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Image by Jakub Pospíšil, Cellim Lab, Centre for cellular imaging, CEITEC-MU, Brno
S5015
Light microscopy methods in biology
Lecture 3: Superresolution Microscopy
Jakub Pospíšil
Cellular Imaging Core Facility, Ceitec MU
jakub.pospisil@ceitec.muni.cz

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Resolution ranges of Biological Imaging techniques
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PET, MRI and Ultrasound
Widefield and TIRF fluorescence micrscopy
Electron micrscopy
Confocal Microscopy
Fluorescence microscopy
Abbe`s diffraction limit (200 nm)

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Difraction limit
The diffraction limit defined by Abbe corresponds to the radius of the spot where the light is
diffracted.
4
Lens
(NA)
500 nm
200 nm
Verdet (1869)
Abbe (1873)
Helmholtz (1874)
Rayleigh (1874)
Resolutionz = 2λ / [η • sin(α)]2

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Difraction limit
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Problem: molecules (features) within <200 nm not recognizable
The Abbe diffraction limit depends on:
-The light wavelength (λ)
-
-refractive index of the medium (n)
-
-half-angle of the converging spot (ϴ)
200 nm

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Gustafsson M G 1999 Extended resolution fluorescence microscopy Curr. Opin. Struct. Biol. 9 627–34
“Even though the classical resolution limits are imposed by physical law, they can in fact, be
exceeded and the limitations are true only under certain assumptions.
1) Observation takes place in the conventional geometry in which light is collected by a single
objective lens;
2) That the excitation light is uniform throughout the sample;
3) Fluorescence takes place through normal, linear absorption and emission of a single photon”

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Expansion Microscopy (ExM)
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Ed Boyden, the leader of the Synthetic Neurobiology Group at the Massachusetts Institute of
Technology
http://syntheticneurobiology.org/videos
ExM
4,5x

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Resolution ranges of Biological Imaging techniques
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PET, MRI and Ultrasound
Widefield and TIRF fluorescence micrscopy
Superresolution micrscopy
Electron micrscopy
Confocal Microscopy
Fluorescence microscopy
Abbe`s diffraction limit (200 nm)

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SR microscopy clasification
Challenges and trade-offs in super-resolution fluorescence microscopy. Although the nominal lateral
(xy) and axial (z) resolution of a microscope is the most prominent system parameter, the
usefulness for broader or routine application depends on a wealth of additional criteria. This
includes the ability to image time series of living samples and multidimensional imaging (3D
sectioning with multiple wavelength), as well as soft criteria, such as the easy applicability and
the reliability of the results. Notably, none of the currently available super-resolution
technologies fulfill all criteria.
https://rupress.org/jcb/article/190/2/165/35915/A-guide-to-super-resolution-fluorescence
Challenges and trade-offs in super-resolution fluorescence microscopy.

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Superresolution microscopy strategies
Diagram Description automatically generated

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Superresolution microscopy strategies
https://www.sciencedirect.com/science/article/pii/S106358231830019X#f0020

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Characterization of the effect of pinhole size on resolution and SNR in confocal microscopy
Airyscan microscopy
Confocal imaging with improved signal-to-noise ratio and super-resolution
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Axial and lateral resolution of a confocal microscope improves with smaller pinhole (below 1 AU).
But the signal decreases quickly!
Airyscan consists of an array of detectors where each element acts as a small pinhole. Each
detector element provides its own signal, and the software builds the image from a combination of
these signals. The array is able to collect more light from the microscope’s open pinhole. This
greatly improved light efficiency even comes with higher resolution.
https://www.researchgate.net/publication/318287874_Exploring_the_Potential_of_Airyscan_Microscopy_f
or_Live_Cell_Imaging

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Airyscan microscopy
Sample preparation
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Standard fixation and sample handling
Common fluorescence labelling
Airyscan detector acquisition
Ariscan processing
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•Advantages
•Compatibility with various samples
•
•Useful for any photostable fluorophore
‒Little adaption for sample preparation
•
•Good live cell imaging condition
•
•Resolution improvement
•
•Low phototoxicity
•
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Airyscan advantages and challanges
Challenges
•Speed
•
•Limited sample thickness
•
•Subject to algorithmic effects due to required mathematical post-processing

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Airyscan microscopy
Applications
Comparison of confocal (left) and Airyscan (right) microscopy
Microtubules labled with Alexa 561, (Zeiss)
Comparison of Airyscan (left) and confocal (right) microscopy
Stalled forks and telomere breakage, (J. Karlseder, Molecular and Cell Biology laboratory)
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Zeiss LSM880 (Cellim-CEITEC)

