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Super-Resolution STED Microscopy and Its Application in Neuroscience

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Super-Resolution STED Microscopy and Its Application in Neuroscience Super-resolution STED microscopy and its application in neuroscience Katrin Willig 18th German-American Frontiers of Engineering Symposium Hamburg 20-23 March 2019 Nanoscale Microscopy and Molecular Physiology of the Brain MPI of Cluster of Excellence 171, Experimental DFG Research Center 103 Medicine 1 Resolution in far-field light microscopy diffraction limit: minimum resolvable distance l l a dmin d 2 nsina numerical aperture (NA) n: refractive index structure image “similar objects closer than about half the wavelength should not be distinguishable in a light microscope” Ernst Abbe 1873 2 Standard (confocal) vs. Superresolution (STED) 3 Confocal (fluorescence) microscopy x 200 nm Abbe‘s equation y λ d 2n sin a Excitation d Fluorescence Detection Dichroic Scanning Mirror 1 Device S1 Excitation Fluorescence S0 4 STED (STimulated Emission Depletion) microscopy x 200 nm Phase Plate y 0 2p STED Excitation d Fluorescence Detection Dichroic Dichroic Scanning Mirror 1 Mirror 2 Device Nobel Prize in Chemistry 2014 to Betzig, Hell & Moerner S1 "for the development of super-resolved fluorescence microscopy." . Excitation Stimul Emission STED beam: keeps molecules non-fluorescent Fluorescence S0 5 Diffraction limited resolution PSF 1 220 nm 0 -200 0 200 1.0 0.5 y 500 nm x Fluorescence 0.0 min max 0.0 0.5 1.0 1.5 I [GW/cm²] STED 6 Subdiffraction resolution Depletion distribution 1 132 nm 0 -200 0 200 1.0 0.5 y 500 nm Fluorescence x 0.0 min max 0.0 0.5 1.0 1.5 I [GW/cm²] STED 7 Subdiffraction resolution Depletion distribution 1 84 nm 0 -200 0 200 1.0 0.5 y 500 nm Fluorescence x 0.0 min max 0.0 0.5 1.0 1.5 I [GW/cm²] STED 8 Subdiffraction resolution Depletion distribution 1 52 nm 0 -200 0 200 1.0 0.5 y 500 nm Fluorescence x 0.0 min max 0.0 0.5 1.0 1.5 I [GW/cm²] STED 9 Subdiffraction resolution Depletion distribution 1 22 nm 0 -200 0 200 1.0 0.5 y 500 nm Fluorescence x 0.0 min max 0.0 0.5 1.0 1.5 I [GW/cm²] STED 10 Diffraction limited resolution PSF 1 220 nm 0 -200 0 200 1.0 0.5 y 500 nm x Fluorescence 0.0 min max 0.0 0.5 1.0 1.5 I [GW/cm²] STED 11 Subdiffraction resolution l l Modified Abbe r r equation 2nsina 2nsina 1 I / Is Confocal STED Is I 1.0 0.5 just physics! Fluorescence 0.0 0.0 0.5 1.0 1.5 I [GW/cm²] STED 12 Synapse: connecting neurons Electron microscopy: Synapse Chemical signal Neuron 1 Synaptic cleft Neuron 2 200 nm Kristen M. Harris lab https://synapseweb.clm.utexas.edu/16-chemical-synapses-11 13 Spine/synapse plasticity requires nanoscale resolution Neuron 1 Strength of synaptic transmission Neuron 2 Spine neck << 200 nm - Synapses are key functional information processing units of neural circuits. - Spines are morphological correlates of synaptic strength - Synaptopathies: Defects in synaptic proteins cause > 100 brain diseases (Autism, Schizophrenia…) 14 Why imaging spines in vivo? State-of-the-art imaging: Cells & connections intact, Two-photon microscopy Natural environment In vivo imaging Zuo et al., 2005, Neuron, 46, 181 Information processing e.g. visual stimulation, learning tasks STED STED 15 STED is ideal to superresolve structures in vivo Two-photon imaging Cells & connections intact, Natural