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 Resolution ranges of Biological Imaging techniques 3 PET, MRI and Ultrasound Widefield and TIRF fluorescence micrscopy Electron micrscopy Confocal MicroscopyFluorescence microscopy Abbe`s diffraction limit (200 nm) Difraction limit The diffraction limit defined by Abbe corresponds to the radius of the spot where the light is diffracted. 4 Lens (NA) Wavelength (λ) 500 nm 200 nm Verdet (1869) Abbe (1873) Helmholtz (1874) Rayleigh (1874) 𝒅 = λ 𝟐[η • sin(α)] Resolutionz = 2λ / [η • sin(α)]2 Difraction limit 5 𝜆 2𝑛𝑠𝑖𝑛𝛼 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 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” Expansion Microscopy (ExM) 7 Ed Boyden, the leader of the Synthetic Neurobiology Group at the Massachusetts Institute of Technology http://syntheticneurobiology.org/videos ExM 4,5x Resolution ranges of Biological Imaging techniques 8 PET, MRI and Ultrasound Widefield and TIRF fluorescence micrscopy Superresolution micrscopy Electron micrscopy Confocal MicroscopyFluorescence microscopy Abbe`s diffraction limit (200 nm) 9 SR microscopy clasification https://rupress.org/jcb/article/190/2/165/35915/A-guide-to-super-resolution-fluorescence Challenges and trade-offs in super-resolution fluorescence microscopy. 10 Superresolution microscopy strategies 11 Superresolution microscopy strategies https://www.sciencedirect.com/science/article/pii/S106358231830019X#f0020 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 12 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_Mic roscopy_for_Live_Cell_Imaging Airyscan microscopy Sample preparation 13 Standard fixation and sample handling Common fluorescence labelling Airyscan detector acquisition Ariscan processing Advantages • Compatibility with various samples • Useful for any photostable fluorophore ‒ Little adaption for sample preparation • Good live cell imaging condition • Resolution improvement • Low phototoxicity 14 Airyscan advantages and challanges Challenges • Speed • Limited sample thickness • Subject to algorithmic effects due to required mathematical post-processing 15 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) Zeiss LSM880 (Cellim-CEITEC) 16 Airyscan microscopy Applications – LSM880 airyscan/confocal (CELLIM) Airyscan moduleConfocal Mitochodria / Actin filament 17 Airyscan moduleConfocal module Airyscan microscopy Applications – LSM880 airyscan/confocal (CELLIM) Mitochodria 18 Airyscan microscopy Modalities: Airyscan FAST module https://www.nature.com/articles/nmeth.f.398 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. 19 Structured ilumination microscopy (SIM) SIM combines fluorescence, widefield-based structured illumination and digital image reconstruction (2002) SR-SIM uses the information contained in the known illumination patternMats Gustafsson Rainer Heintzmann 20https://onlinelibrary.wiley.com/doi/full/10.1046/j.1365-2818.2000.00710.x 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 21 Structured ilumination microscopy (SIM) The Super-Resolution SIM technique principle is to use interference-generated light patterns to create a Moiré effect https://www.degruyter.com/document/doi/10.1515/nanoph-2017-0055/html https://www.semanticscholar.org/paper/An-introduction-to-the-Fourier-transform%3A-to-MRI.- Gallagher-Nemeth/8c65f1e10b198149ac5c8fd2c4d6888b1769ffe6/figure/7 22 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/ 23 Structured ilumination microscopy (SIM) SIM combines fluorescence, widefield-based structured illumination and digital image reconstruction Classic SIM Redundant light exposure 24 Structured ilumination microscopy (SIM) Widefield imaging at super-resolution Classic SIM Lattice SIM Name of the presentation 25 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 Lattice SIM Sample preparation 26 Standard fixation and sample handling common fluorescence labelling SIM acquisition SIM processing 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 27 SIM advantages and challanges Challenges • Limited sample thickness • Phototoxicity (depends on sample type) • Subject to algorithmic effects due to required mathematical post-processing 28 Lattice SIM