Leica EM HPM100

Leica EM HPM100
Operating Manual
Version 167120032 12/10

Important Note
Leica reserves the right to change technical specifications as well as manufacturing
processes without prior notice. Only in this way is it possible to continuously improve
the technology and manufacturing techniques used to provide our customers with
excellent products.
Any copyrights of this document are retained by Leica Mikrosysteme GmbH, Vienna.
Any reproduction of text and illustrations (or any parts thereof) by printing, photocopying,
or other methods (including electronic systems and media) requires express prior
permission in writing.
Issued by:
Leica Mikrosysteme GmbH
Hernalser Hauptstrasse 219
A-1170 Vienna

Leica EM HPM100
Operating Manual
Leica EM HPM100 Serial Number:
Date of purchase:
For the instrument serial number, please refer to the name type label on the
back of the instrument!
Please read this instruction manual carefully
before operating the instrument.

Foreword
This technical documentation is intended to provide essential information about the
proper operation and servicing of the Leica EM HPM100. The system is used for
cryo-immobilization of the ultrastructure of biological material to enable examination
of the specimens under an electron microscope (EM) or a light microscope (LM).
This user manual describes commissioning of the Leica EM HPM100 High Pressure
Freezer, phased testing and adjustment of all components and movement
sequences, and restoring the basic functionality.
Service and operating staff must familiarize themselves with all components of the
system before commissioning. Particular attention must be paid to the aspect of
safety.
This user manual must be retained for future reference.
Texts, schedules and tables may not be copied or reproduced without our express
consent.
It should be noted that this Technical Documentation does not constitute a part of any
existing, prior agreement or covenant or legal relationship. All obligations arise from
the purchase contract, which also is the sole regulator of the warranty conditions.
Contractual regulations are not affected by this technical documentation.
In addition, all generally applicable legal and otherwise binding regulations for
preventing accidents and protecting the environment must be observed and
communicated.
Page 1 Leica EM HPM100 Operating Manual Version 12/10

Tabel of contents
1. Introduction ............................................................................................................................. 4
2. Identification............................................................................................................................ 5
2.1 Product mark and type designation..................................................................................... 5
2.2 Name and address of the manufacturer.............................................................................. 5
3. Product description ................................................................................................................ 6
3.1 Field of application and proper use..................................................................................... 6
3.2 Dimensions and weight (for transport) ................................................................................ 6
3.3 Consumption and emissions............................................................................................... 6
3.4 Environmental conditions for operation and storage........................................................... 7
3.5 Safety information ............................................................................................................... 7
3.5.1 General instructions..................................................................................................... 7
3.5.2 Design safety measures .............................................................................................. 7
3.5.3 Safety measures at the installation site ....................................................................... 7
3.5.4 Safety measures when working with nitrogen ............................................................. 8
3.5.4.1 Physical and chemical properties of nitrogen........................................................ 8
3.5.4.2 Dangers of nitrogen............................................................................................... 8
3.5.4.3 Safety precautions and rules for use..................................................................... 9
3.5.5 Residual hazards ....................................................................................................... 10
3.5.6 Qualification of operating personnel .......................................................................... 10
3.5.7 Emergency procedure ............................................................................................... 11
4. Preparing the system for use............................................................................................... 12
4.1 Transportation and storage conditions.............................................................................. 12
4.2 Installation conditions........................................................................................................ 12
4.2.1 Work area conditions ................................................................................................. 12
4.2.2 Connection conditions ............................................................................................... 12
4.3 Installing, assembling and commissioning........................................................................ 13
4.3.1 Unpacking and assembling the parts......................................................................... 13
4.3.2 Setup ......................................................................................................................... 25
4.3.3 Safe storage of the packing material ......................................................................... 25
4.3.4 Electrical connections ................................................................................................ 25
4.3.5 Commissioning .......................................................................................................... 25
4.3.6 Repacking to prevent damage during transportation................................................. 25
5. Operating instructions.......................................................................................................... 26
5.1 Introduction ....................................................................................................................... 26
5.2 Operating the Leica EM HPM100 ..................................................................................... 26
5.2.1 Switching on the system ............................................................................................ 26
5.2.2 Cooling the system .................................................................................................... 33
5.2.3 Inserting the specimen holder in the loading station.................................................. 35
5.2.4 Inserting specimen and carriers into the specimen cartridge .................................... 37
5.2.5 Starting the automatic freezing process .................................................................... 38
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5.3 Actions following the freezing process.............................................................................. 40
5.3.1 Checking the processing quality based on process data........................................... 40
5.3.2 Documentation of freezing processes ....................................................................... 41
5.3.3 Separating and sorting specimen carriers ................................................................. 42
5.3.4 Transferring the frozen specimens ............................................................................ 42
5.3.5 Switching off the system ............................................................................................ 44
5.3.5.1 Draining the LN 2 Dewars..................................................................................... 45
5.3.5.2 Bake out of the system........................................................................................ 46
5.4 Changing settings on the main screen.............................................................................. 48
5.4.1 Change directory for saving settings ......................................................................... 48
5.4.2 Reset cycle counter ................................................................................................... 49
5.4.3 Changing the temperature of the process chamber .................................................. 50
5.4.4 Turning off the alcohol ............................................................................................... 51
6. Servicing and cleaning ......................................................................................................... 53
6.1 Draining fluids ................................................................................................................... 53
6.2 Cleaning the Leica EM HPM100....................................................................................... 53
7. Messages during operation.................................................................................................. 54
7.1 Standard messages during operation ............................................................................... 54
7.2 Error messages during operation...................................................................................... 54
8. Appendix I.............................................................................................................................. 55
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1. Introduction
In order to ensure the safety of service technicians and operators, and to prevent any
damage to the Leica EM HPM100 High Pressure Freezer, it is essential to read this
manual carefully before beginning any work with the system.
This user manual is intended to help the user to understand the system more
completely, to use it within the specified limits of its working capabilities, and to
maintain and service it in accordance with its physical parameters.
This user manual includes important information regarding proper commissioning,
operation, servicing, troubleshooting and repair. Observing these instructions will
help to prevent hazards, reduce repair and downtime costs, and prolong the system's
service life.
Symbols used in this manual:
Paragraphs with this symbol contain instructions regarding possible hazards.
Failure to observe these alerts may result in serious injury! These instructions
must be complied with in all cases.
Paragraphs with this symbol contain important information which may
endanger staff or result in damage to the system if it is ignored.
Paragraphs with this symbol contain further information relating to a previous
explanation, which does not have a safety-critical function. However, it is
important to observe this information to ensure that the system functions
optimally.
Page 4 Leica EM HPM100 Operating Manual Version 12/10

