r 1999 Wiley-Liss, Inc.

Cytometry 36:224231 (1999)

Single-Molecule Detection With Total Internal Reflection

Excitation: Comparing Signal-to-Background and Total
Signals in Different Geometries
W. Patrick Ambrose,1* Peter M. Goodwin,1 and John P. Nolan2
1Chemical

Science and Technology Division, Los Alamos National Laboratory, Los Alamos, New Mexico
Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico

2Life

Received 3 December 1998; Accepted 13 January 1999

Excitation of fluorescence with total internal reflection

(TIR) excitation yields very low background scattered light
and good signal-to-background contrast. The background
and its associated noise can be made low enough to detect
single fluorescent molecules under ambient conditions. In
this paper, different TIR geometries were compared for
excitation and detection of single rhodamine 6G (R6G)
molecules at airsilica interfaces and single B-phycoerythrin proteins at watersilica interfaces. Through-objective, objectivecoverslip, and prism-based TIR geometries
were investigated. The signal-to-background ratio (SBR)
and the number of photons detected before photobleaching (Nb) were optimum in different geometries. The
greatest image contrast was obtained when using prism-

TIR (SBR 11.5), but the largest number of detected

signal photoelectrons was obtained by using throughobjective TIR for R6Gairsilica (Nb 104). The
results were discussed in terms of the TIR field enhancements and the modified dipole emission pattern near a
dielectric interface. The SBR and total detected photons
are important parameters for designing photon-limited
experiments. Cytometry 36:224231, 1999.
r 1999 Wiley-Liss, Inc.

Single-molecule detection (SMD) is of fundamental and

practical interest because the behavior of the finest constituents of matter can be observed. The fluorescence emitted
by single molecules can be observed when the optical
background and associated noise are made sufficiently
small. When molecules of interest are photolabile (i.e., all
molecules at room temperature), SMD experiments are
limited by the total number of photons emitted before
photobleaching. Increasingly more complex systems will
be investigated at the single-molecule level, such as on or
within living cells, in which the optical background will be
higher. It is useful to explore conditions that optimize
SMD. There are now several hundred papers, reviews, and
books that describe methods for measuring the properties
of individual molecules against a low optical background.
These techniques can be categorized in various ways. The
first demonstrations of SMD were performed with low
temperature spectroscopy in solids (14). At nearly the
same time, single molecules were detected at room
temperature in liquids passing through a stationary focused laser beam [by flowing (59) or diffusing (1012) in
bulk liquids or in droplets (13)]. Soon afterward, detection
of immobilized molecules under ambient conditions was
performed by raster scanning a solid substrate through a
small spot of light [using near-field (1422) or confocal

scanning microscopy (2326)]. Single molecules can be

located at video rates by wide-field excitation and microscopy by using a broad laser beam in external reflection
(27), transmission (2832), or internal reflection (3340)
and by detecting photons with position-sensitive, charge
coupled device (CCD) array or silicon-intensified target
cameras. Pointwise detection with single-element silicon
avalanche photodiodes (APDs) provides high temporal
resolution (100 ps), which is useful for fluorescence
lifetime measurements in the nanosecond time range
(49,12,1618,23,25). The highest signal-to-noise and signal-to-background ratios (SBRs) obtainable under ambient
conditions are found in confocal optical arrangements
(1012,2326) because the optical background from out-offocus objects is spatially filtered by an aperture placed in
an image plane. Obtaining the best SBR by using an
aperture of dimensions corresponding to a single diffraction-limited volume necessitates observing only one mol-

Key terms: single-molecule detection; fluorescence imaging; total internal reflection; photobleaching; B-phycoerythrin; rhodamine 6G

Grant sponsor: DRD funding at Los Alamos National Laboratory.

