, but the variance of an estimator can differ greatly, and
has been studied in a number of works.104,107,114 For this
reason, most development of estimators focuses on minimizing
the variance. However, even given an isolated, well-behaved
point spread function, there is a fundamental limit on the
precision of any localization estimate, the CRLB. In the
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remainder of the work, the localization precision of an
estimator will refer to the standard deviation, such that the
fundamental limit is the square root of the CRLB.
The CRLB is calculated by inverting the Fisher information
matrix, var(θ̂) ≥ I−1(θ). As shown by Ober et al., the
localization precision varies as the inverse square root of the
number of detected photons. 105 When calculating the
λem
PSF, Ober et al. obtain δx = δy = , where λem is the
2π NA Nphoton
wavelength of emitted light, NA is the numerical aperture, and
Nphoton is the number of detected photons. This fundamental
limit cannot generally be reached in experiment, as it is based
on knowledge of photon arrival positions.105
Fisher information can also be used to determine the
optimum magnification for single particle experiments by taking
into account the effects of pixelation. Assuming the number of
photons collected by each pixel of the detector follows a
Poisson distribution with Poisson noise, the Fisher information
matrix is given by
⎡⎛ ⎞
Fisherij = ∑ E⎢⎜⎝ ∂ ln(Poiss(Ik + λ))⎟⎠
k
⎢⎣ ∂i
⎛∂ ⎞⎤
⎜ ln(Poiss(Ik + λ))⎟⎥ for i , j = x , y
⎝ ∂j ⎠⎥⎦ (7)
where Poiss(Ik + λ) is the Poisson random variable, Ik is the
expected number of emitted photons collected by pixel k, and λ
is the average number of photons related to Poisson noise in
each pixel. Ik is calculated by integrating the PSF over pixel k,
which is dependent on magnification and pixel size. When using
a symmetric Gaussian PSF, Fisherxy = Fisheryx = 0, and the
CRLB can be determined by inverting the diagonal elements
separately. Therefore, to determine the localization accuracy of
the x-coordinate estimate of the center of the PSF
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Figure 7. Illustration of particle localization by least-squares fitting to a Gaussian PSF. (A) Simulated image of an isotropic emitter located at the
position of the red cross. (B) Associated histogram of photon counts. (C, D) Results of a weighted least-squares estimate of a Gaussian PSF model to
the data in (A), with estimated localization located at the blue dot.
⎡⎛ ⎞⎞ ⎤
2 computationally slow compared to nonfitting methods.
∂ ⎛ (Ik + λ)
n
⎢
Fisherxx = ∑ E ⎜ ln⎜ exp( −(Ik + λ))⎟⎟ ⎥
k
⎢⎣⎝ ∂x ⎝ n! ⎠⎠ ⎥⎦
⎛ ∂Ik ⎞2 1
= ∑⎜ ⎟
k
⎝ ∂x ⎠ ( Ik + λ)
and
1 position of the PSF in the selected region, and even the relative
std(x) ≥ CRB =
Fisherxx
The theoretical limit on localization precision should be
determined for a given experimental setup in order to test the
performance of the estimator. The parameters of the
experiment should be set such that the localization precision
for the estimator approaches the theoretical limit to ensure
precise localizations. Abraham et al. also created software
packages to estimate the theoretical limitation of localization
accuracy under certain experimental conditions based on Fisher
information.104
2.4. Localization Methods
The choice of localization method for a given experiment
involves a trade-off between computational cost, desired
precision, and knowledge of noise or PSF statistics prior to
fitting. Without a valid localization method, only rudimentary
or correlation based analysis methods will work. In general,
SPT requires a low density of emitters and high purification of
the solution.115 One can track single particles with precision
beyond the diffraction limit because of the prior knowledge that
each observed PSF is the image of a single emitter. Under this
assumption, one can resolve the position of the particle more
than 10 times better than the diffraction limit by finding the
center of the isolated PSF.116,117 Many different methods have
been developed to find the center of the isolated PSF, and can
be broadly categorized by whether or not they fit a PSF
model.106,118 Usually, PSF fitting algorithms are iterative and
Review
However, they can provide higher precision and often give
more information about the emission event, such as the width
of the PSF.119
The most straightforward localization method is to calculate
(8) the intensity weighted centroid of the selected region (center of
mass). Although calculating the centroid is a very fast, single-
iteration method, the results are sensitive to noise, the relative
(9) position in the pixel.114,119−121 However, due to the advantage
in speed, centroid estimation is still useful to estimate particle
positions in active tracking to provide instantaneous feed-
back.122
The most commonly used fitting methods are least-squares
fitting (LS) and maximum likelihood estimation (MLE). A
number of methods exist for solving these optimization
problems, requiring the experimenter to carefully develop the
objective function.104 Though only an approximate representa-
tion of the PSF, the Gaussian model is often used in these
algorithms.106,107,123
Least-squares fitting with a Gaussian PSF model is a standard
method to find the center of a PSF in single-molecule
fields.107,114,124 The least-squares fitting method is an iterative
fitting-based localization method that requires little knowledge
of camera noise and is less sensitive to point spread function
model misspecification, though it lacks precision in low photon
count situations.104,106 An illustration of a weighted least-
squares localization algorithm is shown in Figure 7. The main
principle is to search for the parameters that minimize the
weighted square error between the fit and the data. It can be
written as
K
[xk − fk (θ)]2
arg min ∑
θ
k=1
σk 2 (10)
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Figure 8. Working principle of the radial symmetry method. (A) Simulated recorded PSF with shot noise. The true center of the PSF is labeled by
the red “×”. This image is generated from simulated Airy disk PSF with much smaller pixel size without noise (B). (C) Gradient of the intensity at
each pixel corner is calculated using intensities of four neighbor pixels based on the image in (A). The gradients are represented by orange arrows.
Yellow lines are extensions of the arrows to illustrate how to find the center of the PSF using all the gradients. Circles are the pixel centers. (D) The
center calculated by radial symmetry method (orange circle) matches with the true center (red “×”). The center is calculated by finding the point of
minimal overall distance to all the yellow lines, as shown in (C). Reprinted with permission from ref 119. Copyright 2012 Nature Publishing Group.

for measured pixel intensity values xk, k = 1, ..., K, expected where εxy is the noise. All the unknown parameters in the
variance in pixel k σk2, and prediction of the intensity of pixel k Gaussian function are contained in a1−5. However, this
f k(θ), based on the PSF model with θ as the parameters to be approximation ignores noise and assumes the background is
estimated. The parameters that are required depend on the PSF removed. When the noise level is large noise cannot be simply
model being usedfor the Gaussian PSF model the relevant ignored, and when the local particle density is high the
parameters are the Gaussian width, usually defined either in background is difficult to estimate and remove. Therefore, this
terms of the standard deviation σ, or in terms of full-width at fitting method can replace Gaussian fitting only when the noise
level is small and local particle density is low.127
half-maximum (fwhm = 2 2 ln 2 σ ) of the Gaussian profile;
The most time-consuming part of PSF fitting is the iterative
the mean, or center, position in Cartesian coordinates, (x0,y0); process. A variety of nonfitting methods have been developed
the amplitude (peak intensity of the Gaussian); and the to decrease computational time without decreasing resolu-
background noise parameters. The initial guess of these tion.119,128 The previously mentioned centroid method has
parameters will dramatically influence the performance of the been adapted to a number of fast algorithms;121,129 the fluoro-
fitting. Usually, the initial guess of the Gaussian width for one Bancroft algorithm uses the principle of triangulation130 and the
instrument should always be the same, which is related to the Fourier domain localization algorithm uses fast Fourier
fwhm of the diffraction limited PSF: an initial guess of (x0,y0) transforms.131 The radial symmetry method is one of the
will take the location of the pixel with peak intensity, the peak more promising methods.119
intensity minus the background as the initial amplitude, and use The radial symmetry method calculates the center of the PSF
the minimum intensity as the initial background. Least-squares through one-time linear algebra calculation.119 As illustrated in
fitting is accurate and robust, but is often the most time- Figure 8, first the gradient of the intensity at each pixel corner is
consuming part of an SPT algorithm.119,121,125,126 calculated, which represents a direction in the image. This
Maximum likelihood estimation (MLE) is an iterative fitting- direction and the corner position (xk,yk) give a line:
based localization method that reaches the CRLB (if any y = yk + mk (x − xk) (12)
estimator can)104,105,126 and requires detailed statistical knowl-
edge of the PSF and the noise.106 Rather than fitting the where mk is the slope of this line. Notice this slope can be
collected intensity data to the PSF model, MLE calculates the infinite for vertical lines. In the program, the author replaces the
likeliest set of parameters to generate the observed data. Except infinite with a large number. The distance between the center
for a few cases where the MLE can be calculated analytically,105 of the PSF (xc,yc) to this line is
the MLE estimator is found by calculating the likelihood for a (y − yk ) − mk (x − xk)
set of parameters and then iteratively updating the parameters dk =
to improve the likelihood until the algorithm converges. The 1 + mk 2 (13)
MLE is especially noteworthy due to the fact that it reaches the This center should minimize the weighted overall distance
CRLB if it is possible for any estimator to reach it. For this
∑k dk 2wk , which is the point where the derivatives of this
reason, MLE tends to be more precise than least-squares
estimation,126 though it has been shown that, at high noise objective function over xc and yc are zeros. This condition gives
levels, both methods approach the CRLB.104,119 us two independent equations with two unknowns (xc and
Anthony and Granick noted that taking the log of a Gaussian yc):119
function to generate a polynomial function allows 2D Gaussian −mk 2wk mk wk mk wk(yk − mk xk)
to be simplified as polynomial fitting, which is much faster:127 xc ∑ 2
+ yc ∑ 2
= ∑
k
mk + 1 k
mk + 1 k
mk 2 + 1
⎛ ⎡ ⎛ (y − y0 )2 ⎞⎤ ⎞ (14)
(x − x0)2
ln(Ixy) = ln⎜A exp⎢ −⎜⎜ + ⎟⎥ + εxy⎟
⎜
⎝
⎢⎣ ⎝ 2σx 2 2σy 2 ⎟⎠⎥⎦ ⎟
⎠ xc ∑
−mk wk
+ yc ∑
wk
= ∑
wk(yk − mk xk)
2 2
k
mk + 1 k
mk + 1 k
mk 2 + 1
2 2
≈ a1x + a 2x + a3y + a4y + a5 (11) (15)

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The weight wk is proportional to the magnitude of the
gradient and the inverse of the distance to the center at the
corner position (xk,yk). Knowing wk assumes that we already
know the center. Fortunately, the radial symmetry method is
not very sensitive to the weight. Using the estimated center
based on the centroid of the magnitude of the gradient is good
enough for the calculation. The radial symmetry method has
been shown to be as accurate and robust as Gaussian fitting, but
100 times faster.119
2.5. Simulating Experimental Data
While the precision of a localization method can be estimated
from repeated experimental trials, testing with simulated data is
an important step in determining the localization bias and
precision. Testing with experimental data requires accurate and
precise prior knowledge of the position and orientation of
single particles,106 which is not often feasible. Simulated images
are a benchmark used for most localization108,126,131 and
dynamics-tracking algorithms,125,132−136 though some tests
neglect this step.116,130 Photon counts in each pixel are usually
simulated by a Poisson random variable with a Poisson
parameter set by the PSF, which can be generated using Fourier
optics as discussed in section 2.2 or obtained through
integration of a theoretical PSF model over the pixel region.
This description of photon arrivals as a Poisson random
variable is known as “shot noise”. Shot noise is due to the
uneven distribution of photons,106 where each photon arrives at
a position drawn from the PSF (normalized as a probability
density function), and will be present even under ideal
circumstances with no scattered photons or detector noise.
Due to the discrete nature of pixels, the map of Poisson
parameters (equivalent to the pixelated PSF) encodes subpixel
information as asymmetry. Since localization procedures can
favor certain subpixel positions,120 the PSF used as the Poisson
parameter should be generated with random center positions
across the area of a pixel. Background noise is modeled in
different ways depending on the number of collected photons
and the properties of the camera. The background noise varies
based mainly on the camera and temperature of the sensor
array.120 If the noise distribution of the camera is determined
experimentally, noise intensity values for each pixel can be
chosen directly from the distribution.120 In most circumstances,
noise is modeled as Gaussian or Poisson. Approximations often
use additive Gaussian white noise,137 though for more precise
simulations Poisson noise should be used to model scattered
photons, dark current on the CCD chip, and autofluorescence,
while the Gaussian model is more appropriate for noise arising
from the CCD camera readout.105 An example of simulated
image generation is shown in Figure 9.
2.6. SPT Methods
SPT algorithm development is a relatively mature field but still
has room to grow.35 SPT algorithms can usually be classified
into several independent modules: signal-to-noise ratio (SNR)
enhancement, particle identification and localization, particle
tracking, and post dynamic/kinetic analysis.125 Some published
methods are specially focused on one module, such as the
fitting methods discussed in section 2.4, and the denoising
methods used in more general imaging processing.137 This
classification into modules also matches the recent SPT
competition, as shown in Figure 10.133,138 Of course, this is
only a rough grouping. For example, particle density levels will
influence both particle identification and particle tracking. But a
simplified classification will help researchers to better under-
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Review
Figure 9. Example of image simulation. (A) Pixelated Airy disk PSF
used as the parameters for the Poisson random variable to generate the
shot noise image in (B). (B−D) Noise elements for simulated image.
(B) Shot noise represents the statistical nature of the arrival of discrete
photons. (C) Background Poisson noise uses a constant Poisson
parameter, while (D) Gaussian noise is a zero-mean, constant standard
deviation random variable for each pixel. By randomly generating the
three noise distributions, multiple simulated images can be generated
for the same pixelated PSF. (E) Combined image with the three noise
sources to generate a single simulated image.
stand established methods, and to combine modules with
different algorithms for a customized SPT solution.
The SPT competition published in 2014133,138 considered 14
different algorithms covering 48 different situations in an
attempt to provide an objective comparison. While other
comparisons of SPT methods had been previously pub-
lished,114,139−142 these studies were limited in scope,133 and
the need for a broader comparison was recognized.37 The SPT
competition, as discussed by Chenouard et al., provided
training data with ground truth for four dynamics scenarios
(Brownian motion, directed motion, and random switching
with either random or fixed directed motion components),
three different particle density levels, and four different SNR
levels. Figure 10 shows examples of the four different dynamic
scenarios included in the competition. Ideally, having different
research groups implementing their own algorithm on a
common data set with standard evaluation metrics provides a
more objective comparison than previous studies.133 Even
though not all the algorithms were applied to all the situations,
most of the algorithms were designed for broad applications.
Importantly, it was concluded that no single algorithm
performed best for all situations simulated, which means it is
better to narrow down the target application when choosing or
designing an SPT algorithm.138 Moreover, real experimental
situations are usually more complicated than those presented in
the competition. For example, the competition did not consider
uneven background, which is usually unavoidable due to
astigmatism and tracking in complicated environments.
However, this competition provides a library for future
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Figure 10. Simulated SPT data for different experimental situations for the four transport dynamics used in the 2014 SPT competition. For each
scenario, a typical snapshot image (i−iv) and analyzed trajectories over multiple images (v−viii) are shown. Colors are randomly assigned to different
trajectories. Scenario 1: Brownian diffusion imaged by wide-field microscopy (i, v). Scenario 2: Directed movement (ii, vi). Scenario 3: Randomly
switching between Brownian diffusion and directed movement with random directions imaged by confocal microscopy (iii, vii). Scenario 4:
Randomly switching between Brownian diffusion and directed movement with restricted directions imaged by 3D scanning confocal microscopy (iv,
viii). Not shown here are the four SNR levels and three density levels that were also used for simulating data. Reprinted with permission from ref
133. Copyright 2014 Nature Publishing Group.

