Advances in EM Sample Preparation
Advances in EM Sample Preparation
1. Automated Grid Plunging
2. cryoCLEM
3. Broad Ion Beam preparation
2
1. Plunge Freezing
3
Types of biological specimens
Suspended samples
• Single particle analysis
 Purifed macromolecular complexes
 Viruses
 Liposomes
 Small (non-adherent) cells


Icosahedral reconstruction
Internal structure of small cells by cryo-ET
Cell monolayers grown on grids
4
Leica Workflow
CLEM Workflow
Solution
for cryo TEM
Grid Plunging
Adapter for Cryo TEM
Transfer
Image Analysis
Cryo-Transfer
Cryo-Electron Microscopy
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Leica EM GP Grid plunger
21 March 2014
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The Dewar and the secondary cryogen filling
Liquefier in place over ethane container in Dewar
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Enviromental Chamber
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Enviromental Chamber
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Software Interface
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Plunge Freezing
After freezing the grid remains in or above the
ethane ready for transfer to the
grid box
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EM GP Adapter for Cryo TEM Transfer
21 March 2014
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EM GP Adapter
1
2
5
3
21 March 2014
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Results
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16
Results
Virus-Like Particles of a Fish Nodavirus
 Mag 78000x
Courtesy of Dr. G. Resch CSF Electron Microscopy Facility, Austria
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Results
Yeast RNA polymerase II
 Mag 78000x
Courtesy of Dr. G. Resch CSF Electron Microscopy Facility, Austria
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Results
Microtubules
 Mag 40000x
Courtesy of Dr. Formanek, Leibniz-Institut für Polymerforschung,, Germany
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Results
Liposomes
 Mag 31000x
Pure DPPC liposomes
CS coated DPPC liposomes
Courtesy of Dr. Julia C. Schwarz, Univ. Of Vienna, Austria
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Results
Nanocarriers for dermal drug
delivery
 Mag 31000x
Nano-structured lipid carriers
(NLC)
Solid lipid nanoparticles (SLN)
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2. Cryo CLEM - Correlative Workflow Solution
Introduction
Application

Correlative Light and Electron Microscopy (CLEM) combines fluorescence microscopy and electron
microscopy imaging of the same sample.

A method which allows light microscopy rapid screening of large areas and fast determination of
regions of interest (ROI) in the electron microscope.
W. Baumeister, 2008
Leica Cryo CLEM Workflow Solution
Or
Grid Plunging
Cryo-Transfer
System with Loading
Station
Leica Cryo Light Microscopy
High Pressure Freezing
and
Cryo-Ultramicrotomy
CLEM Software
(Mark and Find Information)
Image Analysis
Cryo Loading Station
Cryo-Electron Microscopy
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CLEM Solution for Cryo Microscopy
The cryo-stage with transfer system is the "missing link" to combine cryo LM with the cryo EM .
•
Safe (sample safety), easy and controlled procedure to maintain cryo conditions from sample
preparation to cryo EM
•
Cryo objective with low working distance (<0,5 mm) for higher resolution, speeds up location of
target structure in EM.
•
Software enables user to find LM-marked structures during EM analysis
Cryo transfer system docked to cryo
stage
Leica Cryo Objectives (50x
and 20x magnification)
Cryo Stage with lid
Cryo Light Microscopy with Leica
Microscope, Camera, Cryo Stage
and Cryo Objectives
Alignment of LM software with EM
software => mark and find function
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Resch et al., in press
Beijing, August 11, 2010
26/127
Resch et al., in press
Beijing, August 11, 2010
27/127
Cryo CLEM system, available 2014
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3. Broad Ion Beam Preparation
Fußzeile
29
21.03.2014
Slope cutting with the EM TIC 3X …..
…biological possibilities?
Ion beam slope cut of the Leica EM TIC 3X
mask
prepared area
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Leica EM TIC 3X “Triple ion beam” slope cutting
Principle
 Three ion beams hitting the sample from
different directions (reduction of curtaining)
 Fixed sample (better heat transfer)
sample
slope
prepared area
Features
 Cutting depth >1000µm
 Cutting width > 4000µm
 Cutting speed >150µm/h
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mask
ion beams
Model of the ion beam slope cut process: Collision cascade
Primary ion can remove an atom if:
• Ione energy > bonding energy of the atom
• Transferred impulse on the lattice atom is directed to the surface of the sample
Ion beam
Sample
area of interest
mask
Application Examples
Application Examples
SiC abrasive paper
Veneer
Ultramicrotomed section on a grid
EM TIC 3X Cooling Stage
 LN2 flow design with external
dewar and pump
 Temperature range
 30°C … -150°C
 Cooled mask
First Animal Experiment – The Gummi Bear
Gummi bear
brain
TIC 020 ~80°C
Cryo (TIC 3X) -150°C
Cross section of Gummi Bear's brain
Marshmallow
 cooling stage -150°C; 5kV
 Heat-sensitive polymer fibers with water-soluble portion
Without cooling (~80°C)
Leica EM TIC 3X (cooling stage -120°C)
Comparison
heat sensitive coaxial polymer fibers with water-soluble portion
FC7 -140°C
TIC 3X -120°C
Cooling stage result
 Heat-sensitive coaxial polymer fibers with water-soluble portion
Coming soon
EM VCT 100
EM TIC 3X
EM VCT100 - connectivity for environmental control
VCT100 Cryo Loading Station
Samples at RT
cooled in TIC
TIC 3X / VCT connectivity





VCT transfer
>-150°C
sample holder with mask
several holder available
ofor EM Pact holders
ofor HPM holders
oindustrial samples
•max. 10x7x4mm
Retrofit for existing TIC 3X
oFSE (first time installation)
Fußzeile
48
21.03.2014
Preparation for ion beam slope cutting the samples
Instruments:
Cryo-loading station with cryo-saw to perform
sample preparation for the TIC 3X under LN2
conditions to fulfil max. protruded length of the
sample above the mask edge
VCT 100 shuttle to transfer the sample
EM TIC 3X with VCT docking station to
perform cryo-slope cut (cross section)
Fußzeile
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21.03.2014
Preparation Workflow




Transfer frozen sample to cryo-loading station filled with LN2
Insert in the VCT holder and pre-prepare under LN2 using the cryo-saw
Attach VCT 100 shuttle onto loading station for transfer to TIC3X
Ion beam mill then transfer to cryoSEM
Fußzeile
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21.03.2014
TIC 3X / VCT connectivity
Application
Fußzeile
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21.03.2014
Samples and Possibilities
Environmentally sensitive samples eg:
1.
Oil shale
2.
Batteries
3.
Polymers
4.
Cryo-transfer needed, min. temperature -150°C
Biological Applications…….?
1.
Ion beam milling pre-FIB to expose large area
2.
Soft biological materials with hard inclusions
3.
Stents
4.
Artificial joints
5.
Cell scaffolds
6.
?
Fußzeile
52
21.03.2014
Thank you
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