*Correspondence to: W. Patrick Ambrose, Chemical Science and
Technology Division, Los Alamos National Laboratory, Los Alamos, NM
87545.
E-mail: wpa@lanl.gov

SINGLE-MOLECULE DETECTION WITH TIR

ecule at a time. Wide-field CCD-array imaging does not

have the high temporal resolution of an APD and does not
achieve the best SBR; more of the background from
out-of-focus sources is collected and detected in a widefield optical system. However, an advantage of parallel
detection in wide-field imaging with a camera is the ability
to acquire statistics rapidly on the millisecond behavior of
hundreds of molecules individually and concurrently.
Exploring conditions that optimize wide-field excitation
and camera detection are useful for obtaining low-temporalresolution statistics on a large number of individual molecules.
Axelrod reviewed a variety of geometries for total
internal reflection (TIR) excitation of fluorescence near a
dielectric interface in wide-field microscopy (41). SMD
with TIR excitation is very effective (3340) because it
tends to have lower optical background than does widefield epi-fluorescence (33,38), presumably because the
optics between the sample and the camera are not
illuminated fully and a thin volume of enhanced intensity
can exist near the interface (41,42) (background contributions from out-of-focus volumes are lower). Two TIR
geometries, prism- and objective-type TIR, have been
compared separately with epi-fluorescence (33,38). In the
present paper, we directly compare results for three
different TIR geometries for molecules at two different
interfaces (airglass and waterglass). The results are
discussed within the context of optimizing wide-field TIR
only and are not compared with other techniques. We
found that one TIR geometry is better for obtaining the
optimum signal-to-background contrast but that another
geometry is better for obtaining the largest total number of
detected signal photons before photobleaching. The results are discussed qualitatively in terms of the field
enhancements available in TIR and the modified directional character of dipole radiation near an interface
(42,43). These data illustrate a few guiding principles that
are useful for designing experiments in which a finite
number of detected photons are available and dispersed
over many time intervals, positions, or wavelengths.
EXPERIMENTAL DETAILS
Figure 1 shows the TIR excitation geometries investigated. The reflection of light is total at an interface of
higher to lower index of refraction (nh to nl) when the
incident angle (, measured from the surface normal) is
greater than the critical angle, cr, given by sin(cr) nl/nh.
The field is not rejected completely from the lower index
region but decays exponentially or evanescently, with a
decay length that changes nonlinearly between infinity
(for cr) and approximately 0.1 m (for 90). The
evanescent field strength also decreases with increasing
angle. In all cases shown in Figure 1, the higher index
medium on the incident side is silica (nh 1.46). For the
lower index medium, we used either air (nl 1.00, cr
43) or an aqueous buffer solution (nl 1.33, cr 66)
adjacent to a silica surface. The axially symmetric dipole
emission pattern in a homogeneous medium is modified
near an interface (42,43). The fractions of light emitted

225

FIG. 1. Various total internal reflection (TIR) excitation geometries. a:

Prism-TIR is performed on the far side of a gap between silica surfaces. b:
Prismless TIR can be obtained by inserting a narrow beam between the
objective and the near-side coverslip. c: Prismless through-objective TIR
occurs for input rays that focus beyond the critical angle at the near-side
silica surface. CCD, charge-coupled device camera; F, dichroic beam
splitter used as a long-pass filter; O, high numerical aperture microscope
objective; C, gap between two silica surfaces i.e., a sample cell; P, silica
prism.

into the two media differ from 0.5 because of optical

interference caused by reflections and by coupling of the
dipole near-field into additional propagation modes beyond the critical angle. We performed experiments to
explore these effects on the fidelity of SMD by collecting
and imaging the light emitted into the silica or into the
adjacent air or water.
We used imaging objectives that were designed for use
with a coverslip and immersion oil. Using a coverslip and
looking through a lower index medium at another silica
surface requires a gap of lower index (air or water)
between two silica surfaces. The lower index gap confined
between silica walls is referred to as a cell, and the walls of
the cell are either the near-side (close to the objective) or
the far-side walls. In Figure 1a, a prism is used to access
angles beyond the critical angle at the far-side wall.
Following previous conventions, Figure 1a is referred to as
prism-TIR (41,33). The prism and objective are optically
contacted to the far-side and near-side coverslips with
immersion oil. The incident angle was 67 for both air
and water in Figure 1a.
The locations of molecules on the near-side wall can be
imaged also. Two means of delivering TIR excitation to the
near-side wall are shown in Figure 1b,c. In Figure 1b, a
trough was constructed that allowed laser light to be
inserted between the objective and a near-side wall. The
trough had glass windows and was filled with an immersion solution of 7% vol/vol water in glycerol that was
index-matched to fused silica (the glycerol had an unknown amount of water initially). Because of spatial
constraints, the incident angle shown in Figure 1b was
83 from the sample-surface normal.
Another method of TIR on the near-side wall is accomplished by sending extreme rays through a high numerical