2. Identification
2.1 Product mark and type designation
Leica EM HPM100 UVN High Pressure Freezer: 100-130V / 50-60Hz
Leica EM HPM100 EVN High Pressure Freezer: 200-240V / 50-60Hz
2.2 Name and address of the manufacturer
Leica Mikrosysteme GmbH
Hernalser Hauptstrasse 219
A-1170 Wien
Tel.: +43 1 48899-0
Fax: +43 1 48899-350
Web: www.leica-microsystems.com
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3. Product description
3.1 Field of application and proper use
The Leica EM HPM100 High Pressure Freezer is used for freezing aqueous
specimens for examination of the specimens with an electron microscope, a light
microscope or other surface-analysis methods, such as atomic force microscopy,
secondary ion mass spectrometry or photoelectron spectroscopy. Additional
preparation stages may occur between freezing and examination.
Specimens are frozen with liquid nitrogen at a temperature of –197 °C under a
pressure of 210 kPa. Only liquefied nitrogen can be used as the cryogen.
Various specimens such as tissue, cell cultures, cosmetic products, food,
suspensions etc. can be processed.
The high pressure greatly reduces the volume of gaseous substances in the
specimen, so specimens should not contain any gas bubbles, which could reduce
pressure transmission and reduce the freezing quality.
When hazardous substances (e.g., radioactive, explosive, toxic or human pathogenic
substances) are processed the substance-specific safety precautions must be
implemented.
The Leica EM HPM100 must not be used beyond the limits specified in Technical
Data.
3.2 Dimensions and weight (for transport)
Dimensions of the shipping container:
Depth:
149cm
Width:
81cm
Height:
146cm
Weight of transport system: approx. 510kg
For detailed information on the HPM100 dimensions see Appendix I.
3.3 Consumption and emissions
Voltage:
Current:
Power consumption:
LN 2 consumption during cool down:
LN 2 consumption in operation:
LN 2 consumption per freezing process:
Ethanol consumption per freezing process:
Volumetric capacity of the LN 2 Dewar:
Volumetric capacity of the ethanol tank:
Nitrogen gas emission:
Noise level during pressure build-up:
Maximum noise level during the freezing process:
100-130V / 50-60Hz
200-240V / 50-60Hz
max. 7A for 230V
max. 14A for 115V
max. 1200W
approx. 20 liters
approx. 5 liters per hour
50 ml
2 ml
20 liters
0.5 liters
approx. 50m 3 per day
approx. 60dB(A)
approx. 75dB(A)
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3.4 Environmental conditions for operation and storage
Operating temperature:
Storage temperature:
Relative humidity:
+15°C to +35°C
+05°C to +40°C
max. 85% non-condensing for operation and storage
Use and store the instrument in an environment free of dust and other destructive
particles.
3.5 Safety information
3.5.1 General instructions
The Leica EM HPM100 High pressure Freezer can be handled safely and easily
provided it is operated in accordance with the instructions in this manual.
Failure to follow these safety instructions may endanger people and the
system.
3.5.2 Design safety measures
All mechanical components are protected by enclosures (doors, panels, etc.). These
enclosures must not be opened except for servicing
The Leica EM HPM100 High Pressure Freezer must not be operated unless all
enclosures are properly in place.
3.5.3 Safety measures at the installation site
The following measures must be implemented to prevent incorrect use at the
installation site:
• The media connections for the system must be connected at the installation
location by qualified technicians.
• Media must not be added or topped up except by authorized and trained
personnel.
• The system must not be operated except by trained and authorized technicians.
• Repairs may only be made to the system by staff of the manufacturer's technical
department.
If the Leica EM HPM100 High Pressure Freezer is installed incorrectly, the
system may be damaged.
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3.5.4 Safety measures when working with nitrogen
3.5.4.1 Physical and chemical properties of nitrogen
Nitrogen gas (N 2 ) is a colorless, odorless and tasteless inert gas and is not
combustible. Nitrogen has no physiological effect, therefore it is not poisonous to
humans and other living things. The nitrogen content of atmospheric air is approx.
78%, along with approx. 21% oxygen (O 2 ). The danger of nitrogen is in the
displacement of atmospheric oxygen as a result of the release of nitrogen. At less
than 17% O 2 depending on the duration of inhalation and the value of the actual
oxygen concentration, symptoms of drowsiness, uneasiness, nausea, high blood
pressure and breathing difficulties culminating in unconsciousness will occur. If an
unconscious person remains in a low-oxygen environment, death will result. In a
virtually pure nitrogen atmosphere unconsciousness is immediate (with no warning
by odor), followed shortly after by death.
Cold liquid nitrogen (LN 2 ) is a very cold liquid (boils at –196°C), it is colorless,
odorless, non-reactive and non-combustible. Escaping cold (~ –190°C) nitrogen gas
(GN 2 ) is initially heavier than air and may accumulate near the floor and in lower
areas. When the gas is released a cold fog is formed by condensation and freezing of
moisture in the air, which can spread out over a wide area. If the gas warms up to
room temperature, it is somewhat lighter than air. If the ventilation is inadequate the
oxygen will be replaced.
Evaporation of one liter of liquid nitrogen generates about 0.75m 3 of nitrogen gas.
3.5.4.2 Dangers of nitrogen
• Cold damage: freezing, frost blisters and serious eye damage on direct contact:
o with liquid nitrogen
o with the escaping cold gas or the resulting fog
o with objects or surfaces that have come into contact with liquid nitrogen or
escaping gas (e.g. pipe joints, tubes, valves, tanks, metallic surfaces etc.).
• Freezing to cold surfaces (e.g. metal) on direct contact.
• Headache, nausea, sleepiness, breathing difficulties
• Unconsciousness with no irritation or warning effect, as a result of oxygen
depletion (i.e., less than 17% O2).
• Ice formation on frozen plugs or other gas-tight Dewar flasks can prevent the
evaporation of nitrogen. If this container is shaken or moved, explosive release of
nitrogen gas (delayed boiling) may rupture the container.
• Uncontrolled boiling and increased vaporization of liquid nitrogen may occur if
damaged or poorly insulated Dewar flasks are used.
• When warm objects are cooled in LN 2 , an insulating gas layer will first form around
the object, and slow the heat transfer (minimal boiling of nitrogen). However, when
the object is cooled beyond a certain point, strong boiling often occurs
unexpectedly and liquid nitrogen may be sprayed out.
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• Accidental release of liquid nitrogen resulting from material damage caused by
some steels and organic materials (plastic, rubber etc.) becoming brittle by
exposure to cold
