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C-CINA.org Single Cell Imaging What can be learned about the single whole cell using EM? Where are we now? What are the challenges going forward? Henning&Stahlberg&

  1. C-CINA.org Single Cell Imaging What can be learned about the single whole cell using EM? Where are we now? What are the challenges going forward? Henning&Stahlberg& Center&for&Cellular&Imaging&and&NanoAnaly4cs&(C8CINA)& Biozentrum,&University&of&Basel,&Switzerland 1 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  2. Multi-Resolution 3D Microscopy 1 mm Super-Resolution Fluorescence Light Microscopy Light Microscopy 100 µm 3View Serial Blockface Sample Diameter (SBF)-SEM 10 µm Focussed Ion Beam (FIB)-SEM 1 µm TEM Tomography 100 nm 10 nm Cryo-TEM 0.1 1.000 100 10 1 Resolution (nm) No single instrument can cover all scales. 2 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  3. File size of a volume at a certain resolution 10 mm 1 PB 1 mm 1 TB 100 µm Sample Diameter 1 GB 10 µm 1 MB 1 kB 1 µm 1 Byte 100 nm 10 nm 0.1 1.000 100 10 1 Resolution (nm) No single instrument can cover all scales. 3 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  4. Data recording time, if recording at 1px/µs 10 mm 1 PB = 31 years 1 mm 1 TB = 2 weeks 100 µm 1 GB = 16 min Sample Diameter 10 µm 1 MB = 1 s 1 kB = 1 ms 1 µm 1 Byte 100 nm 10 nm 0.1 1.000 100 10 1 Resolution (nm) No single instrument can cover all scales. 4 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  5. Specimen(Workflows CLEM CEMOVIS(/(CETOVIS CryoDMicrotome HP(freezing Tokuyasu Tokuyasu Tokuyasu CryoDEl.(Tomography Cryo(Temperature Tissue Room(Temperature Freeze(( subsGtuGon CLEM Chemical( 
 FixaGon Microtome((RT) CLEM El.(Tomography((RT) 3ViewDSBFDSEM((RT) FIBDSEM((RT) 6 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  6. Quanta0200/3View: Christel&& Serial&Block&Face&SEM Genoud& (FMI) Olfactory bulb of zebrafish embryo: 15x15x15 µm: 500 cuts@30nm. 6nm per pixel Saved with isotropic voxel size: 
 volume can be observed in all dimensions RoomDTemperature( Shaved(with(microtome(( Remaining(Block(is(( Block(in(SEM inside(of(the(SEM imaged(with(SEM Collabora@on&with&&Susan&Gasser&&&Rainer&Friedrich,&FMI Denk W, Horstmann H (2004) PLoS Biology 11:e329 7-1 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  7. Specimen(Workflows CLEM CEMOVIS(/(CETOVIS CryoDMicrotome HP(freezing Tokuyasu Tokuyasu Tokuyasu CryoDEl.(Tomography Cryo(Temperature Tissue Room(Temperature Freeze(( subsGtuGon CLEM Chemical( 
 FixaGon Microtome((RT) CLEM El.(Tomography((RT) 3ViewDSBFDSEM((RT) FIBDSEM((RT) 8 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  8. Focussed(Ion(Beam(SEM:(FIB(SEM,(also(called:(DualDbeam HeLa(cell,(72hrs(post(infecGon(with(B rucella'abortus (bacteria 
 ((with(Christoph(Dehio,(InfectX) (c) Jarek Sedzicki, C-CINA 9-2 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  9. &(3View)&Serial&Block&Face&& Scanning&Electron&Microscopy (Christel&Genoud,&FMI/C0CINA) up&to&700µm&diameter& at&10&nm&resolu4on 3View SBF-SEM FIB-SEM Focussed&Ion&Beam& Scanning&Electron&Microscopy& (Jarek&Sędzicki,&C0CINA) up&to&100µm&diameter& at&3&nm&resolu4on 10-1 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  10. • (3View)(SBFDSEM(is(ideal(to(study(the(morphology(of( biological(Gssue.( • Currently(only(on(roomDtemperature,(fixed(and(stained( &(3View)&Serial&Block&Face&& samples.( Scanning&Electron&Microscopy • Can(easily(be(combined(with(thinDsecGoning(TEM.( (Christel&Genoud,&FMI/C0CINA) • Might(be(extended(to(CLEM,(and(combined(with(EDAX,( cathodoluminescense(imaging,(ion(mass(spectrometry,(… up&to&700µm&diameter& at&10&nm&resolu4on 3View SBF-SEM FIB-SEM • FIBDSEM(is(ideal(to(study(the(morphology(of(one(cell.( • Mostly(at(roomDtemperature,(difficult(in(cryo.( • Can(be(combined(with(3View(or(thinDsecGoning(TEM.( Focussed&Ion&Beam& Scanning&Electron&Microscopy& • Might(be(extended(to(CLEM,(and(combined(with(EDAX,( (Jarek&Sędzicki,&C0CINA) cathodoluminescense(imaging,(ion(mass(spectrometry,(… up&to&100µm&diameter& at&3&nm&resolu4on 11 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  11. Multi-Resolution 3D Microscopy 1 mm Super-Resolution Fluorescence Light Microscopy Light Microscopy 100 µm 3View Serial Blockface Sample Diameter (SBF)-SEM 10 µm Focussed Ion Beam (FIB)-SEM 1 µm TEM Tomography 100 nm 10 nm Cryo-TEM 0.1 1.000 100 10 1 Resolution (nm) No single instrument can cover all scales. 