correlative light-electron microscopy (clem)€¦ · on sapphire disc + uac and/ or oso 4 +...

Zoom-in beyond light microscopy:

new approaches for biological structureresearch - correlative light and electron microscopy on one and the same sample

“Small is beautiful....”

Electron Microscopy ETH Zurich (EMEZ), [email protected]

Correlative light-electron microscopy (CLEM)

Fluorescent Light Microscopy fluorescent labels

Transmission Electron Microscopy& Tomography/ Scanning EM

no labels or e- -dense labels

Aim:

A) identify on LM and EM level the identical structure (morphomics) - “retain context information”

B) image and locate specific fluorescent labelled molecules in living cells, record dynamical processes by LM and investigate them at EM level

See lecture: Heinz Schwarz, the “poor mans” confocal way....

The precision depends on the structure preservation .....

“ Imaging Space“ - Time vs. Spacial Resolution:EM...electron microscopy; LM...light microscopy

NMR

X-ray

NMR

X-ray

EM & LM covers a range of x,y -> mm-nm; z -> 10-1µm - <10nm- t -> µsec-min

-> only a correlative approach allows not to get lost in details & keep the large picture „in-focus“


Fluorescent Light Microscopy fluorescent labels

Transmission Electron Microscopy& Tomography/ Scanning EM


Aim:

A) identify on LM and EM level the identical structure

B) the possibility to image and locate fluorescent labelled molecules in living cells and to follow and record dynamical processes.

Needs for correlative microscopy:! the workflow from imaging in a light-microscope (in vivo, ex vivo...) to the EM

level has to be standardized

! 3D information on all structural level have to be integrated on the same specimen area/feature (preselected and best possible preserved)

Challenge:- automatic sample preparation on observation platform (fixation at minimum)- reproducibility and easy to use to reveal stable embedded sample is crucial for

acceptance by life-science researchers...- interfaces between techniques must be standardized and automatic transfered need

to be going side by side with data transfer (ROI, etc..)- electron microscopy (FIB/SEM 3D or TEM 3D Tomography) has to become

automatized for large throughput- specific fluorchrome dyes or in-vivo labeling system=> dedicated preparation & analytic tools need to be introduced...

(there is a lot to do...)

The context matters...

“The Art lies in the context....”

Adapted from: Review: Advanced Correlative Light/Electron Microscopy: current methods and new developments using Tokuyasu cryosections Katia Cortese, Alberto Diaspro and Carlo Tacchetti, 2009 JHC, Vol 57(12)

CLEM is more than 1&1.....

(Ultra-) structural research: the classical approach

question different microscopic techniques

different image data

differently prepared samples

diverse preparation protocols

?answers comparative

microscopy

(Ultra-) structural research: the classical approach

question different microscopic techniques

different image data

differently prepared samples

diverse preparation protocols

?answers comparative

microscopy

Comparative LM&EM

10

The classical way-correlate data from different approaches...

http://www.aqua-fish.net/imgs/articles/goldfish3.jpg

Immunostaining

Extracted, CPD, cryo-coated, cryo-SEM

F-actin

MT

NS, TEM & X-ray

FD, cryo-coatedcryo-SEM NS, TEM & X-ray

unique preparation protocol

different microscopic techniques

correlation of specific data

comparable image

Data quality

answers

One preparation for a microscope-independent approach for tissue & cells....

question

correlativemicroscopy




comparable image

Data quality

answers


question


1973




comparable image

Data quality

answers


question


Thesis: P. Schwarb 1987-90

1973

A) Integrated/ In-Situ solutions:

- FLM/TEM FEI iCorr (cryo-)

- Delmic FLM/SEM

- Jeol ClairScope

B) Off-line solutions (Transformation “2” Coordinate Systems):

- Array Tomography (brutal force)

- Zeiss Shuttle& Find LM-SEM; FEI MAPS CorrSight-SEM; Jeol Map & manual correlation etc...

