Leica EM HPM100 User Manual

Leica EM HPM100

High Pressure Freezer

Superior cryofi xation of biological and industrial samples

High Pressure Freezing

High pressure freezing is by far the most signifi cant sample prep­aration method for morphological and immunocytochemical high
resolution studies for electron microscopy.

High pressure freezing has made it possible to observe aqueous
biological and industrial samples near to native state.

The 2100 bar of high pressure applied to the sample during
high pressure freezing using the Leica EM HPM100 suppresses
ice crystal formation and growth, while cryo immobilization
immediately after pressurization prevents structural damage to
the sample.

High pressure frozen samples can be completely vitrifi ed up to a
thickness of 200 μm, a 10 to 40-fold increase in the depth of amor­phous ice. No conventional freezing method can generate such
large, well frozen samples.

The unique 6 mm diameter carrier system of the Leica EM HPM100
allows even more sample area to be frozen, like no other high
pressure freezing instrument.

The state-of-the-art design of the Leica EM HPM100 enables ex­press sample handling and easy use with perfect freezing results.

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Inspired to Make the Difference

The Unique Leica EM HPM100 Features

• Automatic freezing of samples with one button operation

• Extremely high cooling rates

• Perfect freezing of samples up to 6 mm diameter

• No cryoprotectants required

• Rapid transfer loading device for CLEM work

• Fast, ergonomic sample loading

• Express sample handling for fast freezing

• Graphical data display of actual temperature,

time and pressure for each run

• Integrated work station

• Integrated stereomicroscope and adjustable LED illumination

• Universal and application specifi c sample carriers

• Integrated Dewar with drain outlet

• Automatic sample transfer to LN2 – no manipulation required

• Automatic bake-out cycle

• Thermally insulated process chamber

• Integrated touch control screen with prompt window

• Freezing process cover for ultimate safety

• USB interface for data storage and transfer

• Integrated air compressor

• Compact and mobile

• Uses standard 100–250 V power supply

Leica Design by Werner Hölbl

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Simplicity with Technology

The Leica EM HPM100 is an automatic and easy to handle high pressure freezing instrument. With a one button operation, a cartridge
holding the sample is inserted into the high pressure chamber.

Sample cartridge

Upper cylinder

The sample cartridge is made of high performance insulating poly­mer and consists of three components: two cylinders with a fl ow
channel, and a carrier plate with an opening to hold the sample
carrier assembly.

Carrier plate

Flow

channels

The sample cartridge functions as process chamber and sample
holder at the same time.

The integrated insulating fl ow channels direct the freezing onto
the sample.

Lower cylinder

This results in well frozen samples and low liquid nitrogen con­sumption.

An air-driven intensifi er pressurizes the liquid nitrogen to 2100 bar. The process chamber is fi lled with a small volume of alcohol prior
to freezing. The high pressure liquid nitrogen valve opens after the freezing pressure is reached. The pressurized liquid nitrogen is
injected into the process chamber. The fl ow channels direct the rapid fl ow of liquid nitrogen along the sample carrier surfaces. This
induces extremely high cooling rates.

High cooling rates with the high-pressurized liquid nitrogen system

1 LN2 storage

2 Feed pump

3 Non return valve

4 Air driven pressure intensifi er

5 High pressure LN

6 Alcohol reservoir

7 Alcohol dosing system

8 Non return valve

9 Sample cartridge

10 High pressure chamber

11 Pressure sensor

12 Temperature sensor

13 Exhaust

14 Locking pins

15 Cartridge handling system

valve

2

The vitrifi ed sample is automatically transferred to the LN2-fi lled Dewar. The freezing process is digitally displayed on a graph showing
the actual temperature, time and pressure.

4

Easy Freezing with the Leica EM HPM100

Sample loading

The upper and lower cylinder of the three-piece cartridge are
placed in the sample loading assembly.

➀ Upper cylinder
➁ Carrier plate
➂ Lower cylinder

The carrier plate is placed on top of the lower cylinder.

A sample carrier is placed in the center of the carrier plate. The
user fi lls the carrier with the sample to be frozen and places a
second carrier on top.

With the cartridge lever the upper cylinder is sandwiched onto
the lower cylinder and sample.

➀

➂

➁

One button operation

With a one button operation the enclosed sample is injected
for freezing. The sample is automatically frozen in less than 2.5
seconds after injection.

After cryofi xation, the frozen sample is automatically released
into the LN2 Dewar.

The vitrifi ed frozen sample is transferred to the carrier release
device. The sample is expelled from the carrier plate and is
ready for follow-on procedures.