researchers to search for the method they need for a particular
situation. This is extremely important for both 2D and 3D
tracking, considering all the proposed tracking setups are
different.
The results of the SPT competition show that “the quest for
better particle tracking methods remains,” and provides a
baseline for researchers to use when pursuing them.133 Due to
the modular nature of SPT, different portions of an SPT
algorithm can often be swapped out,142 so algorithms can be
generated by adapting methods that have already been
developed. While a complete summary of all the algorithms
in use today is beyond the scope of this work, a few
representative algorithms are detailed below. The first two
algorithms described are from the SPT competition: the
method submitted by W. J. Godinez and K. Rohr (GR) and the
method submitted by I. F. Sbalzarini, Y. Gong, and J. Cardinale
(SGC). GR was considered the most accurate overall when
counting the number of times it landed in the top three for
different scenarios, though this metric favors methods that
submitted results for all scenarios. SGC was the second most
accurate by this ranking, as well as being the fastest algorithm
based on computation time. It is difficult to compare even two
methods across all metrics, but in general, at the lowest SNR
level GR better determined the particle velocities, while SGC
better determined the particle localizations. This may be due to
the iterative localization technique utilized by SGC.133 Notably,
GR has superior localization accuracy at low SNR for the case
of diffusion with directed motion, which may be due to its use
of a theoretical dynamic model in execution.
GR, submitted to the SPT competition by W. J. Godinez and
K. Rohr, is a probabilistic SPF algorithm that utilizes Kalman or
interacting multiple model (IMM) filters with multiple
measurements.133,143 Probabilistic approaches, which generally
include a filtering step,142 provide a means to handle the
uncertainty of the measurement and movement of particles.143
They make up an important class of SPT algorithms, and
operate in a fundamentally different way from more traditional
deterministic algorithms (of the 14 algorithms in the SPT
competition, only two of them used filtering for particle
7340
linking133). Key concepts for SPT by filtering are the state
vector xt, which describes the particle, and the posterior
distribution of the state vector p(xt|y1:t), which represents how
likely the particle is to be in a certain state given a series of
measurements yt.142 The measurements are obtained by top-
down and bottom-up methods: top-down measurements are
taken from the predicted position x̂t and surrounding points
from the elliptical validation region defined with the innovation
covariance matrix, while bottom-up measurements are based on
deterministic localizations from the image, assigned to the
appropriate filter through a global nearest-neighbor
scheme.133,143 In this application, localization was carried out
by using the spot enhancing filter to enhance Gaussian-like
particles142,144 and intensity-weighted centroid or Gaussian
fitting,133 though other deterministic localization techniques
could be used.
Each tracked particle has its own filter, so determination of
particle position automatically links positions together into
trajectories, rather than separating localization and tracking into
separate modules.143 The filter computes the particle position
from multiple measurements along with their associated
weights, given by the probability that each measurement
generates a Gaussian spot when compared to measured pixel
intensities.143 In order to track particles in close proximity, a
reweighting term is applied by penalizing positions with high
support for neighboring objects.133,143
SGC, submitted to the SPT competition by I. F. Sbalzarini,
Y. Gong, and J. Cardinale, utilizes weighted centroid local-
ization with combinatorial optimization for path linking over
several frames for computationally fast tracking and robust
performance across a wide SNR range.133 One key difference
between SGC and GR (described above) is that SGC requires
no prior knowledge about the motion of the particle, only
requiring assumptions about the size of the particle image,
about the limit on particle speed, and that particle
disappearances occur on short time scales.133,145 Another
difference is that it is fully a deterministic SPT method, and
includes particle localization and tracking as independent
modules. Image restoration is performed by convolution of
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Figure 11. (A) Representative m0, m2 plane where potential particles designated as true particles are shown as crosses and rejected particles are
shown as open circles. (B) Confocal image from virus tracking experiment with reversed image intensities. The crosses represent the location of
particles accepted during the discrimination step; larger or darker spots are internalized virus particles that are excluded from tracking. Inset provides
detail in the indicated region. Reprinted with permission from ref 145. Copyright 2005 Elsevier.

Figure 12. Detection strategy for high-density SPT. (A) Hypothesis test to determine if the selected region contains any PSF. (B) Exhaustive particle
detection process. The first iteration detects and fits the most intense PSF. Subtracting the first fit allows the weaker PSF to be detected in the next
iteration. (C) Detection strategy applied to an experimental image. The left column shows images after last deflation in each iteration. The middle
column shows identified peaks in each iteration. The right column shows the fitting images used for deflation in each iteration. Reprinted with
permission from ref 147. Copyright 2008 Nature Publishing Group.