Angronskaia et al. JSB 164; 2008

Liv, Hoogenboom et al. PLOS; 2013

Prerequisite: Structure preservation & fluorochrome - The idea...

Noran-CLSM: FS UAc/skin in HM20, 1997

0

100

200

300

400

500

600

450 500 550 600 650 700 750

Wavelength (nm)

Fluo

r. In

tens

ity

Aceton Aceton/UAc

UAc shows fluorescence emission....

<700Dalton lost during freeze subst.....

A Compromise: a modified „Freeze Substitution“ (or embedding) for correlative LM/EM

1. transfer frozen sample into substitution media at -110°C or -90°C Media (organic solvent saturated with fluorochrome and Uranyl Acetate)

2. substitution according to tissue experience (-90/-70/-50°C)

3. wash excess fluorochrome and fixative until no colour bleeding (-50°C)

4. start infiltrating with resin (-50°C HM20, 0°C Epon...)

5. polymerization under UV or with heat

6. trimming for CLSM or SEM, sectioning for LM, TEM....

+ Fluorescent dyes e.g. DiIC18 or Safranin O

! Uranylacetate ! Sudan III ! Safranin T! Nile red! Nile blue sulfate! DiIC18

! DiOC6

! Acridin Orange! Nonyl-Acridin Orange! 1,8 ANS! DCVJ! Bodipy 560! Oregon Green! GFP / YFP...?...

Dyes, penetrating or surviving during freeze-substitution...

Fluorescence Protein surviving during freeze-substitution or fixation (GFP, EoS...)

Ultramicroscopy 143 (2014) 3–14

Nat Methods. 2015 Jan 12. doi: 10.1038/nmeth.3225. [Epub ahead of print]

- ..”photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples...”

One biopsy: for LM, CLSM & EMmodified freeze-substition with fluorescence dyes.....

Miriam S. Lucas, Philippe Gasser, Maja M. Günthert, Roger Wepf, Jason Mercer, Ari Helenius, Andreas Schertel: Correlative 3D Microscopy: CLSM and FIB/SEM Tomography: A Study of Cellular Entry of Vaccinia Virus; Imaging& Microscopy, GIT Publ.; EMC2008; Vol. 10, Iss 3, p 30-31

Wepf, R., Richter, T., Biel, S., Schlüter, H., Fischer, F., Wittern, K.-P., and Hohenberg, H., “Multimodal imaging of skin structures: Imagining imaging of the Skin”; In: „ Bioengineering of the skin: Skin Imaging and Analysis“ 2nd Ed. 2007, Informa Healthcare USA, NewYork

S. Biel, K. Kawaschinski, K.-P. Wittern, U. Hintze & R. Wepf; J. Microsc. Oct. 2003, Vol. 212: 91-99 (2003)„From tissue to cellular ultrastructure: closing the gap between micro- and nanostructural imaging“

Katja Kawaschinski; 2000 “Vergleichende Untersuchungen an identischen Hautproben mittels der konfokalen LSM und der Transmissions-Elektronenmikroskopie” FH Hamburg Bergedorf

Correlating light and electron microscopy

One biopsy: for LM, CLSM & EMin-vivo 2photon selection

high pressure freezing; freeze subtituted; HM20 embedded; CLSM 3D imaging on bloc; section staining....LMTEM....Tomo....

Correlating light and electron microscopy

Biel et al., J Microsc 212 (2003)

Wilke et al,; 2008

TEM: - tedious- support grid masking...- higher throughput...???

“thin” section

3D Electron Microscopy10

nm

100nm

1 nm

“thin”e--transparent => “Tomography”

(various angle views..)

1µ

“Thick”not e--transparent=> serial section

real section or “en-bloc”

section projections or bloc-face view

=> Image Stack

The LM/SEM (FIB) world

Cryo-SEM

The LM/TEM world

“thin” => tilt series-> virtual image

stack

=> TEM “Tomo”

serial section LM/SEM

CLEM & Large Volume 3D EM methods...