226

AMBROSE ET AL.

aperture (NA) objective (Fig. 1c) (38,41). Figure 1c is

referred to as prismless or through-objective TIR. In
principle, the reflection can be total if the NA of the
objective is greater than nl. The objective used for Figure
1c was a Zeiss infinity-corrected 63 Plan-apochromat
with NA 1.4. The objective was used in combination
with a 150-mm focusing or tube lens immediately behind
the objective. Kohler illumination was used, with an
extreme off-axis beam focused at the back focal position of
the objective-tubelens combination (38), with either a
300-mm or 750-mm focal-length lens. The incident angles
shown in Figure 1c are estimated to be within a few
degrees of 66 (a small amount of scattered light was
observed on one side of the objective when water was
injected into the cell, which may have been due to
aberrated rays just below the critical angle).
For the cases shown in Figure 1a,b, slightly better image
quality was obtained by using a Leitz Wetzlar 90, 1.3-NA
oil-immersion objective. Except for the glycerolwater
index-matching fluid in the trough shown in Figure 1b,
Zeiss 518c immersion oil was used for optical contacting
and objective immersion. Fluorescence filters used in all
cases were Omega Optical (Brattleboro, VT) 521LP and
525DLRP. Two types of camera were used: for SBR
measurements, a Princeton Instruments (Trenton, NJ) LN2
512- 512-pixel back-thinned SiTe silicon camera was
used with a 50-kHz readout; for photobleaching kinetics, a
Princeton Instruments Pentamax ICCD with Gen IV intensifier and thermoelectrically cooled 512- 1,024-pixel
video-rate frame-transfer CCD was used.
For the purpose of comparing various TIR geometries,
we chose molecules that were immobilized for long
periods of time. Molecules that are highly mobile and
diffuse much farther than a diffraction-limited length (a
few hundred nanometers) during an integration time are
not imaged as distinct spots. For a low coverage of
fluorescent molecules immobilized at the airsilica surface, we spin-coated the silica with rhodamine 6G (R6G) in
methanol, with a starting concentration of 3 1010 M to 3
109 M. Upon evaporation of the methanol (typically in a
few seconds), R6G was immobilized and was observed to
remain in place for at least 8 days (16). R6G is soluble in
water and does not remain at the silicawater interface for
long times. However, many proteins are readily adsorbed
to clean silica surfaces from water. For immobilization at
the watersilica interface, we used B-phycoerythrin (BPE;
Molecular Probes, Eugene, OR) protein at a concentration
of 1011 to 1010 M in Dulbeccos phosphate buffered
saline (1 DPBS; Gibco BRL, Grand Island, NY). The BPE
solution was injected into an 80-m-deep cell and allowed
to diffuse and bind to the silica walls for more than 1 h
before imaging.
To excite BPE and R6G, 514.5-nm wavelength light was
used from a continuous-wave argon-ion laser. The laser
was filtered with an interference notch filter to reject
plasma lines. The incident light was s-polarized in all cases.
Pure fused silica used for these samples was obtained as
130-m-thick coverslips from Esco, Inc. (Oak Ridge, NJ).
The silica was cleaned by sequential sonication in aqueous

