Super-resolution STED 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:

structure image “similar objects closer than about half the wavelength should not be distinguishable in a light ”

Ernst Abbe 1873

2 Standard (confocal) vs. Superresolution (STED)

3 Confocal () 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

1.0 Confocal STED Is I

0.5

Fluorescence

0.0 0.0 0.5 1.0 1.5 I [GW/cm²] STED just physics!

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

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