K.L.Briggman&D.D.Bocks 2012;

SEM

SEMSEM

TEMS(T)EM

FIB

Large Volume 3D EM methods...


SEM

SEMSEM

TEMS(T)EM

FIB

FIB/SEM(focused ion beam SEM)

FIB

SEM

Sample preparation

High-pressure Freezing

Freeze substitution & low temp. embedding

3D FIB-SEM tomography

Katja Kawaschinski; 2000 ; Biel et al., J Microsc 212 (2003)M. Lucas et al., Methods in Cell Biology 2012

e.g.cell culturemicroorganisms tissue (biopsy)plants material …

C. elegans

Soy bean plant

Cell culture on sapphire disc

+ UAc and/ or OsO4

+ Fluorescent dyes e.g. DiIC18

Determination of ROI & 3D imaging

CLSM

40x

Tissue…

October 20, [email protected]

Acquisition of 3D image stacks with FIB-SEM

1. Deposition of protecting C-layer

2. Milling of a trench, milling current 6.5 - 13nA

3. Polish the cross section, milling current 1.5nA

4. Imaging with SEM (ESB)

Jason Mercer/Ari Helenisu IBC and Miriam Droste/ Roger Wepf EMEZ - Anreas Schertel Zeiss

Pre-selection by CLSM & recognition of ROI in SEM

Reflection of surface

Fluorescence signal

SEM image


Dr. Miriam Droste

Acquisition of 3D stack by FIB / SEM Vaccinia interaction with host-cell....

FIB lamella for TEM/STEM Tomography..

FIB-Lamella NVison40 Philippe Gasser/EMEZ & 200kV STEM M.Capers/Hitachi


[email protected] Microscopy Conference 2009 in Graz

Bacteria in legume root nodules

N2 NH3

Mung bean

! Root nodules are colonized by nitrogen-fixing bacteria

! this symbiosis provides the host plant with nitrogen, which in return provides nutrients for the bacteria

Images of plant and roots courtesy of Prof. H.-M. Fischer


Choosing the ROI…

CLSM SEM

FIB-SEM

Soy bean plant


Correlation of CLSM and FIB-SEM stacks

Soy bean plant

Some statistics for system biology...

! Volume: 260 µm3

! 39 % symbiosomes! 12.5 % bacteria

! 31 symbiosomes! Ø 3.5 bacteria per symbiosome

! from CLSM data: ~50% of cells invaded by bacteria

" Tool to study e.g. invasion process of bacteria into root cells ! quantitatively ! in spatial context

Soy bean plant



SEM

SEMSEM

TEMS(T)EM

- array tomography (CAT - correlative/computer controlled)

3D data with an in situ ultramicrotome.....(Denkatome....Denk&Horstmann 2004 PLOS Vol 2)

May be combined with CLSM of pre-embedding antibody labeling, or diffusible dyes....- fast for large volumes- destructive- large amount of heavy metal needed- e-beam induced hardening during operation Gatan 3View....

Tissue…

Fluorescence Protein surviving during freeze-substitution or fixation (GFP...)



- photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples...

GFP, mCherryShort FS protocol:- HM20- K4M- LRW

Correlative Microscopy: on high pressure frozen, substitutedand embedded samples...(passive labeling...)

Resin embedded samples are a “Storage” device for “morphomic data”

-> “Data block” & “Data slices”


Fluorescent Light Microscopy fluorescent labels, live imaging(ideally GFP-like labels)

VideomicroscopyConfocal/Spinning diskMultiphotonFRAPFRET, BRET, FLIMSTEDPALM

Resolution: 16 nm

Transmission Electron Microscopy& Tomography


Classic EM (Room temperature)

and

Cryo-EM (LN2 temperature)

Resolution: few nm - few Å

PALM and CLEM, E. Betzig,et al., Science 313 (2006) 1642

Marker size & distance....