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Airyscan microscopy
Applications – LSM880 airyscan/confocal (CELLIM)
Airyscan module
Confocal
Mitochodria / Actin filament

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Airyscan module
Confocal module
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Airyscan microscopy
Applications – LSM880 airyscan/confocal (CELLIM)
Mitochodria

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Airyscan microscopy
Modalities: Airyscan FAST module
https://www.nature.com/articles/nmeth.f.398
Zeiss LSM880 Fast AiryScan, room 3103 - Department of Biosciences
The Fast module for AiryScan shapes the excitation spot into an ellipse, the AiryScan array
detector is then used to detect 4 pixels simultaneously, increasing scan speed 4-fold (27 fps for
480x480) while still improving resolution significantly compared to conventional confocal (170 nm
lateral).
Users can capture more structural information about highly dynamic processes. This is the highest
speed of any linear scanning confocal microscope. It also has superresolution and sensitivity
modes, which increase its flexibility.

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Structured ilumination microscopy (SIM)
SIM combines fluorescence, widefield-based structured illumination and digital image reconstruction
(2002)
Diagram Description automatically generated
 SR-SIM uses the information contained in the known illumination pattern
Mats Gustafsson
Biological Nanoimaging
Rainer Heintzmann

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https://onlinelibrary.wiley.com/doi/full/10.1046/j.1365-2818.2000.00710.x
What Is Moiré In Photography And How To Deal With It | Light Stalking
Structured ilumination microscopy (SIM)
The Super-Resolution SIM technique principle is to use interference-generated light patterns to
create a Moiré effect
This allows to extract information with higher resolution.
High frequency
Low frequency
pattern

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Structured ilumination microscopy (SIM)
The Super-Resolution SIM technique principle is to use interference-generated light patterns to
create a Moiré effect
New ways of looking at very small holes – using optical nanoscopy to visualize liver sinusoidal
endothelial cell fenestrations
https://www.degruyter.com/document/doi/10.1515/nanoph-2017-0055/html
Fig. 8—Differential filling of k-space. A, When low frequencies are removed from Fourier space of
Lincoln (upper left), sharp edges are preserved in image at the expense of contrast resolution.
When high frequencies are removed, image contrast is preserved; however, it is blurry and
demonstrates Gibbs artifacts (see Fig. 10). Observe how few spatial frequencies are actually
necessary to recreate a recognizable image of Lincoln. FT = Fourier transform, iFT = inverse
Fourier transform. B, By steering frequency- and phase-encoding gradients appropriately during MR
image acquisition, k-space can be filled not only sequentially line by line, but also in a spiral
fashion about the origin. Filling the essential, high-signal-to-noise, central portions of k-space
can save considerable time and result in a recognizable image. This comes at the expense of fine
detail, which is stored in the periphery of k-space (as depicted in A). C, Fourier transform
formula makes use of exponentials of imaginary numbers (ei) to represent simple waves, and as a
result the Fourier transform yields both real and imaginary information displaying complex
conjugate symmetry. Half-Fourier techniques exploit this symmetry by acquiring only half of k-space
and generating a mirror image of the remaining half. Such a time-saving mechanism comes at the
expense of signal-to-noise, however, because only half of the potential signal is actually
acquired.
https://www.semanticscholar.org/paper/An-introduction-to-the-Fourier-transform%3A-to-MRI.-Gallagher
-Nemeth/8c65f1e10b198149ac5c8fd2c4d6888b1769ffe6/figure/7

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Fourier Space: Fourier Transform
Fourier Space: Fourier Transform
This lectures explains the Fourier transform in terms understandable to non-mathematicians, and
explains the relations with microscopy.
Fourier transform is intimately associated with microscopy, since the alternating planes occurring
in the microscope (focal plane – back-focal plane, etc.) are related to each other by a function
very similar to the Fourier transform.
https://www.ibiology.org/talks/fourier-transform/

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Structured ilumination microscopy (SIM)
SIM combines fluorescence, widefield-based structured illumination and digital image reconstruction
Classic SIM
Redundant light exposure

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Structured ilumination microscopy (SIM)
Widefield imaging at super-resolution
Classic SIM
Lattice SIM

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Name of the presentation
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Structured ilumination microscopy (SIM)
Widefield imaging at super-resolution
Classic SIM
Lattice SIM
-Image faster with high image quality and low bleaching
-Better image quality at the same speed and low bleaching
-Image more gently with high speed and image quality

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Lattice SIM
Sample preparation
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Standard fixation and sample handling
common fluorescence labelling
SIM acquisition
SIM processing
Diagram Description automatically generated