Applications Lattice SIM acquisiotion process Tubulin structures labled with Alexa 561, (Elyra7, Zeiss) SIMConfocal 29 Elyra7 Lattice SIM Applications (CELLIM) 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) Mitochodrial membrane stained with anti-TOM20 antibody, (Elyra7, CELLIM) SIMWF 30 Stephan Hell Jan Wichmann (1994) Stimulated Emmision M. Kroug (1995) ground state Depletion Stimulated emission depletion microscopy (STED) PSF shaping with saturated emission depletion 31 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 32 Stimulated emission depletion microscopy (STED) PSF shaping with saturated emission depletion 𝜆 2𝑛𝑠𝑖𝑛𝛼 Problem: molecules (features) within <200 nm not recognizable S1 S0 excitation emission 33 𝜆 2𝑛𝑠𝑖𝑛𝛼 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.html STED microscopy Sample preparation 34 Standard fixation Specific requirement for IF labeling STED acquisition STED processing 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) 35 STED advantages and challanges Challenges • Not suitable for live cell measurment • Point scanning methods = lower scan speed (depends on FOV) • Difficult laser alignment • Intense (5W) depletion laser -> expensive • Very phototoxic, high photobleaching • Photostable fluorophores required • Deconvolution may need to be applied for low signal particularly if sample has high background 36 STED microscopy Applications 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) Comparison of confocal (upper) and STED (lower) microscopy SPY555-tubulin labeled HeLA cells (courtesy of Spirochrome) 37 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. Hippocampal neurons show the characteristic ~192 nm betaII spectrin periodicity. Sample with courtesy from Elisa D’Este (MPIbpc). 38 Single molecule localization microscopy (dSTORM/PALM) PSF optically recostructed Eric Betzig William Moerner Stochastic Optical Reconstruction Microscopy (dSTORM) Photo Activated Localization Microscopy (PALM) 39 SMLM (dSTORM/PALM) PSF optically recostructed 𝜆 2𝑛𝑠𝑖𝑛𝛼 Problem: molecules (features) within <200 nm not recognizable S1 S0 excitation emission 40 SMLM (dSTORM/PALM) PSF optically recostructed 𝜆 2𝑛𝑠𝑖𝑛𝛼 Problem: molecules (features) within <200 nm not recognizable S1 S0 Fluorescence Dark state OFFON T1 D Simplified Jablonski Diagram • 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). 41 Single molecule localization microscopy (dSTORM/PALM) PSF optically recostructed 42 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. 43 (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 SMLM microscopy Sample preparation 44 Standard fixation Specific requirement for IF labeling dSTORM/PALM acquisition dSTORM/PALM processing https://hcbi.fas.harvard.edu/files/hcbidoug/files/en_41_011_065_elyra_sample-prep-quickguide.pdf 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 wellprepared specimens near the coverslide surface (ca. 10 μm from the coverslip surface). 45 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. 46 ~ 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 ~25nm 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. ~50nm 47 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) 48 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) Conclusion 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 thickness, clean mounting, labeling specificity, etc.). Name of the presentation 50 Versatility / live cell imaging Name of the presentation 51 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, noiserich, scattering specimens - quality rapidly decays with penetration depth - laser dosage also on the image What next? • Highest possible resolution • High contrast • Fast acquisition • Lower laser power • Thick samples / live samples • Minimal labelling • phototoxicity • Online functional data processing 52 • Economical viability • Easy to use by non-experts 53 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 3) MINFLUX (abberior) https://abberior-instruments.com/products/minflux/ STEDConfocal 54 http://cellim.ceitec.cz https://twitter.com/Ceitec_CellimCF Cellular Imaging Core Facility - CELLIM https://www.czech-bioimaging.cz https://www.eurobioimaging.eu