frames with a kernel combining background removal and noise
removal. The algorithm operates under the assumption that
particles will overlap over the course of their trajectory, which
could limit the algorithm’s usefulness in scenarios where
7341
particles will be in close proximity.133 Local maxima are used to
initially estimate particle localizations and are refined through
iterative weighted centroid calculation. Nonparticle discrim-
ination is implemented through an algorithm based on the zero
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Chemical Reviews
and second intensity moments about each potential par-
ticle.145,146 The zero and second intensity moments are given
by, respectively
m0 = ∑ Atf (x + i , y + j)
i2 + j2 ≤ w2
and
1 estimate the population MSD as a function of time. The time
m2 = ∑ (i 2 + j 2 )Atf (x + i , y + j)
m0
i2 + j2 ≤ w2
where i and j are pixel indices, x and y are the indices of the
candidate pixel, and Atf is the filtered frame at time t.145 When
viewing the m0, m2 plot of all the potential particles, clustering
will occur based on their intensity distributions. Each potential
particle is assigned a 2D Gaussian profile in the m0, m2 plane,
and each point is then assigned a score which is the value at its
location for the summation of all of the rest of the Gaussian
profiles.145 Assuming that the majority are true particles, the
true particles are identified by scoring over a user-defined
threshold, indicating that they are the most tightly clustered.
Figure 11 A shows an example of clustering in the m0, m2 plane.
Trajectory linking is carried out through a combinatorial
optimization problem, though other methods could be
substituted. For each frame there is an association matrix that
defines linking between particles. The association matrix is
restricted such that each particle is assigned to exactly one
particle or to a dummy particle in the next frame. By calculating
particle linking across multiple frames, the algorithm can deal
with temporary disappearance of particles. Initial particle
linking is determined by nearest neighbors, and a greedy hill-
climbing algorithm is used to find the configuration of the
association matrix that minimizes a cost function based on
location, total intensity, and the second intensity moment. A
technique employed by this method is assigning an infinite cost
to particles that are separated by a set cutoff distancethis
reduces computation time by eliminating the need for
extraneous calculations.145
One of the SPT algorithms not included in the SPT
competition is Sergé’s multiple-target tracing algorithm.147
Sergé et al. applied hypothesis tests in particle detection and
particle tracking to improve the sensitivity of the algorithm,
especially for high-density tracking situations with probes
having a large distribution of brightness, as shown in Figure 12.
Their algorithm first identifies all the local intensity maximums
and then applies a statistical test to each selected region to
determine if there are PSFs in the region (Figure 12A). Once
they determine there are PSFs in the region, they fit the PSF
and subtract the fit from the image. After that, they repeat the
same process (hypothesis test, PSF fitting, PSF subtraction) on
the leftover image until no PSF can be found in the image
(Figure 12B,C). This is a great strategy to analyze high-density
SPT images; however, overfitting problems need to be
considered for uneven background or misspecified PSFs.
2.7. Mean Square Displacement (MSD) Analysis
The diffusion coefficient D is one of the fundamental properties
used to describe particle motion in SPT. A number of methods
have been developed to determine the diffusion coefficient,
with MSD analysis as one of the fundamental tools. MSD can
be calculated a number of ways, but a common definition is
Review
1
MSD(n δt ) =
N−1−n
N−1−n
∑ [r((j + n) δt ) − r(j δt )]2
j=1 (18)
(16)
where r(t) is the position of the particle at time t, n δt is the
time lag MSD is calculated at, and δt is the smallest time
interval resolved.148,149 MSD is calculated for each trajectory to
interval δt between each position measurement defines the
(17)
discrete steps of the MSD. Calculation of MSD for small time
lags includes more trajectories that contribute to the average,
increasing precision.132 In practice, the maximum value of n δt
N
for which MSD is calculated is bounded, such as n < 10 ,149 to
avoid the imprecise estimates for larger time lags. The
increasing overlap of trajectories for increasing time lag also
complicates estimation of MSD due to the dependence of
consecutive trajectories,132 though other definitions of MSD
which only include nonoverlapping trajectories exist to address
this.148 Diffusion is an inherently random process, and as such
any calculation of MSD and diffusion coefficient will have a
statistical variance. The diffusion coefficient and other
applicable parameters can be determined by fitting the MSD
vs time plot to an appropriate diffusion model, which will be
detailed in section 2.8. For an overview of some common
diffusion models and their functional form, see ref 150.
Brownian diffusion, directed diffusion, and anomalous sub-
diffusion are described briefly below.
For Brownian diffusion, the diffusion coefficient is constant
and can be determined from the slope of a linear fit to MSD vs
time using the Einstein relationship, MSD(t) = 4Dt, for
diffusion in 2D. Brownian diffusion is a valid approximation
when diffusion is unhindered and is occurring in a sufficiently
large space. Specifically, the Brownian diffusion model is
appropriate when the total measurement time is much less than
L2/4D for the characteristic length of the space L. When
diffusion is confined to a smaller region, the limit of MSD as t
→ ∞ for 2D diffusion is proportional to the area of
confinement.148,150
It is important to be aware of the pitfalls when visually
inspecting plots of MSD vs time for a small number of
trajectories. Due to the inherent randomness in a random walk,
a particle undergoing Brownian diffusion can easily appear to
undergo confined diffusion or directed diffusion over certain
periods.148,150 Detection methods with a proper null hypothesis
of ideal diffusion can be employed to determine if an alternate
type of motion is present.151
Directed diffusion, or diffusion with flow, alters the transition
probability such that the MSD will be quadratic with respect to
time, given by
MSD(t ) = 4Dt + V 2t 2 (19)
where V is the constant velocity flow rate for directed diffusion
in 2D. Plotting MSD as a function of time will result in a
quadratic curve, with the effect of the flow rate dominating for
larger values of t, while the effect of diffusion dominates for
small values of t. If the motion is identified as directed diffusion,
curve-fitting the plot of MSD vs time can be used to estimate D
and V.152,153
Anomalous subdiffusion can occur due to interactions with
obstacles that alter diffusion, which can be observed as an MSD
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Chemical Reviews
plot with a negative curvature. The time-dependent diffusion
coefficient for anomalous subdiffusion approaches zero for long
times, though certain obstacle structures can result in the
diffusion dynamics shifting to hindered diffusion at large time
scales.150 This is described by Qian et al.148 as a higher,
localized diffusion coefficient that dominates for short time
scales, with the long-term diffusion being determined by the
effective diffusion coefficient. The localized diffusion coefficient
is observed for distances less than the characteristic separation
of the mobile or immobile obstacles, since the diffusing
particles are effectively “in between” obstacles.
2.8. Ergodic Hypothesis in SPT
For the discussion of trajectory lengths and true representations
of dynamic populations and subpopulations, one must consider
the ergodic hypothesis and its applicability to SPT. The ergodic
hypothesis states that it is reasonable to produce ensemble
MSD values of a system from SPT time averages, given the
single particle can be tracked over a long period of time.154
However, the ergodic hypothesis is based on the assumption
that the observation time is infinite. In SPT experiments, the
observation time must exceed the characteristic time of the
diffusion to fulfill the ergodic hypothesis, which is often
challenging to achieve given the photobleaching of emitters and
the finite recording time of detectors. Consequently, there are a
few experimental observations in which the particle diffusion
shows nonergodic behavior in intracellular particle trans-
port.155,156 The first nonergodic behavior was reported by
Bouchaud et al.157 and then Weigel et al.155 Here we highlight
the later one as an example for ergodicity breaking.
The observed anomalous diffusion in intracellular transport is
usually explained by the nonergodicity of the system. Weigel et
al. reported that nonergodic processes exist in the plasma
membrane. By tracking GFP-Kv2,1 (labeled by QDs) expressed
in embryonic kidney (HEK) cells, they observed anomalous
diffusion behavior (Figure 13A), which is indicated by double
exponential curve fitting. By comparing the time averages and
ensemble averages of the single particle trajectories, it is
claimed that the Kv2,1 transport dynamics exhibit nonergodic
behavior (Figure 13B). Weigel et al. showed that the breaking
of ergodicity can be experimentally noticed only when the
behavior of a single particle was compared to the average
Figure 13. Statistical analysis of Kv2.1 channel trajectories. (A) The
cumulative distribution function of square displacement of a clustered
channel with tlag = 0.1 s. The curve is fit with both single and
biexponential distributions, and the biexponential model gives smaller
residuals. (B) Distribution of MSD values calculated from individual
trajectories and ensembles in clustered channels for tlag = 0.1 s.
Adapted with permission from ref 155. Copyright 2011 National
Academy of Sciences.
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behavior of many others, therefore large statistics is the key for
such observations.
3. REVEALING DYNAMIC PROCESSES USING 2D SPT
3.1. Two-Dimensional Interfacial Dynamics
The most basic way to extract dynamics information from a
sample is to directly observe the phenomenon in question.
Typically, in fluorescence measurements, this is done using
wide-field microscopy. In its most basic implementation,
transport is observed as a 2D projection on the sample plane.
For this reason, observation of planar samples is common.
Because of the limited photon budget when using single-
molecule emitters, it is common to use total internal reflection
fluorescence (TIRF) instead of standard wide-field illumination
as TIRF limits the excitation volume to a very narrow depth
(<100 nm), reducing the contribution of out-of-focus back-
ground light.158
TIRF imaging is accomplished using a typical wide-field
epifluorescence microscope. In objective-TIRF the sample of
interest is placed atop a high NA (∼1.4) objective through
which monochromatic laser light is directed to the sample at a
high angle of incidence (with a high NA objective angles of
>69° are usually achievable). The incidence angle is higher than
the critical angle of the sample−solution interface (typically the
interface of glass and water) and thus results in total internal
reflection and the formation of an exponentially decaying
evanescent wave at the interface.159 Emitted fluorescence is
captured by the same objective with filters removing undesired
background such as scattered laser light. Fluorophores are
imaged by capturing sequential images using a high sensitivity
camera. Modern cameras can achieve frame rates in excess of
100 frames per second for 1024 × 1024 pixels. Higher frame
rates are achievable if the readout area of the chip is
reduced,77,81,160 or alternatively using CMOS based SPAD
arrays.77,79,161 TIRF may also be achieved through the use of a
prism. To achieve prism-TIRF, a prism is coupled to the side of
the sample opposite the objective and the excitation light is
coupled into the prism at the desired angle. As in objective-
TIR, an exponentially decaying evanescent wave is generated at
the sample interface with fluorescence emission collected by the
objective. Prism-TIRF requires a more complicated sample
geometry, but does not require the use of an expensive high NA
objective.162 After acquisition, image stacks are processed to
increase the signal-to-noise ratio of each image, identify
fluorescent event locations, and link events across multiple
frames. Additional analyses for SPT and super-resolution image
processing may also be conducted as described in the following
sections.
Confocal imaging is also an effective method of imaging
single fluorophores with minimal background interference.
Confocal imaging involves the raster scanning of a laser focal
volume across the sample and detecting the fluorescence signal
using a single point detector such as an APD or PMT.
Unfortunately, the slow nature of raster scanning makes
confocal imaging useful only for systems devoid of dynamics
on time scales faster than ∼1 s.163
As discussed in section 2.6, there exist a wide variety of SPT
methods and there is no one method that can be singled out as
the best due to the wide variety of potential use cases, but
objective comparisons of these methods exist and suggest that
certain methods work best in the low signal-to-noise regime of
single-molecule measurements.114,139−142,164,165 It was found
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that, generally, methods involving the preprocessing of images
prior to localization performed better.114,125,139−142,164 Selec-
tion of a proper algorithm along with careful experiment design
can allow for new insights using these relatively common
methods.
Even in the simplest case of 2D transport, the diffusion of a
molecule at a solid−liquid or liquid−liquid interface, SPT
measurements have recently provided a new understanding of
the underlying mechanisms at play. It has been known that
interfacial processes are often non-Fickian (their MSD does not
increase linearly with time), but it has also recently been
demonstrated using SPT that even when a process appears
Brownian, it can also exhibit anomalous diffusion.166
Single-molecule tracking analyses from the Granick group
have revealed deviations from Fickian diffusion behavior in
surprisingly simple systems.166 Fluorescent colloidal beads were
measured diffusing along linear phospholipid bilayer tubes and
within F-actin filament networks. The data for beads diffusing
on linear tubes is shown in Figure 14. The MSD as a function
Figure 14. Demonstration of non-Fickian diffusion with linear MSD.
(A) MSD curves (with a slope of 1) for fluorescent particles diffusing
on lipid bilayer tubes without (top line) and with (bottom line) 40%
cholesterol. Curves are plotted on a log−log scale. (B) Logarithmic
plots of the displacement probability plotted against linear displace-
ment normalized by particle diameter for several representative values
of time step: 60 ms (squares), 0.6 s (circles), 3 s (crosses), and 5.8 s
(triangles). Adapted with permission from ref 166. Copyright 2009
National Academy of Sciences.
of time is shown in Figure 14A. The linear appearance would
suggest that the transport process being observed is Fickian.
However, a deeper investigation reveals that this is simply not
the case. When the normalized displacement distributions,
determined from hundreds of trajectories, are examined for
different observation times (Figure 14B), a shift is observed
from an exponential decay (linear in the plot) to Gaussian. This
Figure 15. (A) Desorption mediated diffusion schematic showing intermittent surface association events. (B) Waiting time distributions showing the
waiting time between displacements of >0.2 μm. The data sets were translated vertically to allow easier interpretation (the PEG, Atto6G, and
BODIPY data were shifted by a factor of 101, 10−2, and 10−3, respectively). The dashed line represents a curve with a power law exponent of −2.5.
Adapted with permission from ref 184. Copyright 2013 American Physical Society.
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is not at all representative of classical Brownian diffusion (see
section 2.7 for details) and requires a new model to explain.166
Several models of subdiffusion have been developed to
explain nanometer scale dynamics. Other research groups have
also reported findings similar to those of the Granick group,
and various attempts have been made to determine appropriate
models to explain the data.167−180 Generally speaking,
anomalous diffusion is a nonspecific phenomenon and
describes the case when diffusion deviates from expected
Fickian behavior. There are of course many causes for this
deviation. Activated diffusion is one such case.157 Diffusion in a
lipid bilayer system is often explained as lipid transport
resulting in fluctuating local heterogeneities resulting in
diffusivities that change over time.175 Various other crowding
effects can also result in non-Fickian diffusion.173,179,180 For
characterizing interfacial diffusion, fractional Brownian motion
and the continuous time random walk are often used.181
Because of the generality of anomalous diffusion, it is important
that the model used to explain the results has physical
justifications.
Understanding interfacial transport mechanisms is extremely
important for optimizing the function of membranes and
chromatographic stationary phase materials. Recently Schwartz
and co-workers have used SPT to determine that the primary
mechanism for biological transport at stationary phase materials
is best classified as desorption mediated diffusion.63,182−187 This
desorption mediated diffusion can be modeled as a continuous
time random walk, and is yet another potential candidate when
anomalous diffusion is observed.166,184 This type of diffusion
consists of repeated surface associations punctuated by
excursions of the diffusor into the bulk medium. This is
schematically represented in Figure 15A. Using single-molecule
tracking, the repeated adsorption events from the same emitter
can be monitored and appear as hops across the surface as bulk
excursions tend to occur faster than the frame rate of the
camera being used to observe. When molecules adsorb to the
interface they remain for a period of time, known as the waiting
time. Distributions of the waiting times for a variety of emitters
are shown in Figure 15B. For single-molecule dyes, proteins,
and polymer chains, the waiting time distribution for
adsorption to a hydrophobic silane interface was found to
roughly obey a power law distribution with an exponent of
approximately 2.5.184
These results have been replicated for a variety of interfaces
and diffusor types. Using SPT, desorption mediated transport
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modes have been observed for small molecule organic
dyes,171,184,188 polypeptides/proteins,20,189,190 and long chain
polymers.183,186 While the initial measurements focused on
silanized hydrophobic model surfaces, desorption mediated
dynamics have been observed over polymer matrices,170,183 at
the oil−water interface,20,190 and functionalized substrates for
separations.171 There is also evidence that for certain types of
diffusors, namely long chain polymers, in-plane diffusion occurs
during the adsorption period.183
Desorption mediated transport is also a key factor in more
complicated tunable polymer interfaces. When exposed to a 10
bilayer film composed of alternating layers of poly(allylamine
hydrochloride) (PAH) and poly(acrylic acid) (PAA), small
molecule ionic probes were found to exhibit desorption
mediated dynamics.188 Furthermore, tuning the solution pH
post assembly allowed charge dependent tuning of the
intermittent surface interactions. The single frame displacement
histograms and analyses for this study are summarized in Figure
16. For the anionic probe, Alexa 555, interaction with the
Figure 16. Single frame displacement histograms for Alexa 555 and
Rhodamine 6G at three different pH conditions. (A, B) Displacement
distributions for Alexa 555 and Rhodamine 6G in HCl (pH 3.5). (C,
D) Displacement distributions for Alexa 555 and Rhodamine 6G in
MB water (pH 5.7). (E, F) Displacement distributions for Alexa 555
and Rhodamine 6G in Tris buffer (pH 8.7) Black and gray symbols
represent experimental data and Markov chain Monte Carlo
approximations, respectively. Adapted from ref 188. Copyright 2014
American Chemical Society.
substrate decreased as pH was increased and the long distance
transport mode indicative of hopping was suppressed (Figure
16A,C,E). When a cationic probe (Rhodamine 6G) was used,
roughly the opposite trend was detected (Figure 16B,D,F).
Ionic transport on multilayer films was found to be governed
by complex interaction mechanisms involving electrostatic,
hydrophobic, and steric forces. Electrostatic interactions were
used to explain the interaction dynamics of anionic Alexa 555.
As the outermost film layer became more positively charged at
lower pH values, the anionic probes were more likely to interact
with the surface resulting in the observed desorption mediated
diffusive behavior. In contrast, the interaction of cationic R6G
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with the films as a function of pH likely involves a combination
of electrostatics and sterics. Hydrophobic interactions are also
expected to play a role.188 These studies of tunable interfacial
diffusion were later extended to study the effects of
functionalization on desorption mediated diffusion.171 It was
found that ligand density determined the density and thus the
availability of binding sites for adsorption.
3.2. Imaging 2D Structures with SPT
SPT is particularly useful in the study of diffusion in highly
confined environments and when combined with anisotropy
calculations can yield subdiffraction limited structural informa-
tion. Such tracking experiments typically yield one-dimensional
trajectories. Since the mid-2000s there have been several
studies of confined diffusion in polymer matrixes, liquid
crystals, and mesoporous silica.191−195 Diffusors are also
expected to be rotationally restricted in such structures.195−197
Pramanik et al. recently reported the first quantitative analysis
of lateral and orientational confinement of single molecules in
mesoporous silica.191 In order to quantify the dipole emission
angle of diffusing N,N′-bis(octyloxypropyl)perylene-3,4,9,10-
tetracarboxylic diimide (C11OPDI) molecules within the pores,
circularly polarized excitation light was used to excite all
molecules. Detected fluorescence was split into orthogonal
polarizations and imaged on separate halves of a CCD camera.
This type of detection scheme is known as single-molecule
emission dichroism (SMED).198 The two orthogonal images
detected for C11OPDI diffusing in silica mesoporous films with
two different templates are shown in Figure 17. The one-
dimensional pores can be clearly observed as well as the
dependence of dipole angle on the pore direction. Accounting
for the depolarization of high NA optics (a2) and the average
dipole orientation (ϕ) of each molecule, equations relating the
emission in each channel to the maximum allowed wobble
angle (θmax) within the pore were developed (eqs 20 and 21).
1 2
IV ∝ cos2 ϕ(1 − cos3 θmax ) + (a + sin 2 ϕ)
2
(2 − 2 cos θmax − cos θmax sin 2 θmax ) (20)
1 2
IH ∝ sin 2 ϕ(1 − cos3 θmax ) + (a + cos2 ϕ)
2
(2 − 2 cos θmax − cos θmax sin 2 θmax ) (21)
From the average wobble angle, an estimate of the pore
diameter can be produced. For the pores studied in the
experiment, an ensemble wobble angle of 19 ± 3°
corresponded to an average pore diameter of 1.3 ± 0.2 nm,
much smaller than the expected pore size of around 4 nm. This
is attributed to hydrophobic interactions restricting the dye
molecule to the center of the pore.
A combination of particle tracking and stochastic frequency
analysis has been used to analyze transport in biological
systems. Recent experiments in live cells have demonstrated the
applicability of SPT tracking in these complex and confined
environments, providing new information on DNA replication
and repair. In order to determine how the protein MutS, known
to be involved in DNA mismatch repair, searches for errors, the
Biteen group constructed strains of Bacillus subtilis that natively
expressed MutS conjugated with the fluorescent label
PAmCherry1 schematically represented in Figure 18A.199
Previous experiments targeting these structures were at the
ensemble level and thus were unable to extract information on
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Figure 17. Polarization resolved diffusion in 1D structures. s and p
polarization resolved wide-field images (summed over 100 frames) for
C7OPDI- and C1PDI-doped CTAB-template mesoporous silica films.
The double-ended arrows designate the detected polarizations.
Reprinted (adapted) from ref 191. Copyright 2013 American
Chemical Society.
the location and dynamics of MutS with any specificity.200 By
using a combination of PALM and SPT (Figure 18B,C) the
Figure 18. Location and dynamics of MutS in live B. subtilis. (A) Labeling scheme for MutS−PAmCherry. RBS, ribosome binding site. (B)
Representative frames showing the photoactivation of a single copy of MutS−PAmCherry in a cell. Lines above the images correspond to the initial
activation pulse then imaging laser. (C, lower left) Photoactivated localization microscopy52 reconstruction assembled from localized MutS−
PAmCherry molecules and (C, right) single-molecule trajectories of MutS−PAmCherry in (C, upper left) a liveB. subtilis cell. The red arrow
indicates a region of MutS accumulation. White dashed lines indicate the computer-detected cell boundary (scale bars: 1 μm). Adapted with
permission from ref 199. Copyright 2015 National Academy of Sciences.
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location and dynamics of MutS were determined. It was found
that MutS binding seems to only occur at the replisome, a
phenomenon that is distinctly different compared to the
process in eukaryotic cells.199 These and other examples
demonstrate that, even in the crowded complicated environ-
ment of a bacterium, SPT can reveal useful information with
careful experimental design.201,202
3.3. Enhanced Spatial Resolution as a Stepping Stone to
Improved 2D SPT
Although SPT involves the quantization of dynamic properties
including transport and hopping, the field has benefited from
multiple exciting advances in single-molecule fluorescence
microscopy to improve the spatial resolution of static analytes.
In 2006, several super-resolution imaging methods were
independently developed paving the way for subdiffraction
limited characterization of dynamic processes.10,12,203 In
contrast to STED,84,85 discussed in section 1, these methods
are largely similar in that they all rely on the repeat stochastic
localization of emitters to build up an image with fine detail that
is not detectable using conventional light microscopy. The
techniques differ in the types of probes used and the method of
repeat localization. Point accumulation for imaging in nanoscale
tomography (PAINT) relies on the diffusion of fluorophores
that will stochastically bind to the structure of interest. Repeat
binding events are monitored allowing the creation of a super-
resolved image (Figure 19A,B).203 PAINT requires isolated
binding events and is the most closely related to dynamics
measurements that will be discussed later in this section.
PALM52 (Figure 19C−F) and STORM (Figure 19G) are very
similar in methodology.10,12 The original publications differed
only in systems to which the technique was applied, specifically
the type of probes used. Both use an activation laser to
selectively excite a small number of fluorophores in a heavily
labeled region. The stochastically excited fluorophores are
resolvable individually allowing the subsequent reconstruction
of super-resolution images containing detail at a resolution of
∼20 nm.10,12 Reconstruction is accomplished by localizing
detected PSFs with high precision as discussed in section 2.4.
The aggregated blinking or binding events from many (typically
hundreds to thousands) of sequential frames are plotted to
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Figure 19. Summary of super-resolution imaging techniques. (A, B)
Demonstration of PAINT. Image of a supported lipid bilayer on glass,
probed by Nile red. (A) Standard fluorescence image. (B) High-
resolution synthetic image obtained by locating 2778 single Nile red
probes collected in 4095 frames.203 (C−F) Demonstration of PALM.
Comparative summed-molecule TIRF (C) and PALM (D) images of
the same region within a cryo-prepared thin section from a COS-7 cell
expressing the lysosomal transmembrane protein CD63 tagged with
the PA-FP Kaede. The larger boxed region in (D), when viewed at
higher magnification (E), reveals smaller structures not resolvable in
typical TIRF. In a region where the section is nearly orthogonal to the
lysosomal membrane, the most highly localized molecules fall on a line
of width of ∼10 nm (inset). In an obliquely cut region [(F), from the
smaller boxed region in (D)], the distribution of CD63 within the
membrane plane can be discerned.10 (G) demonstration of STORM.
STORM images of RecA-coated circular plasmid DNA. Indirect
immunofluorescence images with switch-labeled secondary antibody
taken by a total internal reflection microscope (top); the reconstructed
STORM images of the same filaments (bottom). Scale bars, 300 nm.12
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Figure 19. continued
Reprinted with permission from refs 10, 12, and 203. Copyright 2006
The American Association for the Advancement of Science (ref 10),
2006 Nature Publishing Group (ref 12), and 2006 National Academy
of Sciences (ref 203).
create the reconstructed image. These were all originally
imaging techniques, and extensive reviews can be found
elsewhere.13,14,17,204,205
The importance of super-resolution imaging methods such as
PAINT to SPT applications is that their subdiffraction
localization algorithms can be adapted to determine interfacial
dynamics with high temporal and spatial resolutions. Typically,
the PAINT variant mbPAINT (motion-blur points accumu-
lation for imaging in nanoscale topography) is used for this
purpose and is based on the diffusion and adsorption properties
of fluorophores to simultaneously image and extract kinetics at
a nanoscale level. In such methods, small fluorescent diffusing
probe molecules can be selectively observed only when
adsorbed to the target structure at the interface, and are
made unobservable by motion blur when freely diffusing (D ∼
hundreds of μm2/s) in bulk solution compared to the detector
temporal resolution (tens of hertz).
Several research groups have utilized mbPAINT to extract
the kinetics of DNA hybridization.206−211 DNA hybridization
experiments are an excellent test bed for mbPAINT because
probe DNA sequences embedded at the surface will capture
only the corresponding target DNA sequence in solution with
high selectivity. A typical mbPAINT experiment with DNA
hybridization is schematically represented in Figure 20A.
One of the first applications of mbPAINT was to directly
measure the first order kinetics of DNA origami hybrid-
ization.208 The use of mbPAINT allowed for the monitoring of
individual hybridization events. When matching target ssDNA
bound to its corresponding immobilized probe ssDNA on the
surface, it created a bright event. The dark time could also be
monitored (by counting remaining bright spots over time
represented in Figure 20B, inset), allowingd the determination
of the association rate kon (Figure 20C). As would be expected
for a first order process, the reciprocal of the dark time
increases linearly with concentration. A linear fit yielded a rate
constant kon of 2.3 × 106 M−1 s−1, which was comparable to
previously reported ensemble values. After verifying that
excitation laser power did not influence the determination of
koff, it was confirmed that koff did not depend on concentration,
again confirming a first order rate process.208 In addition to the
kinetics determination, the use of mbPAINT allowed for the
simultaneous acquisition of subdiffraction limited images of the
DNA origami structures.
Improving the methodology of single-molecule DNA
hybridization assays is a current area of active research. Two
major obstacles to accurate DNA hybridization kinetics
determination are nonspecific adsorption to the substrate and
the influence of labels on the stability of the hybridized DNA
structures. Nonspecific adsorption can be minimized by using
very high surface coverages of probe DNA as demonstrated by
Peterson and co-workers.209 Ensuring that the concentration of
surface probe sites was at least 100 times more concentrated
than the optically resolvable limit reduced nonspecific binding
to negligible levels.209 Interestingly, the desorption time
histogram from these experiments (Figure 20B) was best fit
with a double exponential decay suggesting an additional
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Figure 20. mbPAINT can be used to determine DNA hybridization kinetics. (A) Schematic showing the method of DNA hybridization detection.
(B) Sample single-molecule residence time histogram (black circles) representing ∼3500 10-mer target ssDNA visits on a probe surface fit to a
double-exponential decay function. (inset) Images showing single-molecule locations. These images represent a time series and show the
disappearance of tracked molecules along with new binding events. (C) The reciprocal of the interevent lifetime τd increases linearly with the
concentration of the imager strand. The linear fit yields an association rate (the rate of new binding events) kon of 2.3 × 106 M−1 s−1 at a salt
concentration of 600 mM NaCl. The dissociation rate (the rate at which the DNA strands unbind) koff is independent of the imager strand
concentration, as expected for a first-order reaction. Reprinted from refs 206, 208, and 209. Copyright 2013 (ref 206), 2010 (ref 208), and 2016 (ref
209) American Chemical Society.