soap, methanol, water, concentrated sulfuric acid, and

copious amounts of water and methanol. The water was
obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA).
RESULTS
Important figures of merit for SMD are the SBR and the
total number of detected photons (Nb) before molecules
photobleach or escape. The SBR is a useful measure of the
quality of a fluorescence detection method for power
levels low enough that optical saturation or the intensity
dependence of other photophysical effects are negligible
[e.g., photobleaching (7,44)]. The total numbers of detected photons (signal and background) then determine
the signal-to-noise ratio (assuming shot-noise-limited detection).
Figure 2 shows examples of the SBR for R6Gairsilica
and BPEwatersilica excited using through-objective,
objectivecoverslip, and prism-TIR. The power and integration times were adjusted to produce measurable signals
(less than several thousand photoelectrons in the largest
peaks) and reduce the fraction of photobleached molecules in one image. Consecutive images were obtained to
verify that most of the molecules survived at least several
frames (severe photobleaching in one integration time
reduces the SBR contrast). Abrupt, stochastic disappearance of signals from frame to frame was observed and is
consistent with single-molecule behavior. The gray scales
shown in Figure 2 were adjusted to range from the dark
current and readout noise level of the camera up to the
highest signals, i.e., the largest signal plus background
values are normalized to white. Results for R6Gairsilica
are presented on the left side of Figure 2 (A,C,E). Results
for BPEwatersilica are presented on the right side of
Figure 2 (B,D,F). The top pair of images (A,B) were
obtained by imaging the near-side wall with throughobjective TIR (see Fig. 1c). Figures 2C,D are also near-sidewall images, but the excitation was changed from throughobjective to objectivecoverslip (see Fig. 1b). Apparently,
changing the geometry and increasing the incident excitation angle from 66 to 83 reduces the SBR. The
bottom pair of images (Fig. 2E,F) were obtained on the
far-side wall with prism-TIR excitation (see Fig. 1a). Table
1 presents values for the different image parameters and
the SBRs for the data shown in Figure 2. The best SBR
contrasts were obtained when using prism-TIR (SBR
11.5 for R6Gairsilica; Fig. 2E).
In addition to the SBR contrast in one image, the total
number of measurements that can be made on a molecule
is of interest. The number of measurements is limited by
the total number of emitted and detected photons; the
total number of emitted photons is limited by photobleaching. We performed experiments to determine the mean
total number of detected photons per molecule until each
was photobleached. Figure 3 shows an example of results
from a photobleaching experiment for BPEwatersilica
using prism-TIR (as in Fig. 1a). The figure shows a tiled
sequence of consecutive images, each with an integration
time of 0.5 s. The first 24 images from this experiment are

SINGLE-MOLECULE DETECTION WITH TIR

227

FIG. 2. Images of the fluorescence detected from single molecules showing different signal-to-background ratios obtained with various total internal
reflection (TIR) excitation geometries. A,B: Images obtained with through-objective TIR excitation. C,D: Images obtained with objectivecoverslip TIR
excitation. E,F: Images obtained with prism-TIR. A, C, and E show the locations of individual rhodamine 6G molecules at airsilica interfaces. B, D, and F
show individual B-phycoerythrin proteins at the interface between a buffer solution and silica. The gray scales are adjusted to normalize the largest signal in
each image to white. The non-zero labels on each gray-scale bar are the largest signals-plus-background and the background level. Scale bar 10 m.

arranged from left to right and from top to bottom. In

Figure 3, the number of fluorescent spots decreases as the
molecules photobleach. Photobleaching occurs as an
abrupt loss of signal from one frame to the next. The
intensities at the watersilica interface also fluctuate, as
they do at the airsilica interface (20). Obtaining a sequence of images is useful for determining when a

sufficient time has elapsed, such that most of the molecules have bleached. Table 2 shows information on
bleaching experiments for BPE and R6G using throughobjective (near-wall) and prism-TIR (far-wall; bleaching
experiments were not performed for objectivecoverslip
excitation, Fig. 1b, because of the low SBR). To obtain
bleaching statistics on a large number of molecules,

AMBROSE ET AL.

228

Table 1
Results for Signal-to-Background Ratio (SBR) Measurements*

Geometry in Figure 1
Results from Figure 2
Excitation power (mW)
Estimated areas (cm2)a
Incident angle
Integration time (s)
Largest signals (photoelectrons)
Background (photoelectrons)
SBR

Through (c)
A
0.03
1.1 106
66
0.5
1,650
264
6.3

R6Gairsilica
Coverslip (b)
C
17
1.2 105
83
2
263
286
0.9

Prism (a)
E
13
9.9 105
67
2
357
31
11.5

Through (c)
B
0.05
1.1 106
66
0.5
3,300
836
4.0

BPEwatersilica
Coverslip (b)
D
6
1.2 105
83
2
510
450
1.1

Prism (a)
F
0.8
9.9 105
67
2
360
35
10.3

*R6G, rhodamine 6G; BPE, B-phycoerythrin.

aIllumination areas were estimated from images of the background scattered light.