5

Complete. Convenient. Preparation.

Not only is the Leica EM HPM100 compact and mobile, it also offes a variety of unique features which enables the user to quickly,
ergonomically and conveniently prepare samples for freezing.

Process parameters can be stored on
a standard memory stick for each run
using the USB interface.

The touch screen displays process
parameters, temperature, pressure
curves, bake-out cycle, and is also
used for service.

All freezing processes are enclosed by
a safety cover for user protection.

The vitrifi ed sample is automatically
released into a conveniently located
LN2-fi lled Dewar. The sample stays
well frozen at all times.

Well-positioned cartridge lever for quick
manoeuvring of the sample cartridge.

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The integrated stereomicroscope can
slide left and right allowing better sample
viewing and access during preparation
and processing. It comes complete with
adjustable LED illumination.

The two-coloured workstation table is
cleverly designed for improved sample
visualisation, such as when working with
cellulose microcapillaries.

The user can operate the Leica EM HPM100
in an ergonomically seated position.

7

Revolutionary in Application

The Leica EM HPM100 offers a variety of sample carriers suit­able for almost any type of application. Most notable is the 6 mm
diameter sample carrier, which no other high pressure freezing
instrument offers.

Once frozen, samples can be placed into the cryo chamber of the
Leica EM UC6/FC6 ultramicrotome for frozen hydrated sectioning
or the Leica EM AFS2 Automatic Freeze Substitution System. In
combination with freeze substitution, high pressure freezing is
an excellent alternative to chemical fi xation for immunoelectron
microscopy as the antigenicity and ultrastructure are both well
preserved.

8

The New 6 mm Diameter Sample Carrier.
Only by Leica Microsystems.

The 6 mm carrier opens up new perspectives for correlative mi­croscopy, as it allows a true pre-selection of a region of interest
by CLSM within large areas and the EM investigation of the same
sample without the drawback of artefacts caused by chemical
fi xation.

3 mm 6 mm

Pressure frozen mouse skin biopsy (diameter 6 mm) after freeze substitution in uranyl
acetate/acetone solution stained with safranin O in a confocal laser scanning microscope.
Images courtesy of Electron Microscopy ETH Zurich (EMEZ).

High pressure frozen mouse skin biopsy (diameter 6 mm)
after freeze substitution in uranyl acetate/acetone.

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Excellent Preservation of Ultra Structure

High pressure frozen mouse skin biopsy (diameter 6 mm)
after freeze substitution in uranyl acetate/acetone solu­tion post-stained with uranyl acetate and lead citrate and
sectioned prior to imaging in a TEM. Electron Microscopy
ETH Zurich (EMEZ).

High pressure frozen Nematode. Freeze substituted in
water-free acetone containing 2% Os04, embedded in
Epon/Araldite, thin sectioned and stained with uranyl
acetate 2% and Reynolds lead citrate. Hexadecene was
used as transmission fl uid. Electron Microscopy ETH
Zurich (EMEZ). Specimen courtesy of M. Gotta, Institute
of Biochemistry, ETH Zurich, Switzerland.

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Alternatively, when using the appropriate sample carriers, samples can be transferred
to the Leica EM BAF060 Freeze Etch System and processed for high resolution cryo­SEM or to make high resolution replicas for TEM, amongst other applications.

High pressure frozen Macrophage monolayer grown on
Sapphire disc. Freeze substituted in water-free acetone
containing 2% Os04, embedded in Epon/Araldite, thin­sectioned and stained with uranyl acetate 2% and Reynolds
lead citrate. Electron Microscopy ETH Zurich (EMEZ).
Specimen courtesy of G. Sumara, Institute of Cell Biology,
ETH Zurich, Switzerland.

Cryo SEM image of a high pressure frozen suspension
of bakers yeast Saccharomyces cerevisiae. Freeze frac­tured in the Leica EM BAF060 at -115°C, etched at -105°C
for 5 minutes, coated by electron beam evaporation with
3 nm of Pt/C. Electron Microscopy ETH Zurich (EMEZ).

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“With the user, for the user”
Leica Microsystems

Leica Microsystems operates internationally in four divi­sions, where we rank with the market leaders.

Life Science Division

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The Leica Microsystems Life Science Division supports the
imaging needs of the scientifi c community with advanced
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The Leica Microsystems Industry Division’s focus is to
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materials science, quality control, forensic science inves­tigation, and educational applications.

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most comprehensive product range. From patient to pa­thologist, the range includes the ideal product for each
histology step and high-productivity workfl ow solutions
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Leica Microsystems creates better patient care through
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