Correlative Light/Electron Microscopy
Dr. Guenter Resch
Leica Beijing HPF Workshop, October 2014
Contents
Overview
Leica EM PACT2 CLEM Workflow
Leica EM HPM100 CLEM Workflow
Leica Cryo-LM Stage
Section 1
Overview
Overview
Imaging Modalities
I
Widefield light microscopy
I
Total internal reflection microscopy
I
Super-resolution light microscopy
I
X-ray tomography
I
Scanning EM
I
Transmission EM
I
AFM
I
...
Each modality has its specific advantages and drawbacks
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 4 / 27
Overview
Correlative Microscopy
Correlative microscopy is the inspection of an identical sample (cell)
with two or more modalities.
CLEM = Correlative Light and Electron Microscopy
(TEM and SEM)
Not a single technique, but a collection of many different approaches
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 5 / 27
Overview
Advantages and Drawbacks of LM
CLEM
HPF Workshop, Oct 2014
I
+ Quick overview
I
+ Live samples, dynamics
I
+ Fluorescent dyes or
endogenous tags (GFP)
I
-- Resolution
I
-- Context (fluorescence)
(C) Nexperion e.U., 2014
Slide 6 / 27
Overview
Advantages and Drawbacks of EM
I
-- Lack of overview
I
-- Fixed (and dehydrated)
specimens
I
+ Resolution
I
+ Cellular context
IMP-IMBA Research Centre, Vienna
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 7 / 27
Overview
Correlative Pre-Fixation LM/EM
I
LM observation
I
Fixation and EM preparation
I
EM observation
I
Wait for event
I
Observe dynamics
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 8 / 27
Overview
Correlative Pre-Fixation LM/EM: Challenges
I
Time resolution observation → fixation
I
Yield
I
Relocation of feature
I
(Alignment of images)
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 9 / 27
Overview
Correlative Post-Fixation LM/EM
I
Fixation and EM preparation
I
LM observation
I
EM observation
I
Localisation of sparse features (’’needle in haystack’’)
I
Functional context of feature
I
Overlay of fluorescent signal
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 10 / 27
Overview
Correlative Post-Fixation LM/EM: Challenges
I
Signal compatible with LM and EM
I
Preservation of fluorescence through EM preparation
I
Preservation of specimen
I
Imaging quality and flexibility
I
Relocation of feature
I
Alignment of images
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 11 / 27
Section 2
Leica EM PACT2 CLEM Workflow
Leica EM PACT2 CLEM Workflow
Workflow
I
I
Samples grown on sapphire
discs with index pattern
EM PACT2 RTS designed to
shorten transfer times
I
I
I
Paul Verkade
Thomas Müller-Reichert
Special transfer holder on
light microscope
Cveta Tomova, Leica Microsystems
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 13 / 27
Leica EM PACT2 CLEM Workflow
Workflow
I
I
Samples grown on sapphire
discs with index pattern
EM PACT2 RTS designed to
shorten transfer times
I
I
I
Paul Verkade
Thomas Müller-Reichert
Special transfer holder on
light microscope
Leica Microsystems
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 13 / 27
Leica EM PACT2 CLEM Workflow
Preload Rapid Transfer System
Leica Microsystems
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 14 / 27
Leica EM PACT2 CLEM Workflow
Live Cell Carrier
Leica Microsystems
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 15 / 27
Leica EM PACT2 CLEM Workflow
Live Cell Carrier
Leica Microsystems
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 15 / 27
Leica EM PACT2 CLEM Workflow
Light Microscopy
I
Live cell carrier mounted in
CLEM rapid loader
I
Specimen in petri dish with
glass bottom
Leica Microsystems
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 16 / 27
Leica EM PACT2 CLEM Workflow
TEM Preparation
I
Quick transfer of sample to
EM PACT2 RTS
I
Freezing (5 s)
I
Freeze substitution
I
Target trimming
I
TEM
Leica Microsystems
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 17 / 27
Section 3
Leica EM HPM100 CLEM Workflow
Leica EM HPM100 CLEM Workflow
Overview
I
Live sample on sapphire discs
I
3 mm (→ Monday) and 6 mm
system available
I
Sapphire discs with pattern
for relocation
I
Readyly assembled for
freezing
Leica Microsystems
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 19 / 27
Leica EM HPM100 CLEM Workflow
Overview
I
Live sample on sapphire discs
I
3 mm (→ Monday) and 6 mm
system available
I
Sapphire discs with pattern
for relocation
I
Readyly assembled for
freezing
Cveta Tomova, Leica Microsystems
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 19 / 27
Leica EM HPM100 CLEM Workflow
Components of CLEM System
Cveta Tomova, Leica Microsystems
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 20 / 27
Leica EM HPM100 CLEM Workflow
6 mm CLEM System
Cveta Tomova, Leica Microsystems
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 21 / 27
Leica EM HPM100 CLEM Workflow
3 mm CLEM System
Cveta Tomova, Leica Microsystems
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 22 / 27
Leica EM HPM100 CLEM Workflow
Light Microscopy
Only dry objectives, 15-20 minutes
Andres Kaech, University of Zurich -- Cveta Tomova, Leica Microsystems
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 23 / 27
Leica EM HPM100 CLEM Workflow
Light Microscopy
Only dry objectives, 15-20 minutes
Hela GFP-Tubulin, H2B mCherry; Andres Kaech, University of Zurich
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 23 / 27
Leica EM HPM100 CLEM Workflow
TEM Preparation
Andres Kaech, University of Zurich -- Cveta Tomova, Leica
I
Locate certain feature, wait
for certain stage
I
Quick transfer of sample to
EM HPM100 <Video 1:55>
I
Freezing (min. 7 s)
I
Freeze substitution
I
Target trimming
I
TEM
Microsystems
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 24 / 27
Section 4
Leica Cryo-LM Stage
Leica Cryo-LM Stage
Separate Presentation
CLEM
HPF Workshop, Oct 2014
(C) Nexperion e.U., 2014
Slide 26 / 27
Thank you for your attention.
Nexperion e.U.
Solutions for Electron Microscopy
http://www.nexperion.net
[email protected]
+ 43 664 94 17 210
nexperion
guenterresch
@nexperion