Figure 21. Ligand clustering effects on protein adsorption kinetics and separations. (A, B) Cartoons of protein interacting with (A) engineered
clusters and (B) individual ligands immobilized on a porous support. (C, D) Super-resolution images showing adsorption is only detectable at ligand
clusters. (E) Relating rate constants through a theoretical model predicts that engineered clusters would induce more efficient protein separation.
Adapted with permission from ref 27. Copyright 2014 National Academy of Sciences.

adsorption pathway and was attributed not to bleaching, which
was ruled out in control experiments, but rather to desorption
of the double stranded DNA duplex following binding. This
suggests that consideration must be given to the chosen
immobilization scheme. The stabilizing effects of labeling DNA
with fluorescent dyes have recently been demonstrated.212
Competitive single molecule binding assays have been
demonstrated as a solution to this potential issue.207
Stochastic based super-resolution imaging methods have
been adapted to understand the mechanisms driving the
adsorption of proteins onto chromatographic substrates (Figure
21).27 We studied the dynamics of adsorption of a model
protein, α-lactalbumin, to clustered charge vs individual
argininamide ligands (Figure 21A, B) on agarose substrates.27,29
Super-resolution images are produced by localizing multiple
adsorbed dye-labeled proteins. A low protein concentration
ensures that only a few fluorophores are adsorbed at a time
within a single frame. The previously described 2D super-
localization methods can thus achieve simultaneous ∼30 nm
precision as well as dynamic information about desorption and
adsorption statistics.27,206 Potentially important conclusions can
be drawn from super-resolved spatial and kinetic character-
7348
ization. For example, by combining adsorption and desorption
kinetics with a statistical mechanical model of chromatography,
these experiments suggest that much of the broadening that
occurs in typical protein ion-exchange separations occurs due to
stochastic ligand clustering (Figure 21E).27
3.4. Correlation Analysis
It is important to contrast SPT with other methods for
determining transport from single-molecule image series.
Correlation methods can be applied to existing movies taken
on conventional wide-field microscopes with little to no
modification of the instrument or data collection protocols.
Modern correlation-based methods for determining transport
dynamics at the single-molecule level are rooted in fluorescence
correlation spectroscopy (FCS), originally an ensemble analysis
technique developed by Elson and Magde.49,213 Image
correlation spectroscopy (ICS) was developed for confocal
scanning microscopes and operated in the spatial domain.214
Later it was extended into the temporal domain and was used
to characterize dynamics in live cells.215,216 While ICS allows
for the measurement of dynamics over larger areas than
conventional FCS, the temporal resolution is low with
accessible time scales of tens of seconds to minutes. The
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Figure 22. (A) Imaging of DiI on C18-modifed surface (30 ms acquisition time, 128 × 128 pixels). The expanded inset shows the various imaging
FCS observation areas, and the 3D intensity plot shows the molecular PSF of an emitter over the 8 × 8 pixel area. Molecular spots had an average
signal-to-noise (S/N) ratio of ∼4. (B) Normalized autocorrelation functions for varying probe region sizes fit to eq 20. An example residual plot is
from the 8 × 8 pixel autocorrelation included. (C) Plot of 1/τ versus 1/ω2 showing the expected linear dependence of the diffusion time extracted
from the fits in (B) to the width of the observation area. Adapted from ref 219. Copyright 2014 American Chemical Society.

Gratton group extended ICS by accounting for the fast raster
scanning motion of the focal volume in a typical commercial
confocal imaging system allowing access to much faster time
scales, on the order of the maximum scan speed.217,218
In recent years, ICS has been modified to capture faster
dynamics by combining ICS concepts with fluorescence
correlation spectroscopy analysis methods.219−221 Called
imaging FCS, this technique is similar in concept to ICS but
uses frames of data acquired consecutively on CCD or CMOS
chips. Because raster scanning to generate an image is not
required, faster time scales are accessible than would be
achievable with confocal imaging. Typically, EMCCD cameras
can image with integration times on the order of tens of
milliseconds. By decreasing the readout area of the CCD chip
used for the experiment, faster time scales (∼1 ms) are
accessible.
The theoretical framework for this method was adapted from
ICS and FCS for wide-field imaging. For a typical experiment, a
subregion of pixels varying in size from 8 × 8 to 2 × 2 pixels
(Figure 22A) was imaged for ∼40 000 frames. The signal in this
region was averaged over all pixels for each frame to generate a
fluctuating intensity trace over time. Following autocorrelation
of this trace, the resulting curve was fit with a Brownian
diffusion equation adapted from FCS. Generalized as
1 diffraction limit (300 nm, Figure 23F). The diffusion coefficient
G (τ ) = A * +B
1 + τ /τ1/2
where τ1/2 is the diffusion time. From this, the diffusion
coefficient can be determined. Sample autocorrelation curves
and fits for the hydrophobic dye DiI diffusing on a silanized
glass interface are shown in Figure 22B. The extracted diffusion
time corresponds to the imaging area that is modeled, not as a
Gaussian focal volume as in traditional FCS, but as a
convolution of the microscope PSF and the imaging area.221
The linear relationship between the imaging volume and
diffusion time is shown in Figure 22C.
In 2009 Dertinger et al. developed super-resolution optical
fluctuation imaging (SOFI), which generates super-resolved
spatial information from the autocorrelation analysis of
sequential wide-field images. SOFI uses wide-field image series
and high order correlations to improve spatial resolution of
images by up to a factor of 5. Autocorrelation is performed on
the signal transient from each pixel in the image stack over
time. The resolution enhancement scales with the square root
of the correlation order. The analysis of images can be
performed iteratively allowing a balance of computation time
and image quality to be determined.222
We have combined SOFI and an imaging FCS based method
to map super-resolution spatial information combined with
diffusion dynamics in porous structures with fluorescence
correlation spectroscopy SOFI (fcsSOFI).50 fcsSOFI allows for
super-resolution spatial mapping of diffusion on a surface or
within confined pores by using the diffusion of molecules near
the surface or within pores as the source of fluctuations, which
are subsequently analyzed using correlation analysis. The
process is illustrated in Figure 23. Diffusion was simulated in
side-by-side pores separated by a distance less than the
(22) was different in each pore with Dleft = 1 × 105 nm2/s and Dright
= 1 × 104 nm2/s. Five hundred frames of diffusion were
simulated and sample images are shown in Figure 23A.
Intensity traces for sample pixels in each pore are shown in
Figure 23B,C. A second order autocorrelation is performed on
the intensity transient from each pixel. The correlation curves
for the intensity traces in Figure 23, parts B and C, are shown in
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Figure 23. fcsSOFI analysis of 1D pores. (A) Two emitters are
simulated diffusing in two different pores shown by the colored lines.
(B, C) Example intensity traces from a single pixel in each pore. (D, E)
Autocorrelation of each intensity trace is fit to extract D. (F) Ground
truth pore locations. (G) An intensity-averaged image, showing pore
locations are within diffraction limit. (H) Sample trajectories from
conventional SPT. (I) fcsSOFI image maps the value of G2(τ) from
(D,E) as the source of the contrast. (J) Resolution comparison
between diffraction limit, SPT image, and fcsSOFI vs ground truth
shown in (F−I). (K) Dcalc for each pixel. (L) Final fcsSOFI image
generated by fusing (I) and (K) Scale bars = 300 nm. Reprinted from
ref 50. Copyright 2015 American Chemical Society.
Figure 23, parts D and E, respectively. Each autocorrelation
curve is fit with the standard model for Brownian diffusion
modified for wide-field analysis. The value of the first time lag
in G2(r,τ) for each pixel is plotted to produce the fcsSOFI
image (Figure 23I). A comparison of the fcsSOFI image with
conventional particle tracking (Figure 23H) and the average
intensity image (Figure 23G) are also given, and sample cross
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sections are plotted in Figure 23J. The resolution enhancement
is clearly apparent. The extracted diffusion coefficients from
each pixel were mapped to a color and plotted to produce the
diffusion map in Figure 23K. The images were then fused
producing the super-resolution diffusion map in Figure 23L.50
Second order correlation was used to produce the fcsSOFI
images shown in Figure 23, but further resolution enhance-
ments can theoretically be achieved by using higher order
correlations at the expense of computational resources. For the
second order correlation, the expected resolution enhancement
is ≲√2. A blind deconvolution would increase the resolution
enhancement further to ≲2. For higher order correlations, the
resolution enhancement is expected to scale with the
correlation order n and fall within the range σxy/√n < σn <
σxy, where σ is the width of the Gaussian PSF of a diffraction
limited emitter. Higher correlation orders come with increased
computational cost, which is expected to scale with the
correlation order squared.50
3.5. Differential Interference Contrast (DIC) Microscopy
DIC microscopy is often used in conjunction with other 2D
fluorescence imaging and tracking techniques, or can perform
tracking without the use of other techniques. Unlike
fluorescence based methods, DIC microscopy probes the
effective optical path length of the sample.223 DIC microscopy
creates an image by using two orthogonally polarized, spatially
offset light rays. As the rays pass through the sample, the
relative phase will drift as they encounter different optical
environments. Upon recombination, this phase difference is
used as an image contrast and allows DIC microscopy to
resolve objects that would be transparent in other forms of
optical microscopy.223 DIC is often used to colocalize cells and
probes when fluorescence tracking is used, such that the
position of the probe is known relative to the rest of the
cell.224−226 It is especially convenient because, as a transmission
technique, it can be implemented on the same microscope
assembly as an epifluorescence tracking experiment. DIC
microscopy is highly sensitive and as such was used in some
of the earliest single particle tracking experiments. It was used
to study the motion of kinesin coated latex beads along
microtubules as early as 1988.227 Nanometer spatial resolution
and microsecond temporal resolution are possible with DIC
tracking, but its usefulness is limited by the fact that it requires
enough material to change the effective optical path length of
the sample; therefore, single molecule resolution has not been
achieved.227,228 Even relatively recent iterations of this
technique use relatively large (>100 nm in diameter)
beads.228 Additionally, the optics involved tend to complicate
data analysis;229 therefore, tracking using DIC microscopy
remains a challenge.
4. REVEALING DYNAMIC PROCESSES USING 3D SPT
Recent developments offer exciting possibilities to understand
more complex structure−function dynamics in 3D, but also
present new challenges. For example, fast data collection rates
and the ability to transfer, save, and process large amounts of
data are important. Modifications of wide-field methods are
attractive because they intrinsically capture events in a large
area (∼10 μm) in one focal plane, and thus are inherently high-
throughput. Moreover, as discussed earlier, TIRF microscopy
significantly suppresses background signal and noise from
defocused events not in the focal plane. TIRF microscopy has
been a powerful tool to study events near the plasma
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Figure 24. Design of bifocal plane microscopy. (A) Layout of bifocal plane microscopy. Usually, two excitation laser beams with different frequencies
are used. Two beams are focused in different planes. In the original design, the high frequency beam (488 nm laser) is focused at the glass−water
interface in TIRF mode and the corresponding signal is recorded by camera 1, and the low frequency beam (543 nm laser) is focused at a focal plane
above the glass−water interface in wide-field epifluorescence mode and the corresponding signal is recorded by camera 2. Target probes are double
labeled to be detected in both excitations. (B) Simplified light path to explain the working principle of bifocal plane microscopy. Two lenses
represent the objective (left) and the tube lens (right). The focal plane in light color is the standard focal plane, and the corresponding image plane
represents camera 1 in (A). The focal plane in dark color is the adjustable focal plane, and the corresponding image plane represents camera 2 in (A).
Adapted with permission from ref 236. Copyright 2004 IEEE.