Perfused cell membrane are helpful for import of “Marker” ...but what does it mean for the localisation

3D views of a IgG molecule160kDa

GFP (3-5nm)238AA; 26,9kDa

miniSOG (3nm)106AA; 15kDa

Llama AB (3-5nm)238AA; 15/30kDa

Is two more than one? Or greater then the sum....

Review: Advanced Correlative Light/Electron Microscopy: current methods and new developments using Tokuyasu cryosections Katia Cortese, Alberto Diaspro and Carlo Tacchetti, 2009 JHC, Vol 57(12)

Correlative Life cell LM - EM CLLEM

Paul Verkade - “Moving EM: the Rapid Transfer System as a new tool for correlative light and electron microscopy and high throughput for high-pressure freezing “ J. Microsc. 2008, Vol 230;Issue 2

Some slides with different approaches..

! 3D LM/TEM - Data from bloc....quantum dots pre-embedding labeling..

Ben N. G. Giepmans ; Bridging fluorescence microscopy and electron microscopy; Histochem Cell Biol (2008) 130:211–217

Ben N G Giepmans, Thomas J Deerinck, Benjamin L Smarr, Ying Z Jones & Mark H Ellisman; (2005) Nature Methods 2Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots

1. Chemical Fixation2. QD-antibody staining3. Dehydrated4. embedded in epoxy resin

5. bloc-face imaging6. ROI selection and LM imaging7. Ultrathin section8. TEM

! 3D LM/ EM Tomo - sections (GFP, YFP, photoconversion)

Grabenbauer M. et al., Nat Methods. 2005 Nov;2(11):857-62; “Correlative microscopy and electron tomography of GFP through photooxidation”

1. in vivo observation LM...2. chem. fix with succrose3. photooxidation...4.wash5. poststaining 6. embedding in resin7. TEM at <200-300nm thin sample areas

G. record tomogram....

! New fluorescence protein (miniSOG, photoconversion)

1. in vivo observation LM...2. chem. fix with succrose3. photooxidation...4.wash5. poststaining 6. embedding in resin7. TEM at <200-300nm thin sample areas


GFP et al. at cryo-LM conditions much more stable -> C-Cryo-LEM...

! 3D LM/ EM Tomo - Data from cryo-layers/sections.. (GFP, YFP, in-vivo marker....)

Sartori et al., J. Struct. Biol. 160 (2007) 146-156“Correlative microscopy: Bridging the gap between fluorescence light microscopy and cryo-electron tomography”

A. in vivo observation LM...B. rapid freezing...C. cryo-FLM (ROI)D. Cryo PhakoE. ROI identification - select thin areaF. TEM at <200-300nm thin sample areas


Furhter see also: 1) J Microsc. 2007 Aug;227(Pt 2):98-109.; “Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching”; Schwartz CL et al.2) J Struct Biol. 2008 Jul 11. [Epub ahead of print] ; “Integrated fluorescence and transmission electron microscopy.”; Agronskaia AV, et al.

Correlating FM and cryo-ET: Full Correlation Cycle

LM EM

µm (10-6 m)

mm (10-3 m) nm (10-9 m)

200 nm

200 nm

50 nm

1 µm

50 µm

50 µm

NG108 neuroblastoma/glioma hybrid cell line

Plunge-freezing

Transfer coordinates area of interest

Sartori et al., J. Struct. Biol. 160 (2007) 146-156

Can SEM replace classical TEM application for ultrastructure research?



SEM

SEMSEM

TEMS(T)EM

- array tomography (CAT - correlative/computer controlled)

Conventional SEM

Cryo-SEM

S-900/199150nm 10nm

J. Woodward 2009

! 3D LM/SEM - Data from sections....Serial section array SEM imaging:K. D. Micheva, S.J. SmithNeuron 55, 2007

1. Chemical Fixation2. Dehydration3. Embedding4. Serial sections

5. Post-embedding labeling6. Multilabeling...7. Histo or SEM-> Au-BSE localisation???

# 3D - Data from sections....

Serial section array SEM imaging:K. D. Micheva, S.J. SmithNeuron 55, 2007

A good biological EM Lab needs:

- a Fluorescence LM (FLM)- a HR-SEM- a Ultramicrotome

do you realy need a TEM!!???Jeff Lichtmann et al., Harvard & www.rmcproducts.com

TEM or SEM???