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•Advantages
•Compatibility with various samples
•
•Useful for any photostable fluorophore
‒Little adaption for sample preparation
•
•Good live cell imaging condition
•
•High throughput and fast acquisition
•
•Resolution improvement
•
•Lattice SIM up to 100 μm  distance from cpverslip surface
•
•Straightforward data analysis
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SIM advantages and challanges
Challenges
•Limited sample thickness
•
•Phototoxicity (depends on sample type)
•
•Subject to algorithmic effects due to required mathematical post-processing

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Lattice SIM
Applications
Lattice SIM acquisiotion process
Tubulin structures labled with Alexa 561, (Elyra7, Zeiss)
SIM
Confocal

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Elyra7 Lattice SIM
Applications (CELLIM)
A picture containing text, indoor, floor, desk Description automatically generated A picture
containing outdoor object Description automatically generated
Zeiss Elyra7 (CELLIM)
Lattice SIM demonstration (CELLIM)
Growth cone of MEFs cells
(Elyra7, CELLIM)
Actin filament (white), FAKs (red)
Staphyloccocus (Michaela Procházková, Pavel Plevka, Ceitec MU Brno)
Cell mebrane (green), Nuclei (purple)
A picture containing outdoor object Description automatically generated A picture containing dark
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Mitochodrial membrane stained with anti-TOM20 antibody, (Elyra7, CELLIM)
SIM
WF

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Diagram Description automatically generated
Stephan Hell
Jan Wichmann (1994)
Stimulated Emmision
M. Kroug (1995)
ground state Depletion
Stimulated emission depletion microscopy (STED)
PSF shaping with saturated emission depletion

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Stimulated emission depletion microscopy (STED)
PSF shaping with saturated emission depletion
In STED microscopy:
•Focal plane is scanned with two overlapping laser beams
•
•Typically being pulsed with a mutual deley
•
•The first laser excitates the flourophores
•
•The second longer wavelength laser drives the fluorophores back to the ground state by the process
of stimulated emmision.
•A phase plate (phase mask) in the light path of the depletion laser generates a donut-shaped
energy distribution, leaving only a small volume from which light can be emitted that is then being
detetcted.
•
• Thus, the PSF is shaped to a volume smaller than the diffraction limit
Fig. 1

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Stimulated emission depletion microscopy (STED)
PSF shaping with saturated emission depletion
Problem: molecules (features) within <200 nm not recognizable
S1
S0
excitation
emission

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Solution: keep some molecules (features) dark
Stimulated emission depletion microscopy (STED)
PSF shaping with saturated emission depletion
Problem: molecules (features) within <200 nm not recognizable
S1
S0
excitation
STED
https://www.bcp.fu-berlin.de/en/biologie/arbeitsgruppen/genetik/ag_sigrist/forschung/sted/index.htm
l

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•STED pulse is modified to feature a zero-intensity point at the center of focus with strong
intensity at the periphery.
•When the two laser pulses are superimposed, only molecules that reside in the center of the STED
beam are able to emit fluorescence, thus significantly restricting emission.
•This action effectively narrows the point spread function and ultimately increases resolution
beyond the diffraction limit.
•To generate a complete image, the central zero is rasterscanned across the specimen in a manner
similar to singlephoton confocal microscopy

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STED microscopy
Sample preparation
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Standard fixation
Specific requirement for IF labeling
STED acquisition
STED processing
Diagram Description automatically generated

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•Advantages
•Imaging resolution improved by directly optimizing the point spread function, not during
post-procesing
•
•Multicolor imaging
•
•Applications when biological question requires <100 nm resolution, but cells must be fixed to
achieve this
•
•The depletion beam can also be shaped along the z-axis, giving resolution in z of about 80 nm (at
a slight expense of lateral resolution)
•
•
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STED advantages and challanges
Challenges
•Not suitable for live cell measurment
•
•Point scanning methods = lower scan speed (depends on FOV)
•
•Difficult laser alignment
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•Intense (5W) depletion laser -> expensive
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•Very phototoxic, high photobleaching
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•Photostable fluorophores required
•
•Deconvolution may need to be applied for low signal particularly if sample has high background

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STED microscopy
Applications
Integrated Microscopy Technologies - SciLifeLab
Comparison of confocal (upper) and STED (lower) microscopy
HeLA cells stained against nuclear pore complex protein NUP153,
(http://jcb.rupress.org/content/190/2/165.fu)
SPY555-tubulinArtboard
Comparison of confocal (upper) and STED (lower) microscopy
SPY555-tubulin labeled HeLA cells
(courtesy of Spirochrome)