membrane230,231 and observe interfacial transport dynamics of
biological macromolecules.170,171,231 However, combining the
two techniques cannot provide depth information about single
emitters in 3D especially in cellular matrixes. Wide-field
microscopy combined with electronically controlled piezo
stages and fast detectors provides a simple solution: scanning
over different z depths and recording the corresponding
images, as discussed in section 5.58,232,233 However, the
movement of the piezo stage can effectively reduce the SNR
of the recorded images due to hysteresis.234 In contrast, lock-in
methods that rely on piezo stages to actively track a single
emitter are low-throughput and can easily lose a fast moving
emitter due to temporal limitation in the feedback systems.235
In the following sections, we discuss a range of solutions that
are aimed at the ultimate goal of 3D SPT: high-throughput
particle localization in 3D efficiently and accurately.
4.1. Multifocal Plane Microscopy (MPM)
MPM, developed by Ober and co-workers,236 is one method of
3D SPT that has been widely adopted, and records
simultaneous images at different focal planes in sample space
without sacrificing the temporal and spatial resolution of the
resulting trajectories. The initial design of MPM recorded
events in two different focal planes simultaneously to extract a
particle’s axial location, and was coined “biplane 3D
microscopy” (Figure 24A).236 In a typical microscopy system,
each focal distance has a corresponding image distance (Figure
24B). In biplane 3D microscopy, two cameras are arranged at
different imaging distances allowing signal to be recorded at
two different focal planes, as depicted in Figure 24B. In the
initial design, two excitation lasers were used with different
wavelengths (488 and 543 nm). The 488 nm laser was utilized
to image glass−water interfaces in TIRF mode, while the 543
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nm laser was focused above the glass−water interface in a wide-
field excitation geometry with adjustable focal distance. The
novel microscope design of simultaneous detection in both
TIRF mode and epifluorescence mode provides more
sensitivity in the setup. Accordingly, the target probe is labeled
with two fluorescent molecules. Mixed fluorescent signals are
split equally into the respective detection paths by a beam
splitter (Figure 24A). Signal excited by the 488 nm laser is
recorded by camera 1, while signal excited by the 543 nm laser
is recorded by camera 2 on an adjustable translational stage in
order to ensure precise calibration of the system. Emission
filters are used in each light path to eliminate the laser reflection
and signal cross talk. It should be noted that this experimental
design is very similar to two-color single molecule Förster
resonance energy transfer (FRET) setups;237 therefore using a
long pass beam splitter would save more photons when
separating the signals excited by two lasers. During the early
stages of biplane microscopy, the photon budget was less of a
concern prior to the single-molecule explosion after 2006.10,12
Biplane microscopy provides better resolution in all three
dimensions compared to conventional wide-field microsco-
py.236,238 The theoretical spatial resolution can be determined
by Cramér−Rao lower bound (CRLB) simulations, which
involve calculations using the Fisher information matrix (see
section 2.3 for details).238 For a typical separation of two planes
(∼500 nm) and considering the magnification at the camera
and noise, biplane microscopy shows a better and more stable
resolution (∼20 nm in x and y, and ∼100 nm in z) in 3D when
the fluorescent probe is near or in-between the two focal
planes, as compared with standard Gaussian PSFs.238 This can
provide an ∼1 μm depth detection range for 3D SPT.
However, for these spatial resolutions to be achieved, precise
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Figure 25. Using bifocal plane microscope to observe transport process from sorting endosomes to exocytic sites. The lower focal plane is focused
on the glass−water interface to detect the membrane. The higher focal plane is focused 0.6 μm higher than the membrane plane. The MHC class I
related receptor, neonatal Fc receptor (FcRn), is shown in green. The detected tubule is highlighted in orange. Cartoons in the third row explain the
corresponding cellular events. By detecting in two focal planes simultaneously, tubule extending (0.51−2.38 s, leftward arrows), partial fusion with
the plasma membrane (2.38−2.89 s, upward arrows), and retracting and detaching (3.23−8.33 s, leftward arrows) are observed. Adapted with
permission from ref 239. Copyright 2007 National Academy of Sciences.
calibration of the system is necessary. This is also true for other
techniques that will be discussed in later sections.
Although biplane microscopy is powerful in its ability to
study 3D processes, it does involve strict experimental criteria
that must be met in order to explore complex biological
environments in 3D. Prabhat et al. elegantly utilized biplane
microscopy to study 3D cellular processes, specifically the
highly debated dynamic process of intracellular transport
carried out by sorting endosomes.239 The dynamic intracellular
transport inside the plasma membrane is a popular target for
wide-field microscopy, which was probed in Prabhat’s
experimental setup using a TIR geometry given its advantage
of high SNR at a given interface (Figure 25). The second focal
plane in this work was within the intracellular volume (0.6 μm
from the membrane, Figure 25) to track the dynamic process of
endosome fusion and exocytosis carried out by FcRn receptors.
As shown in Figure 25, the details observed in the intermediate
plane can reveal the location of the endosome as it undergoes
transport to the cellular membrane. However, if this was the
only plane that was imaged, important mechanistic 3D
information would be lost. More specifically, the details of
exocytic fusion, exocytosis, and detachment, including the time
scale over which these dynamic events occur, would have been
lost without simultaneous imaging of the membrane plane. This
highlights the tremendous insight and advancements biplane
microscopy have made for probing important and complex
intracellular trafficking events. It must be noted in the outlined
experiments that the lateral position of the emitter in both
image planes should be spatially correlated.
In principle, MPM is not limited to only two planes. Ober
and co-workers have developed three- and four-plane
microscopies to enhance the 3D detection capabilities.239−241
However, in their four-plane microscopy, only two lasers and
two fluorescent labels were used: one for the TIRF mode
detection at the glass−water interface and the other for three
focal planes that were focused in the bulk sample environment.
The crossover among different light paths (corresponding to
signal from different focal planes) would highly diminish the
respective SNRs without the proper separation of wave-
lengths.239 The same group also simplified the instrumentation
by using only one laser and one fluorescent label,241 as shown
in Figure 26A. When using four detectors, the excitation path
effectively becomes a typical wide-field excitation path with one
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laser, in comparison to the original bifocal plane microscopy.
Compared to the double plane microscopy discussed earlier
(Figure 24), only one emission filter is needed to block the
laser scattering and reflection. After the emission filter, the
signal is split into four paths and collected by four cameras. The
four cameras are placed behind tube lenses with increased
distance to capture signal from different detection planes. In
this setup, an objective with small NA is utilized, effectively
decreasing the photon flux transmitted through the detection
path and lowering the SNR at camera 4.
As a result of the aforementioned advancements in MPM,
the study of complex intercellular processes within large
detection volumes has been achieved by producing 3D single
particle trajectories.242−244 An example of these results can be
seen in Figure 26B−D, where single QD-labeled transferrin
molecules dynamically transport between adjacent cells. The
transferrin was initially observed in one cell (highlighted by the
red arrow in Figure 26B), and then undergoes exocytosis,
quickly moving to the target plasma membrane of another cell.
This process is then followed by endocytosis into the recipient
cell (Figure 26C,D). From these observations it is noted that
transferrin has a very short travel time between cells and short
resident time before being internalized by the recipient cell.
The 3D detection capability reveals the vertical distance
traveled by the transferrin while uncovering specific interfacial
regions of the plasma membrane at which endocytosis occurs.
MPM is a powerful tool for tracking 3D events in cellular
environments. Recent developments in MPM instrumentation
have focused on detection path modifications to enhance 3D
detection capabilities, leaving the excitation path free to be
adapted with other microscopy techniques. This has given rise
to methods such as biplane FPALM super-resolution
microscopy.245 The main drawback of MPM is a great loss in
the photon flux at a detector when the emission is split to a
large number of detectors, which is a problem particularly when
using organic fluorescent molecules with low quantum yields.
The next two techniques discussed in sections 4.2 and 4.3
improve upon the photon budget in the detection path and
achieve 3D SPT in a unique and novel manner.
4.2. Astigmatic 3D Detection Using a Cylindrical Lens
More recent 3D imaging techniques such as astigmatic imaging
allow for higher photon counts through PSF engineer-
ing.91,92,246−248 These methods only detect one focal plane of
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Figure 26. Four focal plane MPM scheme and a 3D tracking
application. (A) Principle of MPM. The fluorescence signal is split into
four different light paths (represented by different colors) using three
50:50 beam splitters. Different light paths correspond to different
detection planes. Each detection plane matches with one detector
(CAM1 to CAM4) at a calibrated focal distance from the tube lens.
(B) Example tracking images of quantum dot labeled transferrin
traveling between adjacent cells (scale bar = 5 μm). Four images are
recorded by four detectors at the same time, and overlapped with the
segmented plasma membrane profiles (in green). The red arrow in the
second image highlights the quantum dot labeled transferrin receptor
that transports between cells during the measurement. The trajectory
of the transferrin receptor is depicted in 2D (C) and in 3D (D), with
time dimension being color-coded. Adapted with permission from ref
241. Copyright 2012 Elsevier.
the sample space, but through the use of a cylindrical lens
encode the depth information of the emitter within the shape
variation of the PSF. As a result of the inherent nature of this
technique, photon loss can be greatly minimized. Traditional
Gaussian PSFs are centrosymmetric, thus not allowing the axial
location of the emitter to be encoded in the PSF. In astigmatic
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imaging non-Gaussian PSFs allow the z position information to
be extracted from the shape of the PSF.
Astigmatic 3D detection techniques use a weak cylindrical
lens to slightly shift the focal plane in one lateral dimension
thereby encoding the particle’s z position in the shape of the
recorded PSF. As shown in Figure 27, a cylindrical lens with a
focal distance much larger than the tube lens is mounted in the
detection path.91 The cylindrical lens causes shifts of the focal
distance along the x and y directions. Usually, the focal distance
of the cylindrical lens is about 1 m, while the focal distance of
an imaging lens is about 10 cm.91 To make our discussion
easier, we follow Figure 27 and assume the focal distance in the
x direction has been changed by the cylindrical lens. In some
studies, the focal plane remains unchanged with the addition of
a cylindrical lens.246 Therefore, the corresponding Gaussian
PSFs have different widths in two directions at the focal plane.
However, other studies also suggest that the focal plane is the
image plane where the corresponding Gaussian PSF is
symmetric in shape, meaning that the focal plane is above the
current image plane shown in Figure 27.91 The difference
between these two cases is subtle, corresponding to a difference
of a few millimeters in the camera’s location.
In the development of astigmatic 3D detection, multiple
methods to perform 3D SPT have been proposed. In 1994, Kao
and Verkman were the first to develop astigmatic 3D detection
to track rhodamine-labeled beads. They calculated the center of
mass to determine the x and y positions of emitters, and the
widths of the PSF in the x and y directions to determine the z
position.246 Later in 2007, Holtzer et al. applied the same
technique to track the 3D diffusion of quantum dots in a
cellular environment.247 At that point, Gaussian fitting had
been demonstrated to be one of the most accurate and precise
methods to calculate the lateral positions of a standard 2D
PSF.119,127 In Holtzer’s work, 2D Gaussian fitting was used to
extract the x and y positions and the fwhm in both x and y
dimensions (wx and wy). wx and wy are compared with the
fwhm’s of standard calibrated PSFs to determine the z
position.247 As mentioned in section 2.4, even though 2D
Gaussian PSF fitting achieves relatively high precision, it often
requires intensive computational time in comparison to
determining an emitter’s center of mass (section 2.4).119,127
Huang et al. developed another approach based on calibration
data to determine the z position.91 Typical calibrated wx,calib and
wy,calib at different z positions are shown in Figure 27B. The z
position of a new emitter can be found by minimizing the
following:
(wx ,new 0.5 − wx ,calib 0.5)2 + (wy ,new 0.5 − wy ,calib 0.5)2
The PSF will be rejected if the minimized distance is larger than
the predefined threshold.91
Astigmatic 3D detection allows researchers the freedom to
combine it with the excitation geometries of other techniques.
A cylindrical lens can be used to produce non-Gaussian PSFs,
such as the diamond-like PSF used by the Wang group to probe
the 3D motion of fluorescent polystyrene beads in cylindrical
pores.249 As the PSF is not Gaussian, more complicated
correlation based analysis algorithms are implemented to
extract the 3D localization information from the corresponding
images. Huang et al. have coupled the detection techniques of
astigmatic 3D detection with the excitation setups used in
STORM for 3D super-resolution imaging.91 The complicated
excitation setups utilized in STORM are readily coupled with
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Figure 27. Principle of using a weak cylindrical lens to encode an emitter’s 3D position. (A) Detection path of the 3D microscope using a cylindrical
lens. A weak cylindrical lens is added in the typical detection path so that the focal points in both the x (green light path) and y (blue light path)
directions are shifted. PSFs corresponding to different z positions are shown in the right panel. Two-dimensional Gaussian function with different
widths in x and y directions can be used to calculate the 3D center position of the emitter. (B) Gaussian widths in the x and y directions (wx and wy)
as a function of z. Details about measuring the calibration data and the fitting are described in ref 91. Reprinted with permission from ref 91.
Copyright 2008 American Association for the Advancement of Science.
an astigmatic based 3D detection setup.91 Lien et al. has used a
cylindrical lens for 3D detection with temporal focusing of
multiphoton excitation microscopy, which provides deeper 3D
imaging in thick tissues.250 Spille et al. combined the astigmatic
3D detection path with light sheet excitation and a feedback
controlled three-axis translation stage, which can provide long
time 3D tracking capability deep inside living tissues.122,251
Here we will highlight the work by Spille et al. (Figure
28)122,251
Figure 28. Application of 3D microscopy combining light sheet
microscopy and astigmatic imaging. (A) Profile of a C. tentans salivary
gland cell nucleus (in green) and trajectories of fluorescent beads, with
different color labeling different trajectories. (B) One representative
trajectory that is tracked more than 4500 frames. Adapted with
permission from ref 122. Copyright 2012 Optical Society of America.
Light sheet microscopy combined with an astigmatic 3D
detection path can simultaneously achieve a large detection
range and nanometer scale spatial resolution. Light sheet
microscopy utilizes a thin light sheet to excite only one layer of
specific depth such that the background signal from fluorescent
emitters in the bulk solution is suppressed. Light sheet
excitation provides approximately uniform illumination in the
sample for a large horizontal area. The x−y−z translation stage
provides coarse 3D tracking capability as deep as 100 μm, and
the cylindrical lens further provides ∼10 nm resolution along
with fast feedback in the z direction. This method was
demonstrated to track particles up to 200 μm inside live tissues
and provides a useful tool to study the intranuclear
dynamics.122,251 Spille et al. tracked 20 nm fluorescent beads
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around the nucleus in Chironomus tentans salivary gland cells
covering 100 μm in depth (Figure 28), which cannot be
accomplished with traditional 3D microscopy.122 However, the
particle tracking relies on feedback and as such can only follow
one or a few particles for an extended period of time. The
feedback voltage is based on the 3D position of one selected
particle. The z position of the particle is roughly determined by
the ratio between wx and wy for a fast estimation enabling real-
time image processing taking only 50 μs per frame. The 3D
position of the target particle can be determined in high
resolution later by post data processing. Because of the careful
instrument design, they were able to track one targeted particle
for 800 frames over 12.8 s for an axial distance of 60 μm.122
Later, they focused on Balbiani ring (BR) mRNA and rRNA
diffusion near the nuclear envelope to investigate the kinetic
properties of the two types of RNA molecules (Figure 29).251
For certain time steps and positions the mRNA molecules
exhibited reduced mobility, which was believed to be a direct
observation of the mRNA sampling different nuclear pores
along the nuclear membrane. Diffusion types, MSD, and dwell
time distributions can be extracted from the long-lived SPT
trajectories.
Overall, in comparison to MPM, astigmatic 3D detection is
versatile and can be used in many excitation designs, has no
additional photon loss due to signal collection, and uses one
detection path and one detector. However, overlapping of PSFs
from different depths can introduce challenges in the data
analysis (“decoding” process). Because only a weak cylindrical
lens is added in the detection path, the detection range is
similar to the diffraction limit of the standard PSF in the axial
direction. Later we will see that the detection range can be
further improved through manipulating the phase information
on the signal. Cylindrical lenses break the axial symmetry of the
standard PSF generated by spherical lenses,91 and this resulting
asymmetry encodes the axial information. We will see in section
4.3 that a cylindrical lens is just one type of depth encoding
technique for 3D detection. Yet, broad applicability and
convenient data analysis make the use of cylindrical lenses a
useful and cheap alternative to other more complicated and
expensive 3D detection methods.
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Figure 29. mRNA diffusion paths near the nuclear envelope produced by 3D SPT with a cylindrical lens. (a) Three-dimensional representation of an
mRNA diffusion trajectory. (b) Overlay of the nuclear envelope (green), maximum intensity projection of mRNA signal (red), and trajectory data
(white) over a time course of 15.4 s. Scale bar, 2 μm. Adapted with permission from ref 251. Copyright 2014 Oxford University Press.