Tissue…

! 3D LM/SEM - Data from sections....Preparation....& Localisation....

Wilke et al,; 2008TEM (Grid...)

Roger Wepf/; Miriam Lucas; Falk Lucas, Maja Günthert...

LM#–#Dark#field

Serial section vs. FIB/SEM.....

2kV - BSE 2kV - BSERoger Wepf/; Miriam Lucas; Falk Lucas, Maja Günthert....Microbiol/ INI/ Pharma...

Prerequisite transparent, conductive support -> ITO

7kV 4kV

2kV 1kV70-100nm:1.8 - 2 kV ideal

2kV

C

1kV

C

Casino 3.2.01 (freeware)

Topview

Sideview

Roger Wepf/; Miriam Lucas; Falk Lucas, Maja Günthert....Microbiol/ INI/ Pharma...

kV adapted to Section Thickness - induced conductivity7kV 4kV

2kV 1kV

2kV

C

70-100nm HM20/ Epon:1.8 - 2 kV ideal

cover slide C/S - 150-170µm (LM)

ITO-coating (grainless)

► Shuttle & Find: Transfer

Data transfer

Sample transfer

Correlative Microscopy for Materials Analysis

- Different shuttles (HW)- Ideal sample support (transparent and conductive)- Preserving fluorochromes (LM->SEM->LM)- Easy data transfer and (re-)location

Bridging Microscopes....http://www.zeiss.de/corrmiclsekvzeiss

A new HW & SW solutionsfor large scale correlative LM&SEM

FEI Confidential

FEI Solution - MAPS: Correlative Microscopy

Light Microscopy

• Corr. Workbench

Electron

Microscopy

• SEM

MAPS

LM Acquisition EM Acquisition

- compatible with any kind of LM brand’s images..

Bridging Microscopes....

Shuttle & Find Zürich, 22.04.2010

Fluorescence after Fixation a. Dehydration EtOH

after CPD after FD

DAPI ++ ++ + ++

Alexa 488; FITC

+ - - -

Cy3 ++ ++ + ++


Bridging Microscopes....

Shuttle & Find Zürich, 22.04.2010

Text


3h: 40-50 different ROI on 10 different samples - matching SEM found & vice versa

CLEM: Fluorescence LM & Cryo-SEM ....

F-actinDAPICatherin

Correlative Microscopy 2 - Array Tomography: - combine Shuttle&Find with automatic section detection - ROI definition and

automatic section imaging in a SEM

Correlative Microscopy 2 - Array Tomography: - 60 Sections (80nm)- 310µm x 230µm; 5nm pixelsize; 4µsec dwell time...- Matrix of 4 x 3 images each 16384 x 16384pixels

=> 3’221’225’472 pixel per ROI ! => Acquisition time is per ROI 4h 9min!

310µm x 230µm x 4,8µmGemini 1530LM Stack: 440MB

SEM Stack: 197 GBCloud Microscopy: www.cubicice.de

Array Tomography & Immunolocalisation (lipids): - primary antibody against Glucosyl-Ceramid 3; secondary antibody Cy3

PSF correction....: - superresolution in Z (50-70nm)

PSF corr500nm

Correlative microscopy: advantages unique preparation protocol

" all samples viable for LM, CLSM, TEM, REM

" number of necessary samples is decreased

" more time for imaging

" comparable image data from different microscopes

" parallel imaging on identical structures possible

" combination with super-resolution possible...