12-2 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  12. • Electron(tomography(is(the(best(possible(method(to(study( thin(cells((<1(µm)(by(EM((thinner(is(be_er).( • Can(be(applied(to(secGons(of(cells((CETOVIS),(can(be(done( in(cryo.( TEM(Tomography • Can(easily(be(combined(with(LM((CLEM).( • Can(be(applied(to(serial(secGons((e.g.,(Brad(Marsh’s(work)( • Can(be(extended(by(subDvolume(averaging. up&to&1µm&in&diameter& at&2&nm&resolu4on • STEM(Tomography(allows(to(image(slightly(thicker( samples(than(TEM(tomography.(( • Only(amplitude(contrast(can(be(recorded.(( • The(resoluGon(is(be_er(in(the(upper(half(of(the(sample.(( STEM(Tomography • Technically(challenging. up&to&2µm&diameter& at&3&nm&resolu4on 13 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  13. Prionoid fibril strains and Neurodegeneration Investigations at the level of - Tissue, - Cellular, - Membrane, - and Fibrils. Goedert et al. , Trends in Neurosciences 33 (7), 317-325 (2010) Diseases related to 흰 -Synuclein fibrils Diseases related to tau neurofibrillar tangles • Alzheimer’s Disease (AD) • Parkinson’s Disease (PD) • Agyrophilic Grain Disease • Dementia with Lewy Bodies • Corticobasal Degeneration • Lewy Body variant of AD • Frontotemporal Dementia (Pick’s Disease) • Multiple System Atrophy • Progressive Supranuclear Palsy • Neurodegeneration with Brain Iron Accumulation • Tangle-only Dementia (NBIA) Type I • White matter tauopathy with globular glial • Parkinson’s Disease with Dementia • Pure Autonomic Failure (PAF) Disease inclusions 15 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  14. Following the Spreading Process at the Single Cell Level Microfluidics-based co-pathological neuronal cell cultures LUHMES cells (Marcel Leist, Konstanz): 
 E human mesencephalic cells that can be differentiated 
 into neuron-like dopaminergic cells The single-cell visual proteomics platform α -synuclein tau 
 (Stahlberg lab) (Villars, Stahlberg, et al. , PNAS 2008) 16 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  15. The single-cell visual proteomics pipeline Microfluidics-based co-pathological neuronal cell cultures Cell culturing Stabilisation Conditioning Live cell imaging X-linking TEM RPPA E MS MicroViso Cell lysis Protein fishing* Hand-over 5 pliter 5-100 nliter 200 mg/ml 2 µg/ml 17 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  16. Negative stain EM grid preparations “Classical” Total proteomics Single cell 5 pl @ 200 mg/ml Sample 5µl @ 0.1 mg/ml Cell lysis, mixing with heavy metal salt Sample preparation in µ-fluidics. Selective adsorption. Huge protein loss during blotting. Droplet-deposition/Writing (5 to 100nl) Staining with heavy metal salt. Drying of complete droplet. Up-concentration on grid. Removal of excess stain. 100% of proteins on grid. 0.1% of proteins on grid. No selective adsorption. Selective adsorption. Kemmerling et al., 2012 18 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  17. Set-up 19-1 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  18. Set-up 19-2 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  19. OpenBEB – LabView-based instrument control 
 with database integration 20-1 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  20. Pipeline Stabilisation Cell culturing Conditioning Live cell imaging X-linking E Cell lysis Protein fishing* Hand-over 21-1 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  21. Cell cultures E 30µm LUHMES (Lund Human Mesencephalic) cells, can be differentiated into dopaminergic, neuron-like cells. Cells express α -synuclein and tau, and cytosolic GFP. 22 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  22. Single cell lysis E Electro-lysis & “Writing” onto 
 aspiration of cytosol EM grid or substrate Volume handled: 5-100nl 23 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  23. Single cell lysate Before FEM Simulation of Electrical Field Strength A 5 ] m 4 c / V k [ d 3 l e i F l a 2 After c i r t c e l E 1 20µm 0 ] Arnold SA, et al. Single-cell lysis for visual analysis by electron microscopy. J. Struct. Biol. 2013; 183(3):467–73. T 24-1 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

  24. Single cell lysate Microganglion cells, collaboration with Petr Broz, Biozentrum Basel Arnold SA, et al. Single-cell lysis for visual analysis by electron microscopy. J. Struct. Biol. 2013; 183(3):467–73. 25 NRAMM-2014-Stahlberg-2014-11-10.key - November 10, 2014

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