Figure 30. Microscope detection path with 4f system and a phase mask for 3D detection. A 4f system (in dashed box) is added in a typical
microscope detection path. Lens 1 and lens 2 are identical with comparable focal distance as the focal lens (FL). A transparent phase mask is
mounted in the middle of lens 1 and lens 2 (the Fourier plane). The phase mask for the double-helix PSF is shown at right. Different colors
represent different phase delays caused by the thickness of the material. The rotated PSFs corresponding to different z positions are shown in the
right panel. Adapted with permission from ref 261. Copyright 2016 Nature Publishing Group.

4.3. Engineered 3D Point Spread Functions Using Phase
Masks for 3D Imaging and SPT
By manipulating phase via Fourier optics, as discussed in
section 2.2, we can acquire more freedom and options in
encoding depth information in the detection path compared to
the previously covered 3D imaging methods. According to the
optics wave equations, the depth information is embedded in
the phase of the wave. Depth encoding using Fourier optics
couples phase information via intensity interference. Earlier
developments in Fourier optics led to the invention of
holograms,252 phase contrast microscopy,253 and DIC micros-
copy.254,255 In Fourier optics, the function of a lens is
mathematically equivalent to taking a Fourier transform of
signal from the focal plane (see section 2.2 for more details).
Therefore, the signal can be manipulated in the phase domain
after a lens. The ideal application of this model is the 4f
system.256 A 4f system is usually composed of two identical
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lenses placed a distance equal to 2f, with f being the focal
distance, as highlighted in Figure 30. The plane in the middle of
the two lenses is called the Fourier plane, because wave
functions of the corresponding signals at this plane are Fourier
transforms of the signal in the focal plane of the sample.
Emitters at different depths exhibit different phase distributions
at the Fourier plane. If a phase mask is added with a specific
phase pattern designed to break axial symmetry, then the
corresponding asymmetric PSFs can be obtained in the final
image of a system of interest. These asymmetric PSFs better
indicate the axial positions of emitters. The use of phase masks
that produce a double-helix (DH) PSF, and related improve-
ments, is one of the most successful examples of 3D
SPT.257−260
DH PSFs use the relative orientation between the two lobes
to indicate the axial position of fluorescent emitters (Figure
30). The idea of a rotating PSF originated from a study using
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Figure 31. Three-dimensional tracking of single mRNA−protein complex in a yeast cell. (A) Overlapping of the particle tracking trajectory in (B)
and (C) (in red box) on top of the bright field image of the cell (in gray scale). (B) Two-dimensional projection of the 3D particle tracking
trajectory. Relative time is shown in different colors as indicated by the color bar. (C) Three-dimensional plot of the same trajectory. The movement
and location of the mRNA−protein complex in the z dimension becomes clear in 3D. (D) Example raw images showing 3D DH PSFs overlapped
with the yeast cell. Different z positions are indicated by the orientation of the two lobes. The relative time of each image is 0, 0.233, 5.78, and 6.99 s.
Scale bar is 2 μm. Adapted with permission from ref 267. Copyright 2010 National Academy of Sciences.

Figure 32. Tetrapod masks and corresponding 3D PSFs for large range 3D tracking. (A, E) Phase mask design for 6 μm (A) and for 20 μm (E)
detection range. (B, F) Corresponding simulated PSFs at different z positions. (C, G) Corresponding experimental measured PSFs at different z
positions. Both scale bar and detection range are larger for the phase mask (E). (D, H) Theoretical resolution calculated based on the CRLB.
Adapted from ref 271. Copyright 2015 American Chemical Society.

finite energy to generate propagation-invariant wave fields.262
In earlier work, Piestun et al. found that certain types of basic
cylindrically symmetric mode (Gauss−Laguerre) combinations
generate waves with a rotating pattern in the final image
plane.262 To generate this exact rotating PSF, both the
amplitude and phase of the signal are required. However,
manipulating the amplitude distribution in the Fourier domain
would result in a loss of photons of more than 90%,258 which is
not suitable for single-molecule studies. Later an optimized
design was developed that only required the manipulation of
the phase pattern of the signal to generate a rotating PSF,
which is called the DH PSF.258 Soon after, this phase mask was
used in 3D super-resolution imaging263 and SPT.257,264,265
Generating DH PSFs is achieved by adding a 4f system in the
detection path and modifying the phase pattern in the Fourier
plane. Initially, reflective spatial light modulators (SLM) were
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used to provide the desired phase pattern. However, SLMs only
reflect linearly polarized light, potentially resulting in a 50%
photon loss.266 Later transparent phase masks, fabricated by
gray-level lithography, provided another option of inducing a
DH PSF with higher photon efficiency.266 Unfortunately, the
phase mask fabricated via lithography is restricted to a specific
optimum wavelength, and the phase pattern cannot be
reprogrammed once fabricated. Considering their complemen-
tary advantages and disadvantages, both types of phase masks
are used nowadays. Figure 30 shows a typical light path used in
a transparent phase mask setup.
Double-helix PSFs allow 3D tracking with a 2 μm range in
the z direction with a 50 nm resolution without any assistance
from a piezo stage.264,267,268 The use of DH PSFs yields better
high throughput tracking capabilities in 3D compared to MPM
and astigmatic 3D detection, giving larger depth detection
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Figure 33. Enhancement of DH PSF microscopy with light sheet excitation. (A) Isolated DH PSF obtained from light sheet fluorescence microscopy
and (B) epi-illumination microscopy. (C) Intensity profile of the cross section for the two white lines in (A) and (B), indicating the SNR
improvement. (D) Three-dimensional trajectory of single fluorescent beads. (E) Linear fitting of the 3D MSD of single fluorescent beads. Adapted
with permission from ref 275. Copyright 2016 IEEE.
ranges as well as higher localization precision.269 Thompson et
al. have used DH PSFs to study transport dynamics of mRNA
in live cells (Figure 31A),267 which is the first step to
understanding the important role of mRNA dynamics in gene
expression and regulation. These details could not be
discovered without the advancements in DH PSF microscopy.
As shown in Figure 31B,C, directed motion in the z direction
could be easily confused as local Brownian motion if this
system was only monitored in 2D (shown in the trajectory
between 6 and 7 s). This work has classified different modes of
transport, such as Brownian motion, confined diffusion, and
directed motion for each single mRNA trajectory, and
quantitatively characterized different types of motion. It has
provided a much clearer picture of how mRNA functions inside
cells by further revealing the specific interactions during
confined diffusion and the mechanism of directed motion.
After the application of the phase engineering for DH PSFs,
other phase mask designs have been proposed to further
improve the 3D detection capability.94,270−274 One of the goals
is to establish a general approach for variations of phase masks
to be designed in the future and to understand the connection
between the phase pattern and the performance of the 3D
PSF.258,272−274 Recently, Shechtman et al. proposed a new
tetrapod phase mask using shape extension as the indicator of
the z position (Figure 32).271,272 The behavior of the tetrapod
PSFs are very similar to the PSFs generated using a cylindrical
lens. However, the intensity profiles of the tetrapod PSFs are
much more complicated. These PSFs are designed for detection
over a large axial range. When including high frequency
components in the phase pattern, the detection range is
increased, as is the size of the PSFs in 2D.271 There is however
a trade-off between peak intensity and detection range, which
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could be problematic if the probes of interest are single organic
fluorescent molecules that exhibit a low quantum yield. For the
purpose of a large detection range, a 3D detection path
combined with an additional feedback tracking system may
offer an alternative solution.122,251
The peak intensity of most current engineered PSFs used to
encode 3D localization is much lower than that of traditional
Gaussian PSFs, leading to low SNR which correspondingly
results in low localization and tracking precision for 3D SPT
measurements.275 To improve the SNR, the phase masks are
designed to maximize the peak intensity of their corresponding
PSFs as long as they can satisfy their basic functions for the 3D
detection.258 When the phase mask efficiency is optimized,
subtle experimental design could help to further boost the SNR.
Light sheet microscopy combined with a feedback circuit can
effectively suppress the background noise from the bulk
solution and has been applied to enhance the SNR in
astigmatic 3D images, as discussed in section 5. Yu et al.
proposed to combine light sheet microscopy excitation method
with the DH PSF 3D microscope to enhance the SNR of the
system. Figure 33A,B compares the DH PSF with and without
light sheet excitation. The 1D cross section of the DH PSF
indicates an SNR increase by a factor of 2.4, which therefore
improves the precision of the particle localization and tracking
results. In the application of 3D tracking experiments with this
setup, a feedback circuit is operated to ensure that the target
particle is excited at all times by the light sheet within an
observation interval. Finally, they demonstrated the ability of
tracking single particles in 3D using fluorescent beads, with one
representative trajectory shown in Figure 33D with fits used to
extract the 3D diffusion coefficients (Figure 33E).275 Even
though light sheet excitation can only be used to track a single
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Figure 34. (A) Multifocus optical elements are appended to a wide-field fluorescence microscope after the primary image plane (at the camera port).
Two relay lenses ( f1 = 150 and f 2 = 200 mm) create a Fourier plane and a second image plane. The multifocus grating (MFG) is placed in the
Fourier plane and followed by the chromatic correction grating (CCG) and prism. A dichroic mirror (purple) splits the color channels onto separate
cameras. (B) Schematic of the MFG. The basic unit of MFG is optimized to distribute light evenly into the central 3 × 3 diffractive orders. The CCG
reverses the dispersion of MFG, and the followed prism directs the images to the camera. (C) The instant focal stack recorded on the camera is
computationally assembled into a 3D volume. (D) Movement in 3D of the two centromere clusters (black and gray). Bottom right, separation
between the centromere clusters over time. Rapid movement (phase I) is followed by slow movement (phase II). (inset) Average speed during
phases I and II (n = 5 cells). Adapted with permission from ref 276. Copyright 2013 Nature Publishing Group.

particle at a time, it provides a potential solution when the
target fluorescent signal is restricted and of low quantum yield.
Phase modulation in the Fourier domain also allows a novel
variant of MPM.276 Abrahamsson et al. proposed a novel phase
pattern to project images from different focal planes onto
different regions of a camera chip. The experimental setup
resembles the DH PSF 3D microscope, as the signal at the
primary image is transferred into the Fourier domain with the
first image lens (Figure 34A). The multifocus grating (MFG)
(Figure 34B) is located exactly at the Fourier plane, and it
encodes the signal in two aspects. First, the basic pattern MFG
evenly splits the signal from a single fluorescent emitter into
nine branches, which correspond to nine regions on a camera
chip, as indicated in Figure 34C. The MFG also provides a
phase aberration such that different regions on the camera chip
correspond to different focal depths in the object space. The
chromatic aberration is corrected by a chromatic correction
grating (CCG) and then finally imaged on the camera by a
second image lens. The advantage of achieving MPM using
phase modulation is that this technique allows for a large depth
detection range (∼4 μm) with fast signal acquisition speeds
compared with other MPM techniques. Abrahamsson et al.
applied this technique to track Saccharomyces cerevisiae
expressing Cse4-GFP to study yeast centromere separations,
and they observed biphasic behavior during cell division. The
fast separation speed (phase I) reaches 20 nm/s over ∼2 min
initially, followed by a slow separation speed (phase II) 3 nm/s
over ∼15 min, as indicated in Figure 34D. This observation is
made possible by the large field of view and fast detection speed
provided by this 3D SPT method.
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Recovery of the 3D position based on complicated PSFs is a
difficult computational problem. For the first two 3D
techniques discussed above, a centrosymmetric 2D Gaussian
is still a reasonable approximation of the generated PSFs.
However, for DH PSFs or tetrapod PSFs, it is difficult to
approximate the PSFs using explicit mathematical expressions.
Therefore, double-helix PSFs were approximated as two
Gaussian PSFs with a certain separation distance.258,277 The
relative orientation between the two Gaussian PSFs was used to
represent the orientation of the double-helix PSFs. However,
the two lobes of the double-helix PSFs are not actually
symmetric as is the 2D Gaussian function,277,278 and the
distance between the two lobes changes at different
orientations. Moerner and co-workers have developed a
corresponding correlation method.260 They calculate the
correlation between the calibration PSFs at different z positions
with the experimentally measured PSFs, with the highest
correlation indicating the corresponding z position. It is
important to note that the correlation method is time-
consuming and computationally expensive. Another family of
proposed methods is 3D deconvolution using sparse sampling
techniques.279 Deconvolution methods are especially useful in
the analysis of images with overlapped PSFs,279,280 but are
computationally intense and vulnerable to overfitting when the
SNR is low. As engineered phase masks become popular and
diversified, reliable recovery algorithms will remain an area of
interest in the near future.
The introduction of the 4f system combined with a phase
mask opened a new era of 3D microscopy and SPT. This
method has no special requirements in the excitation path other
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Figure 35. (A) Position trajectory of transcription protein molecule in a living cell shown by dashed line. (inset) Locations of two residence time
events (τ1 and τ2) and corresponding trajectory pathways. (Scale bars are 500 and 80 nm, respectively.) (B) Displacement (r) per time lapse is the
frame to frame distance the molecule traveled between two consecutive images for trajectory shown in (A). Shaded gray regions correspond to inset
in (A) and corresponding distance traveled. Two microscopic residence times thresholded by r0 = 220 nm (horizontal red dashed line) to determine
binding and unbinding events on a chromosome. (C) Proposed kinetic pathways of specific binding (SB), nonspecific binding (NB), and freely
diffusing (FD) transcription factors generated from single molecule stroboscopic kinetic results. Reprinted with permission from ref 297. Copyright
2015 Nature Publishing Group.

