Leica EM RTS, EM PACT2 User Manual

Leica EM PACT2

High Pressure Freezer

Leica EM RTS

Rapid Transfer System

High Pressure Freezing for Everybody

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The Leica EM PACT2 high pressure freezer serves the needs of
molecular and cell biologists and all researchers who want an “in
vivo” impression of their cellular structures and functions in ques­tion – without the artefacts of chemical fixation but with the
high resolution information of EM immunocytochemistry, frozen
hydrated sections and freeze fracturing.

The Rapid Transfer System EM RTS allows correlative LM/EM
experiments, taking a live specimen from a light microscope (e.g.
a confocal microscope) to freezing in less than 5 seconds. In the
same way, time resolved experiments are possible. Safety and
reproducibility for the specimen are increased while operator
mistakes are reduced.

Structural details of the C. elegans
head in cross-section.
T. Müller-Reichert (MPI-CBG, Dresden,
Germany) and Kent McDonald
(University of California, Berkeley, USA)

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Leica Design by W. Hölbl

Leica EM PACT2 –

High Pressure Freezer

Function: All You Need is LN2…

The specimen is locked (1) and set under pressure (2) just before
freezing (3) via the synchronization mechanism (4). Only the tissue
in the specimen holder is under pressure and not the complete
specimen chamber. As any fluid can be used in the universal
hydraulic system, toxic vapors (e.g. ethanol) are avoided.

Perfect results

– High cooling rates by strong jet of LN

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– 120 samples can be frozen per hour
– Variety of specimen carriers available for all purposes

Easy to install

– Compact and mobile
– Standard electrical supply
– Compressor on EM PACT2 trolley

Easy to use

– Touch-sensitive color screen, menu prompts
– Bayonet loading device for automatic orientation
– Specimen ejection into LN2bath
– LED bath illumination
– 600 ms temperature/pressure curve displayed for each run
– Internal memory for 8 000 freezing runs
– Data download on memory stick

Safe and convenient

– Low LN2consumption
– Dewar with drain outlet
– Universal hydraulic system – can even use water
– Low noise
– Maintenance free “long life pods”

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Perfect results

– Specimen carrier loading in less than 5 secs
– Correlative LM/EM
– Rapid biopsy process

Easy to install

– Factory mounted on EM PACT2
– Control via touch sensitive screen

Easy to use

– Rapid loader for inserting specimen
– Automatic and reproducible specimen freezing

Safe and convenient

– Outlet Dewar
– Safety lid
– Low noise
– Funnel for filling LN

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– Platform for cryopreparation, box, position for accessories

Leica EM RTS –

Rapid Transfer System

Schematic drawing showing the status of all parameters

Freezing data after ejection of the sample

Specimen loading made simple
with the Rapid Transfer System:
insert specimen carrier into rapid
loader. Automatically freeze by
inserting rapid loader into RTS.

Prepare your sample (eg leaf or a
cell monolayer on a sapphire disc) …

… to fit into one of the various
specimen carriers (eg flat carrier
ø 1.2 mm, depth 200 µm or mem­brane carrier: ø 1.5 mm, depth
200 µm). Specimen and carrier are
held in the rapid loader. The long
life pod is connected to the loading
device with a bayonet lock.

Specimen and carrier are held in
the rapid loader.

The long life pod is connected to
the loading device with a bayonet
lock.

The primed loading device then
waits for freezing in the RTS.

By gently pushing the rapid loader
into the RTS the carrier is tightened
securely and then frozen automati­cally in less than 2.5 seconds.

After cryofixation the carriers are
collected in the LN2bath of the EM
PACT2 before freeze substitution in
the Leica EM AFS2.

Leica EM RTS
Application Solutions

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The Tube Holder for every fluid you
can think of … blood, milk, cell sus­pensions ... nematodes – and more!

Directly suck the suspension into
copper tubes (inner ø 350 µm)
already mounted in their holder.

Alternatively, take up sample into
cellulose microcapillary.

With the loading device the
specimen can be inserted into
the EM PACT.

After cryofixation the samples are
stored under LN2in the transfer
box.

Sample Punch: punching out of the
central part of the specimen tube
ready for Frozen Hydrated Sectioning
or ...

... peeling away of the top of the
copper tube for Freeze Substitution.

Storage of the copper tubes is
carried out under LN2.

The specimen tube holders are
recycled by reloading them with
copper tubes.

Leica Design by W. Hölbl

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Taking microbiopsies for EM is now
faster than ever before!

Prepare the Microbiopsy Transfer
Station for RTS under the Optical
Workstation…

… before taking a biopsy with
the biopsy gun.

Insert the biopsy gun into the
transfer station …

… and transfer the tissue into
the biopsy carrier.

The specimen and carrier are held
in the rapid loader …

… while the primed loading device
waits for freezing in the RTS.

By gently pushing the rapid loader
into the RTS the carrier is tightened
securely and then frozen automati­cally in less than 2.5 seconds.

After cryofixation the carriers are
collected in the LN2bath of the
EM PACT2 before freeze substitu­tion in the Leica EM AFS2 or
cryosectioning in the EM FC6.

Leica EM RTS

Application Solutions

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Frozen Hydrated Sectioning
made easy.

Alternatively, the carriers can be
trimmed with a cutter ...

... for Frozen Hydrated Sectioning
in the cryoultramicrotome.

Preparation for follow-on
procedures.

All necessary steps for follow-on
procedures (eg. Freeze Substitu­tion) can be conveniently per­formed in the cryopreparation box
supplied with the Leica EM PACT2.

The Freeze Fracture Holder for
everything that can be fractured ...

Prepare the Freeze Fracture Station
under the Optical Workstation ...

... and preload the freeze fracture
carrier. A copper ring is put on top
of the loaded carrier.

The carrier and copper ring are
sandwiched securely in the pod
with the supplied torque wrench.

With the loading device the
specimen can be inserted into
the instrument.

After cryofixation the carriers are
collected in the LN

2

bath of the

EM PACT2 before Freeze Fracturing.

Accessories are also available for loading EM PACT2 without EM RTS.

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Mouse embryonic fibroblast
grown on sapphire disc.
Soazig Lelay, Jana Maentler and
Paul Verkade, MPI-CBG, Dresden,
Germany

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Yeast frozen in the membrane carrier. Scale bar = 500 nm, 250 nm for insert.
Courtesy of Mark van Breugel, Jana Maentler and Paul Verkade, MPI-CBG, Dresden, Germany.

Insulin granules producing INS-1 cells. Scale bar = 5 µm.
Courtesy of Joke Ouwendijk, Jana Maentler, Melanie Jäger, Michele Solimena and Paul Verkade,
TUD and MPI-CBG, Dresden, Germany

Rat, Langerhans cells. Scale bar = 1 µm Courtesy of Joke Ouwendijk, Jana Maentler,
Melanie Jäger, Michele Solimena and Paul Verkade, TUD and MPI-CBG, Dresden, Germany

@

www.em-preparation.com

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Hernalser Hauptstrasse 219
A-1170 Vienna Austria

Tel. [phone]
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©

Leica Mikrosysteme GmbH

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Vienna Austria

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Leica EM PACT2_EM RTS - E - 6/05 Order No 206002, Printed on chlorine-free bleached paper. Printed in Austria.

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