Innovative Tools for Fluorescence Microscopy

Nanorulers: A Quick and Easy Evaluation of Optical Resolution Beyond the Limit of Diffraction

In recent years the spatial resolution in fluorescence microscopy has seen a dramatic improvement Motivation
due to the invention of several super-resolution techniques which break the diffraction limit. How-
ever, validating and testing these new techniques remains challenging due to the lack of reliable The first decade of the 21st century has
test-structures providing well-defined mark-to-mark distances. Newly developed nanostructures al- seen a dramatic improvement of optical
low to position dye-molecules with nanometer precision and can be produced in a highly parallel microscopy. The optical diffraction limit,
fashion. These structures can be used to test the achievable spatial resolution of super-resolution postulated by Ernst Abbe in 1873, has
but also of conventional fluorescence microscopes. been broken by the invention of several
super-resolution techniques, which offer
spatial resolutions down to six nanome-
ters [1]. The price the users of super-res-
olution microscopes have to pay, in order
to achieve the improved spatial resolution,
is the increased complexity of setup han-
dling and sample preparation as well as
data evaluation. As a consequence, it is not
anymore possible to calculate the achiev-
able spatial resolution for these new tech-
niques just by looking at the hardware
specifications of the microscope. Since the
spatial resolution is essentially the most
important specification of any microscope
it is crucial for users, manufacturers and
developers to measure the achievable spa-
tial resolution experimentally. Convention-
ally, the spatial resolution is demonstrated
and measured by imaging cellular struc-
tures like microtubules or actin filaments.
However, these samples show a couple
of disadvantages like the fact that they
do not provide a large number of identi-
cal samples with defined distances be-
tween fluorescent marks and they are not
very well defined regarding labeling den-
sity and dye-to-dye distance. These disad-
vantages make it almost impossible to use
them as reliable and objective sample to
test the spatial resolution of modern fluo-
rescence microscopes.