than the 4f configuration but is more complicated than simply
using a cylindrical lens, especially for the data analysis. Due to
the complexity of these PSFs, traditional fitting methods cannot
analyze them, and calibration has an even greater influence on
the resolution performance. Although theoretical calculations
show DH PSFs provide better spatial resolution than bifocal
and cylindrical lens techniques,269 reaching this theoretical limit
will be difficult. Advancements in the fields of computational
vision, such as machine learning, could be a promising direction
to further reduce the computational burden in SPT.
In addition to the previously mentioned methods, a less
common technique that is worth mentioning is the use of a
wedge prism to achieve 3D tracking.281 This prism splits the
light from an emitter into two focal spots with a defined offset
in the x direction on the detector. Movement of the emitter in
the z direction creates an additional offset of one of the focal
spots in the y direction on the camera. Thus, the y offset
between the centers of the two spots is linearly related to the
depth of the emitter. This technique was used to determine that
Eg5 and two-headed kinesin molecules produce small amounts
of torque as they travel along microtubules.281 A method using
mirrors instead of a wedge prism to produce a similar effect has
also been developed.282 These techniques are generally not
useful for high throughput tracking because half of the image
space is sacrificed when splitting the image, but they are useful
in certain scenarios where sparse tracking of a limited area can
be employed.
4.4. High Temporal Resolution in Single Particle Tracking
Super-resolution microscopy techniques enhance the spatial
resolution in SPT, while the temporal resolution remains
restricted, mainly by the camera frame rate.283 Temporal
resolution is also influenced by dye photophysics like blinking
and bleaching.284−286 In biological experiments, photodamage
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to cells or tissue should also be considered.287−289 In a typical
well-engineered experiment these processes take place on time
scales longer than the camera integration time and thus set
upper limits to the experimental time scales.284,288 Therefore, it
is crucial to capture emitter signals as fast as possible to reduce
these effects.
Events that occur within one camera frame are assumed to
have occurred at identical points in time, meaning trajectories
cannot be resolved with a temporal resolution higher than the
camera exposure time. Low temporal resolution thus limits the
application of super-resolution microscopy to processes
occurring at or slower than the acquisition time of a single
frame.290,291 Fast, accurate scientific cameras161 have been in
development since 1969, yet high cost and background noise
make it difficult to entirely satisfy the requirements for SPT
experiments. In order to improve the temporal resolution of
SPT without new cameras exhibiting higher frame rates, many
techniques have been proposed and implemented.
One technique developed to overcome these limitations was
shown by Xie and co-workers utilizing stroboscopic excita-
tion.225 Stroboscopic techniques utilize excitation laser pulses
synchronized with camera exposure times in order to increase
the temporal detection of probes in a wide array of
environments.70,292−296 Recently Chen and co-workers utilized
stroboscopic SPT in order to investigate the complex kinetic
process of gene transcription regulation in E. coli cells (Figure
35).297 This work allowed for a temporal resolution of 4 ms to
be achieved, in addition to a 20 nm spatial localization of
transcription regulators in vitro. As shown in Figure 35A,
transcription factors of interest were tracked performing
transcription processes within many areas of the cellular
environment. Single trajectory analyses were implemented
(Figure 35B) to determine residence times and corresponding
termination of binding events on single chromosomes. Further
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analyses of concentration dependent results allowed for
unprecedented kinetic details in the transcription processes to
be uncovered, showing heavy concentration effects on the
transcription processes at varying degrees of chromosomal
condensation. This use of stroboscopic SPT provided kinetic
cues into the complex dynamics occurring in transcription
processes in vitro and allowed for a kinetic model of
transcription to be determined (Figure 35C). This work
highlights the kinetic details that can be uncovered using
stroboscopic techniques; however, the native frame rate of the
camera still limits the achievable temporal resolution of the
system, which can be overcome with other techniques.
Recently we developed Super Temporal-Resolved Micros-
copy (STReM),283 in which the temporal resolution can be
enhanced 20 times greater than the native camera frame rate.
STReM encodes the temporal information in the PSF by
rotating a DH phase mask in the Fourier domain, such that the
orientation of the DH PSF lobes encodes subframe details,
rather than depth, information.283 Figure 36A shows the 4f
Figure 36. Application of STReM to SPT with high temporal
resolution. (A). A DH PSF is generated by installing a DH phase mask
in the Fourier domain. (B). The DH PSF rotates simultaneously with
the DH phase mask. (C, D). Simulated and experimental 2D
trajectories observed in traditional microscopy. The ground truth
trajectory is overlaid in (C). The temporal resolution in (D) is the
camera exposure time, 100 ms. (E, F). Simulated and experimental 2D
trajectories enhanced by STReM. The colored trajectories denote the
recovered emitter movement with high temporal resolution. Figure
adapted from ref 283. Copyright 2016 American Chemical Society.
scheme as discussed in section 4.3, in which a DH PSF is
formed by adding a DH phase mask in the Fourier plane.258,262
Although the DH phase mask was initially designed to extract
information about the depth of an emitter,262 in STReM, the
experiment is constrained to 2D by exciting the samples in TIR
mode, and the DH phase mask is rotated 180° within one
camera exposure time so that temporal information about the
emitter is uniquely encoded in the orientation of the PSF
(Figure 36B). We demonstrated both in simulation and in
experiments that STReM is able to enhance the temporal
resolution in single particle tracking. In the detection of an
analyte in fast 2D motion, only the spatial information can be
resolved in high resolution without STReM, while the temporal
resolution remains the same as the camera exposure time of
100 ms (Figure 36C,D). A complicated trajectory of a fast-
moving emitter is captured with STReM (Figure 36E,F), with
color denoting the temporal coordinates. The trajectories are
recovered by solving an L1 norm constrained optimization
problem with the alternating direction method of multipliers
(ADMM) algorithm.261,298−300 The temporal resolution is
improved 20-fold compared with the native camera frame rate,
which makes it a powerful tool for future SPT applications.
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5. ACTIVE FEEDBACK TRACKING
Active feedback tracking methods utilize real-time particle
position to generate 3D trajectories over several micrometers
within a system of interest. Although active tracking is
inherently low throughput, single particles can be tracked
over large distances in all three dimensions while maintaining
nanometer spatial resolution and submillisecond temporal
resolution. Moreover, feedback based tracking allows the
particle of interest to explore a large variety of local
environments within a system; thus the user is not limited to
a single region of interest as with the previously covered
nonfeedback based systems. Limitations do exist in the range a
particle can be tracked contingent on the experimental method
chosen along with the photophysical effects of fluorescent labels
making it challenging to monitor single particles over long
distances and times. Despite these challenges, advancements in
active tracking can achieve high spatial and temporal resolution
trajectories to help uncover hidden mechanistic details of single
particle dynamics in real time. 3D SPT was first investigated by
Peters et al. to uncover the dynamics of interfacial ligand
interactions with transmembrane proteins.301
5.1. ABEL Trap
The Anti-Brownian ELectrophoretic Trap (ABEL), established
by Cohen et al.,302 is a feedback based method that allows for a
particle to be physically trapped within a microfluidic system
based on a feedback generated potential to offset intrinsic
Brownian motion.303 This applied potential induces electro-
phoretic forces to iteratively counteract intrinsic Brownian drift
via region-specific electrodes assembled within the microfluidic
device (Figure 37).304 The applied potential electric field
directly interacts with the trapped particle via electroosmotic
forces to restore the particle to the center of the microfluidic
cell iteratively after each acquisition period. The location of the
particle was previously determined via camera based (CCD)
fluorescence microscopy; however, to measure smaller bio-
logical particles exhibiting high diffusion coefficients in solution,
high-speed orbiting lasers (discussed in section 5.2) have been
implemented to improve the temporal resolution of
ABEL.304−306 Reconstruction of a particle’s trajectory can be
statistically estimated from feedback potentials applied
throughout the trapping period along with photon locations
by utilizing Kalman filter feedback, ADF, and MLE based
algorithms as previously discussed in section 2.6 (Figure
37).304−307 Trapping nanoscale particles via electrophoretic
forces is advantageous in comparison to optical trapping
methods, given electrophoretic forces are stronger and are not
dependent on second order field components (i.e., polar-
ization). Although induced trapping forces are strong, ABEL is
a noninvasive technique utilized in many applications including
the exploration of DNA dynamics and the ability to perform
single-protein Förster resonance energy transfer studies.308,309
It must be noted that this method is not truly tracking a
particle; however, particles of interest can freely diffuse in
solution and be monitored for longer periods while avoiding
commonly used tethering chemistries, which can alter the
inherent behavior of a particle interacting in a given
environment.310 Additionally, ABEL allows for fluorescent
properties to be observed over long time periods with high
spatial and temporal resolution. This method is unique in its
ability to precisely control the position of a single particle in a
feedback system, thus allowing the particle of interest to explore
its natural dynamics in solution.
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Figure 37. (left) Fundamental setup of the feedback tracking in the ABEL trap with four electrodes (one in each lateral direction). The red laser spot
outlines an orbiting confocal laser exciting a tracked particle; subsequent photons are detected on a single photon counter. The feedback system then
relies on Kalman filtering methods to improve lateral localization of the particle (relies on intrinsic mobility and diffusion of trapped particle). As a
result of this improved localization, restoring electrokinetic forces are applied (indicated by green arrows) to a particle within the microfluidic
chamber in order to return the particle to the center of the trap. (right) Three-dimensional rendering of the microfluidic device in which the depth of
the chamber is roughly 600 nm in area outlined by the red square.311 Adapted from ref 304. Copyright 2011 American Chemical Society.

Figure 38. Schematic of tetrahedral confocal based 3D tracking optical configuration. Tetrahedron detection volume in sample space is created
through coupling of two fiber optic cable pairs before signal is detected on single photon detectors. Roughly 8% of wide-field emission light is
detected on the EMCCD to prior to the fiber optic cables in order to extract contextual information on emitter location. Real-time feedback lock in
detection is achieved by iteratively positioning the sample such that the emitter is in the center of the detection volume through the use of a three-
dimensional piezo scanner.232,315 Adapted from ref 232 and with permission from ref 315. Copyright 2010 American Chemical Society (ref 232) and
2015 Society of Photo Optical Instrumentation Engineers (ref 315).

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Figure 39. (A) Produced single particle 3D trajectory of a quantum dot labeled receptor on a mast cell (color gradient indicative of time further
shown in parts B and D). (B) Photon counts for trajectory in (A) over time. (C) Representative contextual image of emitter location from EMCCD.
(D) Axial localization of emitter showing a range of roughly 4 μm. (E) MSD (blue) of receptor over time fit to single exponential (red), exhibiting
compartmentalization. (F) Photon correlation measurements indicative of antibunching for trajectory in (A) lasting roughly 14 s. (G) Fluorescent
lifetime measurements (∼16 ns) with respect to laser excitation (red) and exponential fit (black). (H) Autocorrelation of photon detection rate
dictated by intrinsic blinking of quantum dot label utilized. Adapted from ref 232. Copyright 2010 American Chemical Society.

after illumination acting as pinholes or by focusing four lasers

Figure 40. (A, B) Image of QD labeled receptor on rat mast cells
without time gate detection (A) and (B) with time gate detection (4
ns). Comparison of two images shows significant decrease in
background emission from intracellular structures. Each image has a
linear scale between minimum and maximum pixel counts applied.
Adapted with permission from ref 315. Copyright 2015 Society of
Photo Optical Instrumentation Engineers.
5.2. Confocal Based Active Tracking
Tetrahedral based active tracking utilizes a stage scanning
confocal microscope, coupled with single-photon avalanche
photodiodes configured in a feedback loop such that particle
location is highly sensitive in the axial and lateral dimensions
while producing ∼5 ms temporal resolution.232,312−315 Utilizing
confocal based detection is advantageous in these applications
given the small illumination volume allows for a high signal-to-
noise ratio to be achieved in complex environments while
minimizing phototoxicity with low illumination powers (mW)
and providing time-resolved fluorescence measurements of
tracked particles. The use of a tetrahedral detection volume for
3D SPT was first used by Berg et al.316 and has recently been
advanced in its capabilities with four coupled fiber optic cables
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into the sample volume of a confocal microscope.232,312−315
Trajectories of actively tracked particles are constructed in real
time from the feedback-driven position of four SPADs, which
can actively track an emitter over ∼20 μm in lateral and axial
directions (Figure 38). Due to the noninvasive nature of this
method, Werner and co-workers have investigated the complex
process of protein transport in ligand induced endocytosis in
live cells while extracting time-resolved information on the
tracked proteins (Figure 39).232 Moreover, photon pair
correlation analyses are representative of photon antibunching,
indicating only one emitter was in the confocal volume
throughout tracking periods (Figure 39).232 This method has
also been able to actively track diffusivity variations in
genetically fluorescent protein oligomers and experimentally
achieve tracking of fast-moving proteins at speeds exceeding
most intracellular protein transport processes.313 Most often
quantum dots are used to label the particle of interest in order
to provide a large photon flux; however, the intrinsic nature of
blinking with these labeling methods can make tracking
challenging. The challenges of photoblinking and large cellular
based autofluorescence were recently overcome by DeVore et
al.315 in live cells through the incorporation of time-gated data
acquisition in the tetrahedral based active tracking setup shown
in Figure 38. This was achieved by using novel nonblinking
quantum dots along with a time-gated detection feedback
system coupled with a pulsed laser excitation (Figure 40).
These advancements allow for 3D SPT to be carried out in
various cell lines where large photon fluxes originating from
cellular autofluorescence are present. Moreover, these techni-
ques allow for further investigation into the complex
mechanistic details in particle transport processes in live cells
(Figure 40).315
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Figure 43. Fundamentals of signal detection using an optical

cantilever. As an emitter is displaced in sample space, the signal
difference between the two detectors is used to determine emitter
location (inset upper left). The plot shows the normalized signal
difference between the two APDs as a function of particle
displacement in sample space. The signal differences were simulated
for optical cantilevers of varying lengths (distance from back aperture
of objective to detector). Optical cantilevers become more sensitive to
smaller displacements as the length is increased. The relationship
shown here was used to optimize the optical response for SPT with a
cantilever length of a few meters. Adopted with permission from ref
83. Copyright 2015 Royal Society of Chemistry.

Figure 41. Orbital lock-in tracking setup. (A) Gray area representative
of orbiting confocal laser beam orbiting and exciting an emitter being
tracked. Emission signal modulation is monitored in two planes to
determine the lateral location and axial location from the difference in
signals at the two image planes. The location of subsequent laser orbits
is determined by a feedback loop driven by a piezo mirror in real time.
(B) Setup of excitation and detection paths for orbital tracking
showing simultaneous wide-field imaging on a CCD and multiple
excitation wavelengths (488, 561, and 633 nm). The two focal planes
monitored in (A) are detected two APDs as shown in (B). Adopted
with permission from ref 38. Copyright 2011 Royal Society of
Chemistry.
An additional active 3D tracking method utilized to
investigate many biological systems of interest while improving
the spatiotemporal resolutions of 3D tracking is orbital
tracking.38 This method was inspired by the work of
Enderlein,317 which originally was designed for tracking
fluorescent single particles in 2D. The evolution of this concept
into the 3D tracking domain evolved from the work of Levi et
al.,318 initially based on scanning FCS319 and was then applied
to study single protein aggregation dynamics in solutions.320
This lock-in technique is based on a laser scanning system
where a confocal laser beam orbits a single fluorescent particle
during tracking periods. A feedback loop then repositions the
laser beam via piezo controlled mirrors, or galvanometric based
mirrors, to re-center the emitter after each time step of the
system’s response. The emitter’s lateral location is determined
in real time using fast FFT calculations on fluorescent intensity

Figure 42. (A) Three-dimensional trajectory of artificial virus (shown in blue) in live eGFP-tagged (green) human hepatome cells. Gray projections
are 2D projections of the blue trajectory to show relative distance traveled in all three dimensions. Included are two wide-field z-slice images from
sample space further shown in (B). Arrows in (B) are indicative of lateral microtubule network motion with blue trace still representing original
trajectory from (A). Adopted with permission from ref 322. Copyright 2009 John Wiley and Sons.