• Glass Dewar flasks are very sensitive to impact and may burst even if lightly
touched by metal instruments. (Temperature tension because of the high
conductivity of metal)
• O 2 enrichment may occur at the exterior walls of non-insulated tanks and
pipes.
o Creates a fire and explosion hazard if a container is open and during use
where organic substances are present. The vaporizing, very fire-promoting
liquid oxygen may cause spontaneous combustion. Oxygen enrichment can
be recognized by a slight bluish coloring of the liquid nitrogen.
3.5.4.3 Safety precautions and rules for use
• Ensure good ventilation at all times.
• Do not actuate isolation valves on Dewar tanks suddenly, check regularly for
leaks.
• Fill liquid nitrogen only into clean, completely dry containers and apparatus.
• Use only intact Dewar flasks: check the insulation of the flasks regularly by the
vaporization rate. Light boiling at the container walls is an indication of poor
insulation.
• Never seal Dewar flasks with gas-tight covers without a safety valve. Use only
easily operated covers designed for Dewar flasks. Never use closures with a
gasket.
• Thaw ice on apparatus and containers with warm air or hot water only, never use a
flame or red-hot objects.
• If possible, do not use glass Dewar flasks, and particularly do not use glass Dewar
flasks with a capacity greater than 2L. Dewar flasks of metal are preferred.
• Use lightly covered Dewar flasks with a sheet metal mantle for small volumes.
• Large storage containers of stainless steel (over 10L capacity) must be fitted with
a tilt mechanism or a lever system.
• Store containers in a cool, well-ventilated place.
• Provide a fresh-air feed in case of possible enrichment of N2 gas. If applicable,
provide an exhaust system.
• Use only freshly prepared colorless liquid nitrogen.
• Do not spill it when filling and transferring.
• Do not fill container past the fill level with liquid nitrogen. Fill Dewar flasks with a
bottleneck only to clearly below the neck (please note that the length of the Dewar
neck may be different from the length of the lagging).
• Allow overfull flasks to empty partially by leaving them open.
• Do not immerse objects at room temperature in liquid nitrogen.
• Cover small barrels and specimen chambers immediately after use with light
covers that are not gas tight to prevent condensation of oxygen (fire hazard). Move
closed unpressurized containers slowly to prevent spray or overflow.
• Transport only in high-quality insulated containers for very cold gases (cryoflasks).
• Secure containers to prevent falling.
• Avoid any damage and unnecessary agitation.
• Only sealed, unpressurized metal Dewar flasks are permitted in elevators.
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• Wear a face shield or safety glasses (open above and below, not enclosed
glasses).
• Wear cryo gloves or leather work gloves with a very long sleeve.
• Always wear a floor-length PE apron when pouring from Dewar flasks.
Do not allow liquid nitrogen to penetrate into or under protective clothing.
Penetrating N 2 will vaporize immediately and cause serious freezing injuries
before the protective clothing can be removed. If possible, keep the sleeve over
the top of the safety gloves and do not use protective clothing that could be
penetrated by liquid nitrogen.
3.5.5 Residual hazards
The Leica EM HPM100 High Pressure Freezer represents the latest technology and
conforms to recognized safety regulations: even so, hazards still exist.
All modifications and conversions to the system are prohibited!
Objects may fall into the system in spite of all possible precautions. In this
case stop the system and switch off the main switch. If the search for the
object that has fallen in is not successful, inform the service department before
continuing to work with the system. Continuing operation may cause damage
to the system.
Install or update software only with data media supplied by Leica
Microsystems.
When handling toxic or human pathogenic substances observe the biohazard
regulations.
3.5.6 Qualification of operating personnel
The operating personnel must be familiar with and follow the recognized rules for
safety at work.
The operating personnel tasked with operating and maintaining the Leica EM
HPM100 High Pressure Freezer must satisfy the specific professional requirements
for their respective duties.
The operating personnel must have received training and must be familiar with the
duties that have been assigned to them and for which they are responsible.
The professional profiles for operating personnel responsible for the system are
outlined in the following.
Operator (user class 2)
Personnel with technical training, able to perform simple tasks, i.e., operate the Leica
EM HPM100 High Pressure Freezer and load and unload samples.
Administrator (user class 1)
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Staff with technical training, able to perform the tasks of qualification 2, and also
typical simple maintenance, adjustment and startup activities with protective devices
switched off as well as restarting the system.
Manufacturer's technician (Leica Microsystems – user class 0)
Trained technician provided by the manufacturer, who is able to carry out more
complex work in exceptional situations or in cases where such has been agreed with
the user.
3.5.7 Emergency procedure
In case of faults, the display shows an error message that describes the
recommended procedure for correcting the fault.
If unusual operating conditions or unaccustomed noises occur, the system may be
switched off using the main system switch.
If firefighting measures are called for, a CO 2 fire extinguisher must be used.
The Technical Service must be consulted before resuming work with the system.
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4. Preparing the system for use
4.1 Transportation and storage conditions
The Leica EM HPM100 High Pressure Freezer is delivered properly packed and in
the semi-assembled state. The customer must check the condition of the system
upon delivery and file a damage report with the shipping company if the equipment is
damaged. The customer must immediately inform the manufacturer of any possible
damage in transit.
The system must be positioned upright for transporting.
The overall weight including packaging is approx. 510 kg.
The packed equipment must be stored in clean, dry areas at temperatures between
5° and 40°C and must not be exposed to aggressive substance that could cause
corrosion.
4.2 Installation conditions
4.2.1 Work area conditions
The system must be installed on a level horizontal base. There must be a specific
clearance around it.
The minimum working distances are 20 cm at the back and to the right and 60
cm at the front and left.
4.2.2 Connection conditions
The following connections must be assured:
• power supply 100-130V / 50-60Hz for Leica EM HPM100 UVN
• power supply 200-240V / 50-60Hz for Leica EM HPM100 EVN
Page 12 Leica EM HPM100 Operating Manual Version 12/10