between five and 14 sets of images were obtained, with

50300 images per set for four different TIR geometries.
The high parallelism of CCD-array detection allowed
statistics to be accumulated for 4103,461 molecules (see
Table 2).
To accumulate all of the detected photons for each
molecule, the sequence of images from one experiment
were added. To distinguish molecules from noise in the
position-dependent background, a position-dependent
threshold was needed. From Table 1, the SBRs for these
experiments were 4.011.5. A threshold function was
constructed by using an image of the background multiplied by 1.21.4. Peaks in the summed image that were
above the threshold were integrated and the background
was subtracted to obtain the total signal photoelectrons
detected from each molecule.
Figure 4 shows histograms of the total detected photons
from R6G and BPE excited with through-objective and
prism-TIR. The roll-off in the histograms at low numbers of
photoelectrons is due to thresholding, as verified by
extracting data from peaks with various threshold multipliers. The decays on the high side are described well by
single exponentials. The mean number of detected photoelectrons are obtained from the inverse slopes of the
natural logarithms of the histograms. Table 2 summarizes
the experimental conditions, numbers of molecules analyzed, and the mean numbers of detected signal photons
per molecule. Figures 4a,b are from BPE on the far- and
near-walls adjacent to aqueous buffer solution. The mean
numbers of detected photons in Figures 4a,b are similar. In
contrast, the slopes of the log-decay histograms for R6G at
the airsilica interface are very different (Fig. 4c,d). As in
Table 2, the mean number of detected photons from
R6Gsilicaair is 10,800/1,830, i.e., 5.9-fold higher when
collected through the silica than when collected through
the air.
DISCUSSION
The main conclusion of this paper is that the conditions
that produce the largest SBR for wide-field TIR are not the
same as the conditions that give the largest numbers of
detected photoelectrons. The largest SBR is obtained for
prism-TIR (far-wall), but the largest total number of detected photons is obtained on the near-wall.

A brief comment is helpful at this point to identify the

main background source regions. In general, the farther a
background source point is from the focal plane at the
interface, the smaller the solid angle collected and the
smaller the contribution at the detector. In a confocal
arrangement, an aperture at an image plane drastically
reduces the solid angle collected from out-of-focus background source points. The background in confocal experiments is considered to arise only from a small diffractionlimited volume (comparable to the wavelength or
approximately 0.5 m in extent). In wide-field microscopy, the collection solid angle is not as strongly reduced
with distance from the focal plane. Consider the geometry
shown in Figure 1a. The background source regions
within the collection solid angle of the objective are the
very thin evanescent region close to the silica surface (less
than a micron in vertical extent) and the volume of silica
illuminated by the laser in the prism. The depth of silica
contributing to the background is more than several
microns and is well beyond the bound-mode region at the
interface. The bulk of the background in this case is
expected to arise from a larger volume, traveling-mode
region in the silica beneath the evanescent mode region
near the interface.
The SBR differences are consistent with a background
from out-of-focus regions and a signal that is affected by
irradiance enhancements or de-enhancements near the
interface (33,38,41,42). Figure 5 shows the calculated
irradiance for s-polarized incident light on the low index
side of the interface using Fresnels equations (41,42). As
the incident angle = cr, the incident and reflected
amplitudes add constructively to produce an irradiance
approximately four times the average irradiance in a
homogeneous medium. For = 90, the phases of the
fields add destructively, resulting in a decrease in irradiance to zero at the interface. In the experiments in which
is close to cr, the irradiance in a thin layer around the
interface is higher than the volume-averaged irradiance
farther away. These out-of-focus regions then contribute
lower background relative to the excitation rate. In the
experiments described using the objectivecoverslip geometry, 83. Unfortunately, the irradiance used to excite
the molecules was diminished compared with the average
irradiance exciting the out-of-focus background. This re-

SINGLE-MOLECULE DETECTION WITH TIR

229

FIG. 3. Sequence of images showing photobleaching and emission fluctuations of B-phycoerythrin (BPE) molecules. Molecules of BPE were allowed to
diffuse and accumulate at a silicabuffer interface for at least 1 h. The interface was then illuminated with total internal reflection (TIR) through a prism and
imaged. The panels are arranged from left to right and from top to bottom, with the first image in the upper left and the 24th image in the lower right corner.
Note (a) the overall trend to blackness (photobleaching), (b) that some molecules are almost constant and turn off in a single frame transition, and (c) others
that blink.

sult is consistent with the observation that the large-angle

TIR in Figure 1b has the lowest SBR. In contrast, prism-TIR
(Fig. 1a) has the highest SBR, probably because the fewest
number of out-of-focus elements are illuminated in the
collection optics (the objective and filter are not illuminated) (33).