Bridging Microscopes: 3D Correlative Light and Electron Microscopy of Complex Biological StructuresMiriam Lucas, Maja Günthert, Philippe Gasser, Falk Lucas and Roger Wepf

3D correlative (SR)LM & SEM -> higher localisation precession - Au-IgG)

Variable surface glycoprotein (VSG)/6nm Au & alpha- Tubulin/12nmAuH.Schwarz/B. Humble (MAPS -FEI)...

SEM

Sample preparation

High-pressure Freezing

Freeze substitution & low temp. embedding

SEM tomography

Katja Kawaschinski; 2000 ; Biel et al., J Microsc 212 (2003)

e.g.cell culturemicroorganisms tissue (biopsy)plants material …

C. elegans

Soy bean plant


+ UAc and/ or OsO4

+ Fluorescent dyes e.g. DiIC18

Determination of ROI & serial sectioning

CLSM

40x

Tissue…

Some slides with different approaches..

Integrated FLM in a TEM (ILEM).....(Agronskaia 2008, J Struct. Biol. 164; 183-189 )

Integrated FLM & SEM: An other solution from a supplier.....

Liv, Hoogenboom et al. PLOS; 2013

Correlating FM, Superresolution (PALM) and SEM imaging Correlating FM (fiducial markers) and TEM Tomography....

W. Kukulski .... J.A.G. Briggs...(Chap. 13)....

Correlating (cryo) FM; cryo-FIB and cryo-TEM Tomo

A. Rigort... J. M. PlitzkoChap. 14





GFP, mCherryShort FS protocol:- HM20- K4M- LRW




Short FS protocol with OsO4:

- find same location - from one imaging system to the other .......

PETXCTMRIOCT

n/µ-CTMRI

n/µ-CTX-cryst.

Bridging Imaging Tools...&Analytical Tools

-> Tof-MS:Radioisotopes; metabolites; drugs...-> File Format:DICOM for Micros.

(Nature; 467/436-440; 2010)

(Bones; 2010 & 2011)

- find same location - from one imaging system to the other .......

PETXCTMRIOCT

n/µ-CTMRI

n/µ-CTX-cryst.

Bridging Imaging Tools...&Analytical Tools

-> Tof-MS:Radioisotopes; metabolites; drugs...-> File Format:DICOM for Micros.

(Nature; 467/436-440; 2010)

(Bones; 2010 & 2011)

Cameca NanoSims/EPFL

The dream...

D.#Schär#et#al.#UniZH/#AGB6M.Güntert

~0.5-1mM Fe

Cameca NanoSims/EPFL

From LM to SEM/ FIB/SEM...to Imaging MS...to S/TEM...to APT

Fe-Cluster(~167 Fe)

correlative under controlled environment & for cryo preparation...

FIB/SEM

HR-SEM NanoSIMS

Atom Probe

Benefit from multimodal correlative microscopy:! 3D information on all structural level can be integrated on the same

specimen area/feature as sub-volume at higher magnification....

You never can image a whole body at max resolution......1x1026 voxels....you need a clever selective working strategy.... (1012 terra; 1015 peta; 1018 exa, 1021 zetta, 1024 yotta)

LM/CLSM

TEM/Xray

TEM/FIB/SEM

Image: Linda Nye; the Exploratorium Visualization Laboratory

CLEM#examples...

U.Suter#et#al./#AGB

G.#Schertler/T.#Ishikawa#et#al./#AGB

Ligh

t&Microscopy

X/ray&&&Electron&Microscopy

T.#Müller#et#al.#(Dresden)#/#AGB

CLEM#examples...