4.3 Installing, assembling and commissioning
The packaged Leica EM HPM100 High Pressure Freezer must be transported with a
forklift truck or a pallet hoist.
4.3.1 Unpacking and assembling the parts
After selecting the installation location, remove the top cover and all side panels of
the wooden crate.
Remove the packaging material in the crate. Do not remove plastic packaging from
around the machine. Place all included boxes in a secure place to the side.
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Remove the screws and remove the wooden brace at the front of the machine.
Remove the screws from the sides of the top wood wedges and remove the wedges
from under the system.
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Use the two wedges as a ramp to roll the Leica EM HPM100 to the floor from the
pallet.
Release the roller brakes and carefully move the system down the ramps to the floor.
Never do this alone. The heavy weight of the Leica EM HPM 100 High Pressure
Freezer may be hazardous to persons if it tilts or when moving it on slopes.
Carefully unpack the boxes with the disassembled parts of the system. Check with
the packing slip that all parts are included and check for damage.
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Remove the plastic packaging from the Leica EM HPM100.
Loosely fasten the metal support to the loading station with 2 bolts and washers
(6mm hex).
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Fasten the worktop to the loading station of the Leica EM HPM100 with the supplied
bolts.
The long bolt attaches through the metal support. Tighten all 7 bolts.
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Undo the loading mechanism from the lever axis by removing the two (2mm hex)
bolts.
Loading
mechanism
Lever axis
Place the loading mechanism into the loading station.
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Insert the lever axis through the worktop and the loading mechanism.
Fix the lever axis to the loading mechanism of the loading station with the two hex
bolts.
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Unpack the box containing the stereomicroscope, two eye pieces, support ring and
ring-light.
Place the stereomicroscope support ring onto the microscope column.
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Place the stereomicroscope into the support ring.
Fasten the stereomicroscope to the support ring.
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Remove the eye piece caps.
Attach the eye piece to the stereomicroscope.
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Attach the ring-light to the outside of the objective lens.
Connect the cable from the microscope column to the ring-light.
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Unscrew the back panel of the Leica EM HPM100 leaving the bottom screws in
place.
Leave the bottom
screws in place
Fasten the handle to the back panel from the inside of the Leica EM HPM100 with
the supplied bolts.
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4.3.2 Setup
The Leica EM HPM100 is moved to the installation location on the integrated rollers.
Release the roller brakes and set them again once the final position has been
reached.
The back rollers have a fixed direction. The front rollers can swivel for steering
the Leica EM HPM100 when it is being moved.
Avoid moving the Leica EM HPM100 over slopes and uneven ground. The
system must never be tilted more than 3°.
The system must be installed on a hard floor or on a floor plate that is at least 100 cm
deep and 70 cm wide.
Sufficient space is required to allow comfortable operation of the Leica EM HPM100
and also for maintenance and service. Reserve a minimum working distance of 20
cm at the back and right and 60 cm at the front and left.
4.3.3 Safe storage of the packing material
The packing material for the Leica EM HPM100 must be kept safely for use if the
system is shipped again, since the system may be damaged if it is not transported in
the original packing. Store the packaging for the parts and the floor of the crate with
the side panels of the lagging.
4.3.4 Electrical connections
The electrical terminal is on the back of the Leica EM HPM100. Power is supplied via
a connector for non-heating appliances. Install a country-specific electrical plug to
connect the power supply at the installation location.
4.3.5 Commissioning
The Leica EM HPM100 is commissioned by employees of Leica Microsystems
and specially trained and authorized technicians.
Before commissioning the system, carry out the following steps:
• unpack the Leica EM HPM100
• install the high pressure freezer
• connect the power
Once the above steps have been completed, commissioning involves following the
operator actions to ensure the machine is functioning properly.
4.3.6 Repacking to prevent damage during transportation
To disassemble and pack the system, carry out the steps for assembly and
unpacking in reverse order.
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5. Operating instructions
5.1 Introduction
The formation of ice crystals can be greatly reduced if the specimen is subjected to a
pressure of 2100 bar before the freezing process has started. Aqueous specimens
can be frozen without ice crystals up to a thickness of 200 µm. Pressure may cause
changes in the physical and chemical equilibrium, such as dissociation or the
reactivity of enzymes. This must be prevented to immobilize the specimen in as close
to a natural state as possible. The pressure should be applied as quickly as possible
and once the specimen is under 2100 bar it should be cooled to at least –140 °C as
quickly as possible. Below this temperature amorphous (vitrified) ice remains stable.
The diagram (Kanno et al. 1975) shows the stable states of water depending on
pressure and temperature. The melting temperature and the temperature for
homogenous nucleation reach a minimum at 2100 bar. At this pressure water can be
supercooled to -90 °C and changes to a very viscous liquid. I, II and III show ranges
in which H 2 O is present as hexagonally oriented ice.
5.2 Operating the Leica EM HPM100
The next section explains the complete freezing process in detail.
5.2.1 Switching on the system
Close all valves at the back of the machine. Switch on the system at the main switch
on the front panel. The compressor starts to build up the operating pressure. This can
take up to 15 minutes if there is no residual air pressure in the machine.
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Close valves at the back of the machine.
Position of main switch
Main switch
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If a USB stick is connected to the port on the front panel, the process parameters for
every freezing cycle will be saved in the selected directory.
If the USB stick is not connected the process parameters will be displayed following
each freezing cycle, but will not be saved.
Connection for
the USB stick
If you use a directory name that has already been used with the USB stick the
older process parameter will be overwritten!
Create a directory name using the touch screen. Press Enter to confirm the name. Up
to 8 characters can be used.
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After pressing “ENTER” on the “directory screen” the following display will appear.
Add isopropanol or absolute ethanol to the Leica EM HPM100.
The tank must be filled with 100% isopropanol or absolute ethanol only!
To add isopropanol or absolute ethanol slide the microscope to the right until it
comes to a stop.
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Open the front safety cover and prop it using the cover support lock.
Cover support lock
Alcohol tank
Unscrew the alcohol tank cap and fill the tank with 40ml isopropanol or absolute
ethanol.
After filling, screw the tank lid closed (hand tight only). Close the safety cover by
pushing the cover support back to release the lock.
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The touch screen shows a “wait for air pressure” message until the compressor has
built up the operating pressure.
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Once the operating pressure has been reached the main touch screen will appear.
Settings can be made with the main touch screen. See Section 5.4.7.
Buttons always have a light background and open a new window when pressed.
2
3
1
4
5
6
7
8
9
10
1 status display(During the cooling process the main screen shows "wait for cool
down“)
2 alcohol On/Off display
3 cycle counter
4 storage Dewar fill level display
5 actual temperature in process chamber
6 button for setpoint temperature in process chamber
7 directory selection button
8 file name display for saving the process data
9 service button (access for service technicians only)
10 system button (includes draining and bake-out)
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5.2.2 Cooling the system
The Leica EM HPM100 is cooled with LN 2 while the system is reaching operating
pressure.
Add liquid nitrogen to the storage Dewar. Use caution as rapid LN 2 evaporation
during the first cooling stages can cause splashing of the LN 2 .
A small pop-up rod in the top cover rises when the Dewar is completely full of LN 2 .
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The display on the main screen shows the “LN 2 Storage” level as a percentage.
The internal LN 2 pump will start when the storage Dewar registers at least 30% full.
The current status is displayed in the top left corner of the main screen.
Fill the specimen Dewar with liquid nitrogen before freezing.
After cryofixation, the frozen sample is automatically released into the specimen
Dewar.
Drawer for removable
specimen Dewar
Note: Freezing can not take place if the specimen Dewar is not filled with LN 2 .
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Fill the specimen Dewar with liquid nitrogen before freezing.
Open the specimen Dewar drawer by pulling the drawer away from the machine.
Position the LN 2 filled specimen Dewar firmly against the metal holder and then close
the drawer.
Specimen LN Dewar
Metal holder
When the system is fully cooled (approximately 30 minutes after the internal LN 2
pump starts) and the small LN 2 Dewar is filled, the main screen shows “ready for
freezing” in the top left corner. The freezing process can begin.
5.2.3 Inserting the specimen holder in the loading station
The loading mechanism must be open to insert the three-piece cartridge consisting of
an upper cylinder, carrier plate and lower cylinder.
Open the loading mechanism by rotating it towards you into the front locking position
with the loading lever.
Hold the bottom half of the closure with the right forefinger and turn the top half to a
vertical position with the loading lever.
Front locking
position of the
loading
mechanism
Loading lever
Page 35 Leica EM HPM100 Operating Manual Version 12/10