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STED microscopy
Modalities: 3D STED with Dynamic Minimum (DyMIN STED)
Live-cell superresolution microscopy with resolutions down to 25 nm* - DyMIN STED dramatically
reduces the light irradiation on your sample (up to two orders of magnitude).
Resolution truly down to 25 nm - As demonstrated by separating two fluorescent point-structures
being 30 nm apart.
Volume / time-lapse imaging with easy3D STED resolution - DyMIN STED substancially reduces
photobleaching and enables long term measurements over volumes or over dozens and dozens of frames.
DyMIN STED is a co-development between Stefan Hell and coworkers and Abberior Instruments.
dymin_1010
Hippocampal neurons show the characteristic ~192 nm betaII spectrin periodicity. Sample with
courtesy from Elisa D’Este (MPIbpc).

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Single molecule localization microscopy (dSTORM/PALM)
PSF optically recostructed
Diagram Description automatically generated
Eric Betzig
Eric Betzig - Biographical - NobelPrize.org
William Moerner
Stochastic Optical Reconstruction Microscopy (dSTORM)
Photo Activated Localization Microscopy (PALM)

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SMLM (dSTORM/PALM)
PSF optically recostructed
Problem: molecules (features) within <200 nm not recognizable
S1
S0
excitation
emission

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SMLM (dSTORM/PALM)
PSF optically recostructed
Problem: molecules (features) within <200 nm not recognizable
S1
S0
Fluorescence
Dark state
OFF
ON
T1
D
Simplified Jablonski Diagram

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•Determines the position of individual fluorescent molecules located at a structure of interest,
rather than resolving them optically.
•
•The positions can be determined with a precision of the order of 10 nm
•
•The resolution depends on the size and density of molecules and the obtainable signal-to-noise
ratio (theoreticaly unlimited).
•
•Typical images, however, provide 10-fold improved resolution in comparison to conventional
microscopy (20 nm in xy and 60 nm in z).
•
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Super-Resolution Microscopy - an overview | ScienceDirect Topics
Single molecule localization microscopy (dSTORM/PALM)
PSF optically recostructed

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Single molecule localization microscopy (dSTORM/PALM)
PSF optically recostructed
Thousands of such positions are gathered and superimposed, then it is possible to generate an image
of a structure with improved resolution.

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(dSTORM/PALM)
COMPARISON
•blinking passively
•interaction of fluorescent molecules with its blinking buffer which cause the molecules to switch
ON and OFF (hence the term “stochastic”).
•Under proper conditions (e.g. pH val-ue, redox states, etc.) only few molecules are ON during the
acquisition of each frame and therefore easily distinguishable
•label molecules emit at random times due to chemical reactions or interactions in their immediate
vicinity
•Fixed samples
•photoactivated (blinking actively) - uses photoactivation to switch the molecules.
•employs photoactivatable dyes (predominantly switchable fluorescent proteins, like photoswitchable
GFP, tdEOS, etc
•The switching of the individual molecules is still random, but the rate with which the molecules
switch on or off can be controlled by increasing or decreasing the intensity of the switching laser
(e.g. 405 nm).
•They can be used in vivo, have a higher specificity and do not require fixation and
permeabilization of the specimen
•Live samples
dSTORM
PALM

https://www.westburg.eu/dstorm

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SMLM microscopy
Sample preparation
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Standard fixation
Specific requirement for IF labeling
dSTORM/PALM acquisition
dSTORM/PALM processing
Diagram Description automatically generated
https://hcbi.fas.harvard.edu/files/hcbidoug/files/en_41_011_065_elyra_sample-prep-quickguide.pdf

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•Advantages
•PALM can be used for live cell imaging
•
•twocolour imaging
•
•PALM / dSTORM deliver the highest resolution of all presented super-reso-lution methods
(theoretically unlimited - typically 20 nm in xy / 60 nm in z) and can deliver molecular detail.
•
•Best results are obtained from transparent and well-prepared specimens near the coverslide surface
(ca. 10 μm from the coverslip surface).
•
•
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SMLM advantages and challanges
Challenges
•dSTORM generally not suitable for live cell imaging
•
•PALM and dSTORM are considered slow because collection of a typical image sequence (>1000 frames)
takes upward of 10s, typically minutes.
•Phototoxicity and photobleaching has to be considered
•Data analysis possibilities that are not easily accessible via other methods
•The biggest challenge for PALM/dSTORM is the need for photoswitchable molecules or addition of
chemistry to bring the labels into an adequate “blinking” regime. Also, PALM and dSTORM have
limited in vivo applications. Long term stability is a crucial concern for PALM/dSTORM equipment.
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~ 15 nm
Fc
Fab
Antigen recognition site
Antibody challenge
Combination of primary and secondary antibody: large `linkage` error (~20nm)
Secondary antibody with
fluorophore
Primary antibody
1°Ab+2°Ab
1°Ab
Nanobodies
Affimers
If we consider the size of antibodies it is preferable to do direct antibody labeling without a
primary and secondary Ab. Smaller antibodies as nanobodies with a size in range of 2 nm may be
preffered. Nowadays new affimers (small ptoeins, with antibody specificity) being produced, which
are even smaller than nanobodies.