A.#Helenius#et#al./#M.Lucas

Ligh

t&Microscopy

Localisa6

on:&M

S/Im

aging&&&Im

mun

o/Electron

&Microscopy

~0.5-1mM FeD.#Schär#et#al.#UniZH/#AGBIM.Güntert

H.Schwarz/B.Humbel#et#al.#UniL/#F.LucasIM.Güntert Immunolabeling

- What are we dealing with... (water, molecules and bioorganisation)- Living “systems” - dynamic and crowdiness...- Consequences for EM...& sample preparation...- 3D & Correlative Microscopy (CL-”S”EM)...- One Biopsy for various Imaging Modalities...- 3D EM techniques & new alternatives...- CLEM alternatives...& Future

Future:- Improvements for data handling & Processing ...- Correlative labels (Click-iT,GFP,EoS,APEX...)...- Automatized interfaces to various imaging modes...- Automatized sample preparation LM-> EM...- Use of histopathology samples

Call to Action - Open opportunity:

What’s required to solve the problem...

-> Instrumentations/ Interfaces-> Computer Science-> Biology-> Chemistry/ Labels - Markers-> Engineering-> Politics & Policy/ Media-> Fund Raising-> Entrepreneurs

ȱ

CenterȱforȱMicroscopyȱandȱImageȱAnalysisȱȬȱUZHȱElectronȱMicroscopyȱȬȱETHȱ|ȱLightȱMicroscopyȱCentreȱȬȱETHȱ

WinterschoolȱinȱAdvancedȱMicroscopyȱȱȱ16.ȱȬȱ21.ȱ1.ȱ2011ȱȱPracticalȱModulesȱȬȱHighȱResolutionȱLightȱMicroscopyȱȬȱLifeȱCellȱMicroscopyȱȬȱFineȱStructureȱPreparationȱinȱElectronȱMicroscopyȱȱȬȱImmunoȱElectronȱMicroscopyȱȬȱCorrelativeȱMicroscopyȱȬȱTEMȱandȱSEMȱTomographyȱIncludingȱCryotechniquesȱȱ

TheoreticalȱBackgroundȱȬȱLecturesȱcoveringȱlightȱandȱelectronȱmicroscopyȱȬȱLatestȱdevelopmentsȱinȱinstrumentationȱandȱȱȱȱtechniqueȱinȱcompanyȱworkshopsȱ

ȱMoreȱInformationȱandȱApplicationȱwww.zmb.uzh.ch/coursesȱDeadline:ȱ14thȱofȱȱNovemberȱ2010ȱ

ȱOrganizedȱbyȱȱUrsȱZiegler,ȱAndresȱKaech,ȱCenterȱforȱMicroscopyȱandȱImageȱAnalysis,ȱUniversityȱofȱZürichȱRogerȱWepf,ȱElectronȱMicroscopy,ȱETHȱZürichȱGaborȱCsucs,ȱLightȱMicroscopyȱCentre,ȱETHȱZürichȱ

ȱSupportedȱby:ȱȱ

-2006 (Beiersdorf AG)

Thank you for your attention

2006 - (ETH)

Prof. H.M. Fischer and N. Maszloebeva Institute of Microbiology ETH Zurich

Special Thanks goes to Zeiss:Dr. Edelmann, Dr. Albrecht, Dr. Boeker, Chr. Dietrich; G. Heuberg; et al..,

Prof. I. Knüsel and J. Döhner Pharmacology&Toxicology Uni Zurich

Prof. R. Hahnloser, M. Kirschmann and D. Oberti Inst. Neuroinformatic ETH Zurich

PD. D. Schaer Univ. Hospital ZurichProf. R. Müller and Ph. Schneider Biomechanics ETH Zurich

a visual comprehensible “Nano-Cosmos”

in its natural context.....

“....we are only a few steps away from studying the same sample in a semi- or fully automated way by using a combination of four-dimensional light-optical live cell imaging techniques, followed by plunge- or high-pressure freezing techniques at a well chosen experimental end point, in order to perform electron tomography of the vitrified sample on the nanoscale in the cryoelectron microscope all in one run, and within one working day....” F. Braet and W. Geerts 2009 J. Microsc. Vol. 235 Correlative Microscopy...

correlative light-electron microscopy (clem)€¦ · on sapphire disc + uac and/ or oso 4 +...

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