Insert the upper and lower cylinder into the loading mechanism. The upper cylinder is
placed in the top closure and the lower cylinder is placed in the bottom closure. A pin
in the bottom closure ensures it is placed correctly.
Place the carrier plate onto the lower cylinder. Note that the side openings of the
carrier plate match with the side openings of the lower cylinder.
Upper
cylinder
Side openings
Lower cylinder
Carrier plate
If the carrier plate is incorrectly oriented it will be damaged when the specimen
is ejected.
Page 36 Leica EM HPM100 Operating Manual Version 12/10

5.2.4 Inserting specimen and carriers into the specimen cartridge
The Leica EM HPM100 requires two specimen carriers for standard freezing. The
following section explains the insertion of the standard 3 mm specimen carriers (Type
A and B). For more detail and insertion of different specimen carriers, e.g. Type C or
others please see the relevant data sheet.
Specimen
carrier and
specimen
Place a type A specimen carrier into the round hole of the carrier plate with the
hollow section of the specimen carrier facing upwards.
Place the specimen into the Type A specimen carrier. Make sure that the specimen
does not extend above the well in the carrier and that the space inside the carrier is
completely filled and there are no air bubbles.
Place a type B specimen carrier with the smooth side down onto the Type A carrier
containing the specimen. Make sure that the carriers are even with the plane of the
carrier plate.
The carrier must be completely filled with liquid (eg Hexadecen, culture media,
cryoprotectant, etc) before freezing
When the carriers are in place, rotate the loading lever so that the top closure locks
(clicks) into the bottom.
Page 37 Leica EM HPM100 Operating Manual Version 12/10