The total number of detected photons depends on

whether a molecule emits after excitation and where the
photons go. Taking into account the excitation irradiance
variation with angle (as in Fig. 5), we estimate that the
peak irradiances were all less than 1 kW/cm2 for all
measurements presented in Table 1. This is well below the

AMBROSE ET AL.

230

Table 2
Parameters and Results for Photobleaching Experiments*

Geometry in Figure 1
Number of experiments
Number of images/experiment
Integration time (s)
Total number of molecules
Mean number of photons detected/molecule (7Nb8)

BPEwatersilica
Through (c)
Prism (a)
14
6
150
50
0.33
0.5
410
3461
1,560 110
1,960 60

R6Gairsilica
Through (c)
Prism (a)
10
5
300
60
0.33
1.0
984
2177
10,800 600
1,830 120

*BPE, B-phycoerythrin; R6G, rhodamine 6G.

FIG. 4. Photobleaching statistics for molecules in various total internal reflection (TIR) excitation geometries.
Sequences of images, such as those shown in Figure 3,
were added together. Background-subtracted signals were
integrated from molecules that were brighter than a
threshold value chosen larger than the noise in the
background. a,b: Probability histograms of the total
signal photoelectrons for B-phycoerythrin molecules at a
silicabuffer interface using (a) through-objective TIR
and (b) prism-TIR. c,d: Rhodamine 6G molecules at
airsilica interfaces for (c) prism-TIR and (d) throughobjective TIR. The roll-off on the low side is a thresholding artifact. The distributions of total photoelectrons
accumulated per molecule follow single exponential
decays.

FIG. 5. Intensity enhancement on the low-index side of a dielectric interface.

Fresnels equations were used to compute the ratio of the intensity on the lowindex side of a dielectric interface to the intensity in a homogeneous medium of
the higher index for various incident angles at (a) a silicaair interface (indices
of refraction 1.46 and 1.0) and at (b) a silicawater interface (indices of
refraction 1.46 and 1.33). The incident light is s-polarized. The enhancement is
nearly equal to 4 on both sides of the critical angle.

high irradiances needed for excited-state transitions and an

irradiance-dependent photobleaching yield [b b(I)]
(7,44), i.e., the different Nb are not expected to depend on
the different irradiances. The large difference in the

numbers of detected photons for the case of R6G on

airsilica for the near-wall and far-wall geometries is
consistent with a redistribution of the dipole emission into
the glass for a dipole laying in the plane of the interface
(43). Incidentally, confocal microscopy at an airglass
interface probably takes advantage of this angular redistribution effect (2326).
Unlike the results for R6G at airsilica interfaces, the Nb
obtained for BPE on the watersilica near- and far-wall
interfaces were similar. BPE is analogous to a bread
pudding composed of protein with multiple imbedded
fluorophores and has physical dimensions of up to 12 nm
(7,45). An unknown factor is the effective index of
refraction of the bread pudding. The emitting fluorophore(s) may see an effective local index that is intermediate between the water and glass. In addition to an
unknown local index, other factors that could contribute
to a reduction of the Nb difference are a greater distance of
the emitter from the interface and an orientational averaging of the emission patterns. Detailed modeling of these
contributions will be needed to account for the similarity
of the fractions of light emitted into the water and glass for
BPE.
These results will be useful for the design of future
experiments. In experiments in which the numbers of
detected photons will be dispersed over a large number of
measurements (e.g., many time channels in a fluorescence
lifetime experiment, many wavelengths for spectroscopy,

SINGLE-MOLECULE DETECTION WITH TIR

or many positions in many consecutive images), a nearwall geometry is likely to be most effective. For experiments in which it is necessary to obtain a single measurement quickly with good SBR, then a far-wall geometry is
likely to be better.
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