environment In vivo imaging Zuo et al., 2005, Neuron, 46, 181 Information processing e.g. visual stimulation, learning tasks STED STED Super-resolution Organotyp. hippoc. brain slice 16 Fluorescent proteins for live-cell STED microscopy First discovered: Green Fluorescent Protein (GFP); 700nm (Shimomura 1961) tagRFP657 mNeptune mPlum mKate2 mRaspberry mCherry 600nm mStrawberry tagRFP mOrange EYFP, Venus, Citrine EGFP 500nm ECFP Aequorea victoria EBFP2 Non-comprehensive E.g. Chudakov et al. (2010) Emission 400nm Physiol Rev 90: 1103 Genetically encoded fusion-proteins for live-cell imaging 17 Challenge of tissue imaging: Penetration depth Problem: refractive index mismatch! spherical aberrations Glycerine immersion objective Corr 1 Corr 10 n = 1,45 NA = 1,3 63x z 200nm x Correction collar ... Compensates spherical aberrations 18 Exploring the brain in vivo 19 Cranial window for in vivo nanoscopy Craniotomie (open-skull) Glass window 20 In vivo STED microscopy Visual cortex of living mouse 2 µm Neurons expressing STED cytoplasmic EYFP Berning, Willig, Steffens, Dibaj, Hell (2012) Science 21 In vivo STED microscopy Visual cortex of living mouse 2 µm Neurons expressing STED cytoplasmic EYFP 23 x 18 x 3 µm, 10µs / px, 800 x 600 x 5 px, interval 5 min Berning, Willig, Steffens, Dibaj, Hell (2012) Science 22 In vivo STED microscopy Visual cortex of living mouse ~20 µm deep 70nm 2 µm Neurons expressing cytoplasmic EYFP 1 µm Berning, Willig, Steffens, Dibaj, Hell (2012) Science 23 In vivo STED microscopy Visual cortex of living mouse STED Neurons expressing cytoplasmic EYFP 128 z-stacks, 5 slices interval 10 sec 1 µm Berning, Willig, Steffens, Dibaj, Hell (2012) Science 24 In vivo STED microscopy of actin STED59nm 100 300 500 [nm] Raw Actin data Lifeact-YFP 1µm Maximum intensity projection Willig et al. (2014) Biophys. J. 106 25 Actin 87nm 55nm 60nm 4µm deep 8µm 65nm 65nm 20µm 1µm Willig et al. (2014) 40µm Biophys. J. 106 4 In vivo STED microscopy of the post-synaptic density PSD95 Scaffolding protein & signalling platform Sheng & Hoogenraad Annu. Rev. Biochem. 2007 76:823 • High complexity: PSD comprises ~1500 proteins • PSD (post-synaptic density) size correlates with synaptic strength Size 200-800nm 50nm thin 27 In vivo nano-organization of PSD95 Side-view: Top-view: Knock-in mouse Spine PSD95-GFP Visual cortex Dendrite PSD95-GFP In vivo confocal In vivo STED min max 500 nm Wegner, Mott, Grant, Steffens, Willig (2018) Sci Rep. 8, 219-219 28 Nano-organization of PSD95: EM vs. in vivo STED STED EM Mushroom spines Perforated Perforated PSD PSD95 200nm fixed In vivo Stewart et al. (2005). European Journal of Neuroscience, 21(12), 3368–3378. 29 Morphological changes of large PSD95 assemblies in vivo Time intervall In vivo STED t=0 1 min 2 min 3 min t=0 1 min 2 min 3 min t = 1 min t=0 0.5 h 1 h 1.5 h t=0 0.5 h 1 h 1.5 h t = 0.5 h t=0 1 h 2 h t=0 1 h 2 h t = 1 h No change t=0 3 h 6 h t=0 3 h 6 h Subtle change t = 3 h t=0 3 h 6 h t=0 3 h 6 h Strong change Wegner, …, Willig (2018) Sci Rep. 500 nm 30 Dissecting the PSD in vivo How can we analyse the shape? 1 Counts 2631 Acknowledgement Heinz Steffens Alexander Mott Waja Wegner Collaboration: Sabine Liebscher, LMU Munich Nanoscale Microscopy and Molecular Physiology of the Brain MPI for Experimental Medicine Cluster of Excellence 171, DFG Research Center 103 32.
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