The loading mechanism is now closed and the sample is ready for freezing.
5.2.5 Starting the automatic freezing process
Rotate the loading lever 180°C until the process button is in position.
Process
button
Page 38 Leica EM HPM100 Operating Manual Version 12/10

Push the process button to start the freezing process.
The microscope must not be used during the freezing process. The motion of
the device may cause injury to the eyes.
After cryo-fixation the three-piece cartridge and specimen carriers are automatically
ejected into the specimen Dewar and are ready for transfer (see Section 5.3.4). The
freezing process is now complete.
Page 39 Leica EM HPM100 Operating Manual Version 12/10

5.3 Actions following the freezing process
5.3.1 Checking the processing quality based on process data
After the freezing process has finished, the following measurement parameters can
be read on the main screen:
Rise Time - The time [ms] it took for the pressure to rise to 2100 bar
Shift p/T - The time [ms] between when pressure rose above 2100 bar and when
the temperature dropped below -100 °C
HP Dwell - The time [ms] from when the pressure rose past 2100 bar until it
dropped back down below 2100 bar
Cooling - The cooling speed [°C/s]
Display of measurement parameters:
The measurement values for temperature and pressure can also be viewed as a
graph. To display the graph press the GRAPH button at the bottom left.
Display of measurement as curves:
Page 40 Leica EM HPM100 Operating Manual Version 12/10