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dSTORM/PALM microscopy
Applications
Microtubules in a cell (Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent
Probes, Science),Confocal microscopy (upper), dSTORM (lower)
Mitochodrial membrane stained with anti-TOM20 antibody and photoswitchable Alexafluor-647, (Elyra7,
CELLIM)

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3D STORM – point spread function distortion based on Z position
Special optical element to shape point spread function of emitters
dSTORM microscopy
Modalities: 3D dSTORM
3D dSTORM of Mitochodrial membrane stained with anti-TOM20 antibody and photoswitchable
Alexafluor-647, (CELLIM)

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Conclusion
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OVERALL
•Super-resolution microscopy requires thorough sample preparation
•
•Image quality is rapidly affected by impurities (dust grains, bubbles, unspecific staining, etc.)
•
•Care must be taken that all parts of the system, from the cover glass to the mounting or embedding
medium are clean and well-defined (e.g. uniform thick-ness, clean mounting, labeling specificity,
etc.).
•
•
Name of the presentation
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Versatility / live cell imaging
Name of the presentation
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Airyscan
SIM
PALM/dSTORM
STED
Live cell imaging
Compatible
Compatible
Not considered
Not compatible
Laser wavelengths
No restrictions
No restrictions
High laser power
/ limited dyes
the highest irradiation dosage / limited dyes
Specific objectives
No restrictions
No restrictions
Specific objectives
Specific objectives
Speed
Limited by FOV – up to 30 fps
Highest acquisition speed – over 100 fps
Thousends of images neccesity
Limited by FOV – up to 30 fps
Depth of samples
Thick specimens
(less sensitive towards changes in RI)
Depends on accurate projection of the illumination pattern - thickness up to 20 um distance from
more challenging when going into thick,
over-labeled, noise-rich, scattering specimens
-quality rapidly decays with penetration depth
-laser dosage
also on the image planes that are not being imaged

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What next?
•Highest possible resolution
•
•High contrast
•
•Fast acquisition
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•Lower laser power
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•Thick samples / live samples
•
•Minimal labelling
•
•phototoxicity
•
•Online functional data processing
•
•
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•Economical viability
•
•Easy to use by non-experts
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What next?
1) Correlative TEM/PALM microscopy
https://www.science.org/doi/10.1126/science.1127344
2) Correlative AFM/STED
https://www.frontiersin.org/articles/10.3389/fncel.2017.00104/full
Correlative stimulated emission depletion (STED)/atomic force... | Download Scientific Diagram
3) MINFLUX (abberior)
https://abberior-instruments.com/products/minflux/
STED
Confocal

Combined super-resolution and atomic force microscopy. (A) Components of a typical atomic force
microscope (AFM) are depicted. AFM employs an exquisitely sensitive cantilever upon which a laser
beam is reflected and a photodetector senses laser beam deflections to map the cell surface at
atomic resolution of 0.1 nm in both lateral and axial dimensions. (B) Representative image of a
live murine astrocyte obtained by sequential, correlated STED-AFM microscopy. Super-resolution STED
images (top inset) reveal polarized F-actin in the lamella and near the leading edge. Actin labeled
with SiR-actin can be seen with corresponding thick filaments in AFM images (bottom panel)
associated with focal adhesions (arrows).
Panel (A): Adapted from Shan, Y., & Wang, H. (2015). The structure and function of cell membranes
examined by atomic force microscopy and single-molecule force spectroscopy. Chemical Society
Reviews, 44, 3617–3638. Panel (B): Reprinted with permission from Curry, N., Ghezali, G., Kaminski
Schierle, G. S., Rouach, N., & Kaminski, C. F. (2017). Correlative STED and atomic force microscopy
on live astrocytes reveals plasticity of cytoskeletal structure and membrane physical properties
during polarized migration. Frontiers in Cellular Neuroscience, 11, 104.

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http://cellim.ceitec.cz
https://twitter.com/Ceitec_CellimCF
Cellular Imaging Core Facility - CELLIM
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https://www.czech-bioimaging.cz
https://www.eurobioimaging.eu