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Figure 44. (A) Overhead view of PS-QD-peptide approaching
protrusion of live cell. Inset is zoomed in on the area where particle
interacts with cell surface. (B, C) High-resolution views of trajectory in
(A) from two different vantage points with color gradients still
indicative of time. (D) Trajectory represented in (A) with color
gradient representing diffusion over the evolution of the trajectory. (E)
Two-photon LSM image of cell expressing GFP-tubulin with overlaid
high resolution trajectory from (A) with scale bar representing 5 μm.
(F) Three-dimensional trajectory of PS-QD-peptide exploring cell
surface uncovering small semicircular protrusions along the cell
membrane. (G, H) Diffusional states analysis and corresponding
diffusional gradient plotted within the trajectory represented in (F).
Adopted with permission from ref 328. Copyright 2014 Nature
Publishing Group.
modulations as the emitter deviates from the center of the
detection volume as previously shown by Kis-Petikova and
Gratton321 (Figure 41A). The orbit of the confocal beam
rotates at a diameter equal to the width of the emitter’s PSF.
Orbital tracking is uniquely advantageous given the FFT
calculations used to produce the location of a tracked particle
are not affected by homogeneous background noise (e.g.,
autofluorescence or shot noise) in a given environment. Lateral
coordinates of the tracked particle can be determined through
various experimental techniques. Levi et al.58 utilized two-
photon illumination coupled with a piezo nanopositioner to
move the objective in order to probe two focal planes,
separated by half the width of the PSF. The fluorescent
intensities at the two focal planes are then compared to
determine the axial location of the emitter. This method
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produced a spatial resolution of 20 nm and a time resolution of
32 ms that could follow an emitter up to 10 μm in all three
dimensions.
Another method shown by Katayama et al.322 uses a one-
photon excitation, while simultaneously monitoring two focal
planes above and below the particle through confocal pinholes;
the resulting signal modulation from these two channels is used
to determine the axial location of an emitter (Figure 41). A
piezo based objective holder is used in this method to center
the emitter in the detection volume between each time step of
the trajectory; it is the confocal pinholes that are adjusted
iteratively in order to extract axial locations. As a result of the
objective not continuously oscillating during measurements,
higher NA water immersion objectives were used to
simultaneously acquire wide-field images of the current focal
plane where the emitter is located (Figure 42).
The ability to extract a trajectory and the contextual wide-
field images of a system can give tremendous insight into the
dynamics within local confinements of biological systems.
Dupont et al.323 utilized the orbital tracking method outlined
by Katayama et al.322 in order to investigate the anisotropic
nature of cellular diffusion of nanoparticles and how that
information is not recovered in traditional 2D measurements of
the same cell types. Moreover, Strano324 and co-workers
tracked single wall carbon nanotubes (SWCNTs) to explore
local cellular viscosities and active transport velocities within
various cellular regions in HeLa cells. The use of SWCNTs is
advantageous due to their large Stokes shift and the inherent
emission in the near-infrared region reducing autofluorescence
background signals and allowing deep tissue detection of
fluorescent signal.325
In addition, recent advances have been made in the
capabilities of orbital tracking to further improve the temporal
resolution and axial tracking range of 3D orbital tracking.
Lanzanò and Gratton were able to achieve SPT in 3D by
designing a piezo feedback controlled system from a
commercially available confocal microscope.326 They demon-
strated the viability of this setup to achieve SPT with the
transport of vesicles within epithelial cells. Furthermore,
Gratton and co-workers were able to improve the temporal
resolution of orbital tracking to 8 ms and increase the tracking
range of the system by 2.5 times.327 This was achieved utilizing
a commercially available electrically tunable lens (ETL) to
replace the use of piezo based stages, effectively eliminating the
movement of the objective in the previously mentioned setups.
The ETL allows for the divergence of the laser to be quickly
changed at the back aperture of the objective by varying the
focal length of the lens with an applied current, thus promptly
controlling the image plane of the system. Moreover, the
tunable range of the ETL is 500 μm in comparison to a piezo
stage actuator with a range of only 200 μm, thus allowing much
longer axial tracking regimes to be probed in a system. As a
result of the objective movement being eliminated, a high NA
oil immersion objective could be used, allowing for more
photons to be captured while not affecting the photobleaching
rates of emitters. This novel setup extracts 3D trajectories of
single fast-moving genomic loci inside the nucleus of live
cells.327 This work highlights the low cost conversion of current
scanning microscopes to achieve unprecedented axial tracking
ranges and temporal resolution for orbital tracking 3D SPT.
In order to push the frontier of the spatiotemporal regimes
(Figure 3) of 3D active tracking, 3D multiresolution
microscopy (3D-MM) was established by Yang and
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Figure 45. (A) Three-dimensional tracking setup using two-photon multiplexing in which four laser beam pulses arrive in sample space every 3.3 ns
iteratively. Lock-in tracking is achieved with scanning mirrors and a piezo objective stage (axial focus). (B) Photon counts of emitter located in the
center of the detection volume showing separation of signal consistent with time delay of laser pulses from beam multiplexer. (C) Detection volume
geometry from various vantage points. (D) Experimental image results of 100 nm bead with four beam excitation using half-wave plate (HWP),
polarizing beam splitter (PBS), dichroic mirror (DM), and beam dump (BD) (scale bar is 1 μm). Adopted with permission from ref 331. Copyright
2015 Nature Publishing Group.
Welsher.83,328 The demand to increase the spatial and temporal
resolutions of these tracking methods is advantageous to
investigate highly dynamic processes occurring on much faster
time scales than previously covered methods. The inception of
3D-MM stemmed from the work of Cang and Yang,329,330
which created a confocal based setup, forming an intensity
gradient in the axial direction of their detection path to extract
the z location of a tracked particle of interest. This method did
suffer from a low spatial resolution of 210 nm; however, it
achieved a temporal resolution of 1 ms. This method was
modified330 to encode the lateral location of an emitter by
splitting the signal with a prism to two SPADs. The difference
in the signal at the detectors was used to determine the lateral
locations, while the axial location was still determined using an
intensity gradient as mentioned.329 This method was most
recently improved with 3D-MM using an elegant combination
of optical cantilevers in the detection path to achieve a 3D
target locking feedback system in combination with simulta-
neous two photon-laser scanning microscopy (2P-LSM)
detection to image large contextual features in the region of
the emitter.83,328 The use of optical cantilevers in the 3D lock-
in tracking portion of the setup allows for the displacement in
lateral directions to be magnified roughly 3000× such that the
system achieves high sensitivity in particle position in sample
space while achieving a large sensitivity in optical response from
diverging photons on the APD in the detection path (Figure
43). 3D-MM has achieved a temporal resolution of 10 μs and a
spatial resolution of 10 nm in both the lateral and axial
dimensions. 3D-MM was used to track polystyrene-QD labeled
Tat peptide (HIV-derived) interacting on the surface of NIH-
3T3 cells (Figure 44).83,328 This study uncovered unprece-
dented 3D details in the anisotropic diffusion of the labeled
peptide near protrusions on the cell membrane. This example
further highlights the powerful information that can be learned
from the use of novel active 3D tracking systems used to
acquire dynamic details from uncharted spatiotemporal
domains while providing insight into the relationships of
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structures to biological interactions in highly dynamic environ-
ments.
5.3. Nonconfocal Based Active Tracking
A recently developed method, coined “TSUNAMI” (Tracking
Single Particles Using Nonlinear and Multiplexed Illumination)
by Dunn331 and co-workers, utilizes two-photon (2P)
microscopy in order to achieve lock-in 3D SPT at large
penetration depths (200 μm) in highly scattering environments.
The excitation beam, focused through a high numerical
aperture objective, is pulsed (13 ns duration) and separated
into four beams to form a tetrahedral shaped detection volume
in sample space (Figure 45A−C). Each separated beam is
delayed by 3.3 ns respectively, shown in Figure 45B, providing
evidence of only one emitter present in the detection volume
during tracking periods. The inception of this method is based
on nonlinear microscopy, in which the implementation of
passive pulse splitters has been shown to reduce the intrinsic
photobleaching rates in 2P microscopy in addition to delivering
a controlled number of equal energy subpulses to the system.332
This is coupled with a beam multiplexer setup used by Cheng
et al.333 allowing for multiplexed 2P illumination to be achieved
with periods of demultiplexing to extract the axial location of
the particle. The range to which a particle can be actively
tracked using this method is limited to the range of piezo
scanners (Figure 45A). The unique capabilities of TSUNAMI
lie in the ability to perform 3D SPT with a single detector while
simultaneously achieving 2P multicolor microscopy; in
addition, time-resolved fluorescent measurements can also be
conducted with this technique. TSUNAMI was used to track
single growth factor receptors tagged with 10 nm fluorescent
beads as they were actively transported across the membrane of
tumor spheroids at depths of up to 100 μm (Figure 46).
TSUNAMI can achieve a spatial resolution of 22 nm laterally
and 90 nm in the z-direction with temporal resolutions ranging
from 1 ms to 50 μs dependent on the photon budget emitted
(i.e., labeling method) from the tracked particle. Lastly, the axial
resolution information extracted from TSUNAMI was
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Chem. Rev. 2017, 117, 7331−7376
Chemical Reviews Review

Figure 46. Deep 3D SPT in cancer spheroids. (A) Three-dimensional contextual image of spheroid captured utilizing 2P scanning microscopy with
red stain for the plasma membrane and blue stain for the nuclei. The green image plane 50 μm into the spheroid is indicative of the plane in which
tracking will be carried out with white circle highlighting area of subsequent trajectory. (B) Cross-section representation of location of trajectory in
black circle which is zoomed in within the inset showing time dependence of the trajectory. (C) Zoomed-in image of trajectory in (B) originating
from within the cell. (D) Trajectory plotted with no contextual background from same perspective as shown in (C). (E) Instantaneous velocity over
the evolution of trajectory in (C) and (D). Adopted with permission from ref 331. Copyright 2015 Nature Publishing Group.

improved with the application of an MLE based algorithm.334
From the Monte Carlo simulations executed using this MLE on
TSUNAMI trajectories the axial resolution was increased 1.7-
fold.
Advancements in the field of 3D SPT have been
indispensable in probing complex biological systems in situ,
further elucidating diffraction-limited mechanistic details of
biologically important molecules carrying out their functions.
The aforementioned techniques have allowed for dynamic
processes to be tracked at the single-molecule level in 3D,
which has uncovered rich contextual structures and anisotropic
diffusional details within single-molecule trajectories. In our
review of 3D SPT we covered two main categories of
techniques: intrinsically high throughput tracking techniques
followed by active tracking methods, which are inherently low
throughput. Although both types of 3D SPT have their
advantages and drawbacks, improving the spatial and temporal
resolutions of 3D SPT is imperative to expanding the systems
where SPT is feasible.
6. CONCLUSION AND PERSPECTIVE
We hope we have shown that SPT is a powerful tool for
interrogating complex dynamic processes in a wide variety of
7366
systems. The evolution and development of SPT in the past
three decades is the result of continuous efforts from chemists,
biochemists, physicists, and engineers to further advance its
utility and applicability. As a result, SPT has become a rapidly
growing and interdisciplinary field where no single review can
cover all the relevant topics. We aimed to provide both enough
depth through a brief description of underlying theories and
enough breadth by highlighting a range of methodologies
utilized in emitter localization and trajectory reconstruction,
recent instrument developments to achieve 2D and 3D
subdiffraction tracking, as well as some of their applications
in cell biology and other materials.
As for future directions, it is almost certain that SPT as a field
has not reached full maturity. In particular, more efforts are
needed in the search for brighter and more photostable tagging
species that induce minimal perturbations to the analyte or host
matrix. As we have discussed in previous sections, QDs seem to
be the best choice to date given their high quantum yield and
relatively small physical sizes. However, it is well-known that
most QDs suffer from photoblinking,335 which makes trajectory
reconstruction in later stages of data analyses more difficult.
Moreover, QDs also require capping ligands for studies
conducted within cellular environments, thus setting limitations
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Chemical Reviews
on their applications. Breakthroughs in the development of
better fluorescent reporters would greatly benefit the entire
field of optical microscopy,336−338 including SPT studies.
Alternate excitation/detection schemes that do not rely on
linear fluorescence microscopy also present opportunities for
improvement.339,340
We also predict the incorporation of modern signal
processing methods in SPT. It will be important to address
the “big data” issues plaguing the newest 3D SPT applications
due to the challenges introduced by transferring, saving,
reconstructing, and analyzing large volumes of data that are
intrinsically sparse in information. At the same time, as was
found for the earlier SPT challenge,138 we do not foresee that
there is a single one-size-fits-all solution. We introduced a few
representative algorithms in section 2, and hopefully with the
aid of this review, readers can find or develop the most suitable
algorithm based on their unique sample conditions. In the
future, the integration with compressive imaging meth-
ods341−345 and machine learning algorithms125,346,347 might
allow SPT researchers to reach the ultimate goal of real-time
analysis.
Finally, we suggest that the types of analytes studied using
SPT will continue to expand. In section 3, we focused on the
most common applications of SPT including studying diffusion
in biologic systems, and on inorganic and polymeric surfaces.
Additionally, we reviewed various diffusion models and the
important ergodicity assumptions and their roles in this section.
To date ergodicity breaking has been reported mostly in
biophysical systems; whether it is a universal behavior that also
exists in other systems remains a fundamental question to
address in future studies. Recent examples of new applications
include a study of the free-radical polymerization mechanism
and a review of how nanoscale structure governs mass transport
in a range of materials.348,349 Achieving a mechanistic
understanding of a broad range of nanoscale chemical, physical,
and biological processes will be possible in part by continuing
advances in SPT.
AUTHOR INFORMATION
Corresponding Author
*E-mail: cflandes@rice.edu.
ORCID
Hao Shen: 0000-0002-2798-5861
Nicholas Moringo: 0000-0002-0044-255X
Christy F. Landes: 0000-0003-4163-6497
Notes
The authors declare no competing financial interest.
Biographies
Hao Shen is a postdoctoral research associate working with Prof.
Christy F. Landes in the Department of Chemistry at Rice University.
He received his Ph.D. from Cornell University in 2014 under the
supervision of Prof. Peng Chen. During his Ph.D., he studied
heterogeneous catalysis and electrochemical catalysis at the single
molecule level. His current research is mainly focused on exploring
protein−synthetic polymer interactions using single molecule spec-
troscopy.
Lawrence Tauzin is a postdoctoral research fellow working for Dr.
Stephan Link at Rice University studying the mechanisms of noble
metal photoluminescence. He received his Ph.D. from Rice University
in 2016 under the supervision of Dr. Christy F. Landes. His Ph.D.
7367
Review
work focused on understanding the mechanisms of interfacial
transport at tunable polymer interfaces at the single molecule level.
He received his B.S. in chemistry from Louisiana State University,
where he performed research for Dr. Bin Chen and Dr. Jayne Garno.
Rashad Baiyasi was born in Michigan, where he received his B.S. in
physics from Saginaw Valley State University. He is currently a
graduate student at Rice University in Houston, TX. His work focuses
on improving computational analysis techniques for super-resolution
imaging and single particle tracking. He is particularly interested in the
shifting and splitting of fluorescent emission in the vicinity of
plasmonic nanoparticles.
Wenxiao Wang is a graduate student in the Landes research group in
the Department of Electrical and Computer Engineering at Rice
University. His current research is focused on applying computational
method to improve super-resolution microscopy, including both the
spatial resolution and temporal resolution.
Nicholas Moringo obtained his B.S. in chemistry from the University
of California, Irvine, in 2014. He joined the Landes group at Rice
University in 2015, where his work has focused on single protein−
polymer interactions for modeling chromatography based protein
separation systems. As a recent Ph.D. candidate he was awarded the
NSF GRFP Fellowship in 2017 and plans to further pursue his interest
of protein separation systems at the single molecule level.
Bo Shuang graduated from Rice University in 2016 with a Ph.D. in
physical chemistry under the supervision of Prof. Christy F. Landes.
His research focused on strategies of data analysis for 2D and 3D
single molecule microscopy. He is working for Dow Chemical Co. in
Freeport, TX, focusing on chemical process optimization.
Christy F. Landes is an associate professor in the Departments of
Chemistry and Electrical and Computer Engineering at Rice
University in Houston, TX. Her research group develops next-
generation spectroscopic tools to understand dynamics at soft
nanoscale interfaces.
ACKNOWLEDGMENTS
The authors acknowledge the Welch Foundation (Grant C-
1787) and the National Science Foundation (Grant CHE-
1151647) for support of this work.
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7376 DOI: 10.1021/acs.chemrev.6b00815

Chem. Rev. 2017, 117, 7331−7376