The first 500 ms are shown. At the left is the axis for the temperature and on the right
the axis for the pressure. The top curve shows the pressure trend, the bottom the
temperature trend. To display the first 20 ms press the DETAILS button.
Display of measurement curves in detail:
Press VALUES button to return to the main screen.
5.3.2 Documentation of freezing processes
If a USB stick was connected before starting the freezing process, all processing data
are saved to the selected directory under the displayed name (e.g. 002.xls). These
files can be opened with Microsoft Excel and other programs. The following picture
shows an extract from such a file. The header contains general information such as
date and time. Pressure and temperature data are shown below. A value is saved
every 50 µs for the first 50 ms, then every 2.5 ms.
Overview of saved data:
Leica
Microsystems 17/10/2010 08:21
HPM100 Version V4.06
Directory name hpm100
Filename
001.xls
Rise time 4.0 ms
Shift p/T 1.1 ms
hp Dwell 310.4 ms
Cooling rate 13986 °C/s
============ ============ ============
time [µs] pressure [bar] temperature[°C]
0 60 18
50 65 18
100 72 20
150 77 20
Page 41 Leica EM HPM100 Operating Manual Version 12/10

5.3.3 Separating and sorting specimen carriers
At the end of the freezing process the upper and lower cylinder can be removed from
the specimen Dewar and heated. Clean the cylinders with a lint-free paper towel.
Handling the carrier plate with the frozen sample in it is explained in the following
section.
5.3.4 Transferring the frozen specimens
Place the inset for sample carrier release (3 mm, 4.5 mm or 6 mm respectively) and
the specimen container in the metal bath. Connect the lever by screwing it to the
punching device.
Fill the bath with LN 2 and allow all parts to cool before transferring the specimens.
The container has six numbered storage positions.
Page 42 Leica EM HPM100 Operating Manual Version 12/10

Position the carrier plate (arresting the frozen specimen) in the slot of the inset using
cold forceps. Push the carrier plate into the guide until it stops.
Punching
tool
Carrier plate slot
Page 43 Leica EM HPM100 Operating Manual Version 12/10

Push the lever forward.
The specimen carrier is ejected from the carrier plate into a small specimen container
within the insert where they can be retrieved from.
Transfer the released specimen in specimen container under the desired
number.
5.3.5 Switching off the system
Once all freezing is complete, the Leica EM HPM100 can be shut down. If the
machine will not be used for several days it is safe to simply turn off the power and let
the LN 2 evaporate and the machine gradually warm to room temperature. If the Leica
EM HPM100 is to be used within the next 24 hours, the following drain and bake-out
protocol should be followed.
Page 44 Leica EM HPM100 Operating Manual Version 12/10

5.3.5.1 Draining the LN 2 Dewars
The Leica EM HPM100 operates with two LN 2 Dewars, one storage Dewar (20 ltr)
and a process Dewar (10 ltr).
Two valves at the back panel of the instrument allow draining of both Dewars.
Storage Dewar
drain valve
Process Dewar
drain valve
Place a small Dewar or LN 2 bucket under the hose and open the storage and/or
process Dewar valves to drain the LN 2 . The LN 2 can be used for other purposes or
disposed of.
Before commissioning the instrument again, close both the storage and process
Dewar valves.
Page 45 Leica EM HPM100 Operating Manual Version 12/10

5.3.5.2 Bake out of the system
Once the storage Dewar has reached a level of below 20%, select SYSTEM from the
main screen.
Select AIR HEATER ON display button to activate the heating cycle.
The 80°C heating cycle will only start when the chamber temperature warms to 10°C.
Page 46 Leica EM HPM100 Operating Manual Version 12/10

When the heating cycle is activated the status display reads “air heater working”.
The screen displays the cycle time left (starting at 8000 seconds), and the
temperatures of the air inlet, draw tube, storage Dewar and the connecting pipe
(in °C).
The bake-out will automatically terminate after reaching the end of the cycle leaving
the instrument ready for the next use. Once the cycle is complete, the machine
switches back to the standard screen.
Note: Leave the main power on for the heating cycle to run.
The heating cycle can be terminated at any time by pressing “Air Heater Off”.
During the bake-out cycle DO NOT turn the system and main power off for this
will terminate the bake-out and the bake-out cycle.
Page 47 Leica EM HPM100 Operating Manual Version 12/10

5.4 Changing settings on the main screen
Certain parameters and settings can be changed on the main screen and are
explained below.
5.4.1 Change directory for saving settings
To change the current directory press the current directory name on the main screen.
The new directory is ready for input.
Confirm your input by pressing Enter.
Page 48 Leica EM HPM100 Operating Manual Version 12/10

5.4.2 Reset cycle counter
The cycle counter starts from zero when the system is switched on. The counter can
also be reset manually during operation.
To reset the cycle counter press the “cycle no.” counter button in the top right corner.
Confirm the reset by pressing YES.
Resetting the cycle number does not influence the file name in which the process
data are saved when a USB stick is connected.
Page 49 Leica EM HPM100 Operating Manual Version 12/10

5.4.3 Changing the temperature of the process chamber
The temperature of the process chamber can be controlled. The current nominal and
actual values of the cryo chamber can be read on the main screen.
To change the temperature of the process chamber press the nominal temperature
button.
Increase or decrease the temperature using the two arrow buttons.
Confirm the temperature by pressing the nominal temperature and the main screen
will appear.
Page 50 Leica EM HPM100 Operating Manual Version 12/10

5.4.4 Turning off the alcohol
A freezing process can be carried out without alcohol if needed.
To turn off the alcohol select SYSTEM from the main screen (see 5.3.5.2) and press
ALCOHOL ON.
The display button changes to ALCOHOL OFF.
Page 51 Leica EM HPM100 Operating Manual Version 12/10