Nanorulers Based
on DNA Nanotechnology

A technique which allows the creation

of test-samples satisfying all require-
ments defined above is the so-called DNA
origami technique [2]. Simplified, DNA
origami structures can be seen as na-
noscale molecular breadboards which
allow to position dye-molecules, fluores-
Fig. 1: Nanorulers for different imaging techniques: (a) Diffraction limited confocal imaging. Distance cent proteins or essentially any object
between the two fluorescent marks: 350 nm. Dye: Alexa488 (b) Structured Illumination Microscopy. which can be coupled to single-stranded
Distance: 160nm Dye: Alexa568 (c) STED Microscopy. Distance: 71nm. Dye: ATTO647N (d) Localization DNA. DNA origami structures can be
based sample for dSTORM. Distance: 94 nm. Dye: Alexa647 (e) Localization based sample using the produced highly parallel and self-as-
DNA-PAINT technique. 3 dualcolor-spots in a row. Distance between two neighboring spots: 80nm. sembled which makes it possible to use
Dyes: Atto655 and Atto550. This dualcolor sample can also be used to check for chromatic aberrations. them as ubiquitously available samples.
A schematic view of a typical test-sam-
ple, called nanoruler, based on DNA ori-
gami can be seen in the subsets of fig-
ure 1. Usually two to three fluorescent
marks, each consisting of several organic
dye-molecules, are positioned on each
DNA origami structure. The distance be-
tween these fluorescent marks can be
adjusted at will in the range of 6-360 nm.
The number of dye-molecules per flu-
orescent mark is in the range of 10-20
and essentially every commonly used
sort of dye can be attached to the nanor-
ulers. The nanoruler test-samples can
be prepared ready-to-use by immobiliz-
ing the DNA origami structure on a cov-
erslip with densities of around 1 struc-
ture per µm², which means that within
a typical field of view hundreds of iden-
tical nanorulers are visible. This allows
to measure the distances between the
fluorescent marks, thus to test the spa-
tial resolution of the microscope and to
calculate the statistical errors for these
measurements. Due to the large number
of identical structures it is possible to use
efficient algorithms which automatically
detect the nanorulers in super-resolved
images and measure the distances be-
tween the fluorescent marks (fig. 2).
Ready-to-use samples of nanorulers
can be stored for months in the fridge
and allow measuring the spatial reso-
lution on a daily basis [3]. DNA origami
based nanorulers can be optimized for es-
sentially every common super-resolution
technique, like STED [4], SIM [5] or lo-
calization based techniques like dSTORM,
PALM, GSDIM or DNA-PAINT [6,7,8,9], as
well as for conventional confocal imag-
ing, as shown in figure 1. Nanorulers are
used by scientific labs to optimize imag-
ing, to teach the basics of super-resolu-
tion, to decide whether the microscope
is performing well or in order to decide
which commercial setup fits best to the
specific needs of the lab. Manufacturers of
commercial super-resolution and fluores-
cence microscopes are using nanorulers
to demonstrate that their products fulfill
the technical specifications and claimed
spatial resolution but also for developing
new techniques and products.
Brightness Standards
Beside the control over the position of
dye molecules and by this over the posi-
tion of fluorescent marks, also the num-
ber of dye-molecules on a DNA origami
structure and by this within one diffrac-
tion limited spot can be controlled very
precisely. Furthermore, also the distance
between dye-molecules on a DNA origami
structure can be controlled which can be
Fig. 2: Software for semi-automated data analysis of measurements with nanorulers. (a) The software
(GATTAnalysis, GATTAquant GmbH) identifies correctly imaged nanorulers. (b) The distances between
the resolved fluorescent marks are measured by the software. The theoretical distance between two
neighbouring spots is here 80 nm. (c) Histogram of measured distances. The measured value of
(79.3±1.8) nm corresponds very well to the theoretical value of 80 nm.
used to prevent undesired photophysical standards can be used in order to count
effects like quenching, which would po- absolute quantities of fluorescent mole-
tentially decrease the fluorescence inten- cules, particularly in biological samples.
sity. It was shown that the fluorescence
intensity scales linearly with the number
of dye-molecules on a DNA origami struc- References
ture [10]. These properties allow it to use [1] Raab M. et al.: Chemphyschem 15, 2431–2435
dye-labeled DNA origami structures as (2014)
brightness standards, in order to count [2] Rothemund P. W. K.: Nature 440, 297–302
absolute quantities of fluorescent dye- (2006)
molecules in fluorescent samples. [3] Schmied J. J. et al.: Nat Protoc 9, 1367–1391
(2014)
[4] Klar T. A. et al.: Proc. Natl. Acad. Sci. U.S.A. 97,
Conclusion 8206–8210 (2000)
[5] Gustafsson M. G. L.: Journal of microscopy
New methods of optical fluorescence im- 198, 82–87 (2000)
aging provide a ten-fold increase of spatial [6] Heilemann M. et al.: Angewandte Chemie In-
resolution. However, sample preparation, ternational Edition 47, 6172–6176 (2008)
microscope handling and data-analysis [7] Jungmann R. et al.: Nano letters 10, 4756–
are very demanding and it is not anymore 4761 (2010)
possible to calculate the achievable spatial [8] Betzig E. et al.: Science 313, 1642–1645
resolution. DNA origami based nanorulers (2006)
are ideal test-samples which offer defined [9] Fölling J. et al.: Nature methods 5, 943–945
distances between fluorescent marks and (2008)
can be immobilized in huge quantities on [10] Schmied J. J. et al.: Nat. Methods 9, 1133–
a standard cover-slip. Nanorulers allow to 1134 (2012)
measure the achievable spatial resolution
of modern super-resolution microscopes
but also of conventional confocal micro- Contact
scopes. Furthermore, the DNA origami Dr. Jürgen Schmied
technique allows the production of bright- GATTAquant GmbH
ness standards with very well defined Braunschweig, Germany
numbers of dye-molecules within one dif- schmied@gattaquant.com
fraction limited spot. These brightness www.gattaquant.com
More information about Read more about DNA
super-resolution microscopy: origami: http://bit.ly/
http://bit.ly/IM-SRM rothemund