The machine is ready to freeze without alcohol, which is indicated on the main
screen.
Page 52 Leica EM HPM100 Operating Manual Version 12/10

6. Servicing and cleaning
Servicing and cleaning
• preserves the perfect condition and operating readiness of the system,
• increases the system's rate of use by avoiding downtime;
• enables efficient planning of maintenance work and materials.
6.1 Draining fluids
There are three valves on the back lower panel of the Leica EM HPM100
The FRESH ALCOHOL OUT valve drains the pure (clean) alcohol from the alcohol
tank.
The WASTE ALCOHOL OUT valve drains the collected waste (used) alcohol and
should be drained into a beaker and disposed of in accordance with local chemical
disposal guidelines. The valve should be opened slowly and carefully.
The AIR PRESSURE OUT valve releases the air pressure from the reserve tank. The
power should be turned off before opening this valve. It is only necessary to release
the air pressure if the instrument is going to be shipped
6.2 Cleaning the Leica EM HPM100
It is recommended to periodically wipe the machine with a mild cleaning solution and
a soft cloth. Use of solvents will damage the finish.
Page 53 Leica EM HPM100 Operating Manual Version 12/10

7. Messages during operation
7.1 Standard messages during operation
Message Explanation Solution
Close safety cover Safety cover is open Close safety cover
Wait for air pressure
Wait for storage
pressure
The machine has not
reached operating
pressure
The machine has not
reached storage pressure
Wait for the machine
to reach operating
pressure
Wait for the machine
to reach storage
pressure
7.2 Error messages during operation
Message Explanation Solution
INIT Rotor The rotor is out of position Press the Init button
Check specimen holder
INIT HP Cylinder
Refill Alcohol
Close LN 2 storage
Dewar
Refill LN 2 storage Dewar
Check Drawer LN 2
Dewar
Check Process
Chamber
Cartridge is not loaded or
the specimen was lost
before the freezing
process
The cylinder is out of
position
The alcohol level is low
The LN 2 specimen Dewar
drawer is open
The specimen Dewar is
empty or the level of LN 2
is low
The specimen Dewar is
missing
The cartridge is lost after
the freezing process
Open the safety cover.
Remove the lost
cartridge pieces from
the rotor. Press exit.
Press INIT
Open safety cover and
refill the alcohol tank
Close the LN 2
specimen Dewar
drawer
Fill the specimen
Dewar with LN 2 or
position the specimen
Dewar firmly against
the metal holder
Place the specimen
Dewar in the drawer
and fill with LN 2
Open the safety cover.
Remove the lost
cartridge pieces from
the process chamber
or the rotor.
In the event other error messages appear on your Leica EM HPM100 touch screen,
please contact your local service representative.
Page 54 Leica EM HPM100 Operating Manual Version 12/10

8. Appendix I
Numbers show dimensions in millimeters.
Page 55 Leica EM HPM100 Operating Manual Version 12/10

Numbers show dimensions in millimeters.
Page 56 Leica EM HPM100 Operating Manual Version 12/10

EC Declaration
EC Declaration of Conformity
EG Konformitäts-Erklärung
Déclaration CE de Conformité
We/Wir/Nous
Leica Mikrosysteme GmbH
Hernalser Hauptstrasse 219
A-1170 Wien, Austria
declare in exclusive responsibility that the product
erklären in alleiniger Verantwortung, dass das Produkt
déclarons sous notre seule responsabilité que le produit
Model
Modell
modèle
EM HPM100
EM HPM100
EM HPM100
Type/Typenbezeichnung/type
HPM100
to which this declaration relates is in conformity with the following standards:
auf das sich diese Erklärung bezieht, mit den folgenden Normen übereinstimmt:
auquel se réfère cette déclaration est conforme aux normes :
EN 61010-1
EN 61326-1
following the provisions of directive
gemäss den Bestimmungen der Richtlinie
conformément aux dispositions de directive
2004/108/EC
2006/95/EC
2006/42/EC
87/404/EWG
(Electromagnetic compatibility)
(Elektromagnetische Verträglichkeit)
(Low Voltage Equipment)
(Niederspannungsrichtlinie)
(Machinery)
(Maschinen)
(Pressure Vessel)
(Druckbehälter)
Wien, 15. Februar 2008
Dr. Reinhard Lihl
Entwicklungsleiter
R & D Manager
Chef du service développement
Leica Microsysteme GmbH Tel. +43 1 48899
Hernalser Hauptstrasse 219 Fax +43 1 48899-350
A-1170 Wien www.leica-microsystems.com

Minutes
LMS Holdings GmbH Tel. +49(0)6441/29-2327
Ernst-Leitz-Strasse 17-37 Fax +49(0)6441/29-2211
D-35578 Wetzlar www.leica-microsystems.com

www.leica-microsystems.com
Copyright © Leica Mikrosysteme GmbH, Hernalser Hauptstrasse 219, 1170 Vienna, Austria · Tel. +43 1 48899, Fax +43 1 48899350 LEICA and the Leica logos are registered trademarks of Leic IR GmbH.
Order nos. of the editions in: Operating Manual Leica EM HPM100, Version 167120032 12/10 Printed on chlorine-free bleached paper.