Leica LN22 User Manual
Instructions for Use
Leica LN22 – Liquid Nitrogen Freezing Device
V1.2, English – 10/2012
Order No.: 14 0373 80101 RevB
Always keep this manual near the instrument.
Read carefully prior to operating the instrument.
Leica LN22
Liquid nitrogen freezing
device
NOTE
The information contained in this documentation
represents the state of the art in science and
technology.
We are under no obligation to update the present
manual periodically and on an ongoing basis according to the latest technical developments, nor
to provide our customers with additional copies,
updates etc. of this manual.
For erroneous statements, drawings, technical
illustrations etc. contained in this manual we exclude liability as far as permissible according to
the national law applicable in each individual
case. In particular, no liability whatsoever is accepted for any financial loss or consequential
damage caused by or related to compliance with
statements or other information in this
manual.
Statements, drawings, illustrations and other information as regards contents or technical details of the present manual are not to be considered as warranted characteristics of our
products.
These are determined only by the contract provisions agreed between ourselves and our customers.
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 in our products.
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© Leica Biosystems Nussloch GmbH
Published by:
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D-69226 Nussloch
Germany
Telephone: +49 (0) 62 24 143-0
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Internet: http://www.LeicaBiosystems.com
Leica LN22
3
Table of contents
1. Important information ................................................................................................................ 6
1.1 Symbols in the text and their meanings ................................................................................... 6
1.2 Intended use of instrument ........................................................................................................ 6
2. Safety ............................................................................................................................................ 7
2.1 Safety instructions ...................................................................................................................... 7
3. Instrument components and specifications ....................................................................... 15
3.1 Technical data ............................................................................................................................ 15
3.2 Overview and instrument components .................................................................................. 16
3.3 Standard delivery ...................................................................................................................... 17
4. Startup ........................................................................................................................................ 18
4.1 Installation site requirements.................................................................................................. 18
4.2 Unpacking ................................................................................................................................... 18
4.3 Preparing the microtome for attachment of the LN22 ......................................................... 19
4.4 Installing the cryochamber ...................................................................................................... 20
4.5 Connecting to mains ................................................................................................................. 21
4.5.1 Checking the voltage selector setting ................................................................................... 21
4.5.2 Changing the voltage selector setting and inserting the fuses ......................................... 22
4.5.3 Plugging in the connection cables ......................................................................................... 23
4.5.4 Grounding the Dewar ................................................................................................................ 23
4.5.5 Connecting the mains cable .................................................................................................... 24
4.6 Switching on the instrument.................................................................................................... 24
4.7 Control unit ................................................................................................................................. 25
4.8 Specimen holders...................................................................................................................... 27
4.8.1 Specimen holder for block embeddings ................................................................................ 27
4.8.2 Universal specimen holder and flat specimen holder ......................................................... 29
4.9 Knife holders .............................................................................................................................. 30
4.9.1 Knife holders for tungsten carbide knives............................................................................. 30
4.9.2 Glass knife, CryoDiamond knife, trimming knife Histo ......................................................... 30
4.10 Preparing the pumping system ............................................................................................... 33
4.11 Refrigerating .............................................................................................................................. 33
4.12 Refilling the Dewar vessel ....................................................................................................... 34
4.13 Heating up the chamber ........................................................................................................... 35
4
Instructions for Use V1.2, RevB – 10/2012
Table of contents
5. Cleaning and maintenance .................................................................................................... 36
5.1 Cleaning ...................................................................................................................................... 36
5.2 Maintenance .............................................................................................................................. 39
5.2.1 General maintenance instructions ......................................................................................... 39
5.2.2 Exchanging the mains fuses .................................................................................................... 39
5.2.3 Exchanging the bulb .................................................................................................................. 40
5.2.4 Removal of the pump valves .................................................................................................... 41
6. Appendix .................................................................................................................................... 42
6.1 Ordering information ................................................................................................................. 42
7. Warranty and service .............................................................................................................. 43
Leica LN22
5
1. Important information
1.1 Symbols in the text and their meanings
Warnings
appear in a gray box and are marked by
a warning triangle .
Notes,
i.e. important user information, appear in
a gray box and are marked by an infor-
mation symbol .
Solvents and reagents that are flammable are identified by this symbol.
This warning symbol identifies surfaces
of the instrument that are hot during operation.
(5)
Numbers in parentheses refer to item
numbers in illustrations.
1.2 Intended use of instrument
The Leica LN22 is a liquid nitrogen freezing device to quickly and effortlessly change from sectioning at room temperature to freezing sectioning applications and vice versa.
Any other use of the instrument is
considered improper!
ENTER
6
Function keys that must be pressed on the
input screen are boldfaced in the text and
capitalized.
Instructions for Use V1.2, RevB – 10/2012
Be sure to comply with the safety instructions and warnings provided in this chapter.
Be sure to read these instructions, even if you are already familiar with the operation and use
of other Leica products.
2.1 Safety instructions
2. Safety
These operating instructions contain important
information for the operational safety and maintenance of the device and are an important component of the device.
If additional requirements on accident
prevention and environmental protection exist in the country of operation,
this Instructions for Use must be supplemented by appropriate instructions
to ensure compliance with such requirements.
Device type
All information provided in this manual applies
only to the instrument type indicated on the cover
page.
An identification label with the serial number is
attached on the underside
of the control unit.
This instrument was built and tested in accordance with the safety regulations for electrical
measuring, control, regulating and laboratory
devices as specified below:
• EN 61010-1:2001,
• EN 61326: 1997+A1: 1998 +A2: 2001
.
The protective devices on both instrument and accessories must neither be
removed nor modified.
Only service personnel qualified by
Leica may repair the instrument and
access the instrument’s internal components.
Leica LN22
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2. Safety
Safety measures in cryopreparation
Provided that the safety measures outlined in the present chapter are
observed, using all safety devices of the instrument as described, there
is no significant accident risk involved in cryopreparation. For this reason, please make sure at all times when working with the LN22 system
that the few, but decisive, rules for safety listed in this chapter are followed.
When handling liquid nitrogen (LN
•LN2 is extremely cold and boils at -196°C. As a consequence, also nitrogen gas (GN2) escapes from boiling LN2 at a very low temperature.
Bear in mind, that LN2 and/or GN2 as well as objects which are in contact with LN2 / GN2 (pipe joints, valves, pipes, containers or stoppers)
can cause severe frost bite on skin and eyes.
• When evaporating, LN2 expands at a ratio of approximately 1 : 700,
i.e. 1 liter of liquid nitrogen produces almost 1 m3 of nitrogen gas
(GN2). Therefore, please make sure that the working area is kept well
ventilated when larger quantities of liquid nitrogen evaporate (e.g.
when pouring LN2 from one container into another). LN2 residues from
containers may only be emptied outdoors: Useful for disposing of liquid nitrogen is a gravel-filled container or pit, where LN2 will evaporate quickly and safely.
• Nitrogen gas (GN2) is odorless and has no taste so it cannot be distinguished from normal air when inhaled. GN2 is nontoxic as a substance. Still, a higher GN2 content (normal ratio: about 78 % GN2 to
21 % of oxygen) in the air will cause the oxygen level to diminish, thus
causing sudden fainting and deep unconsciousness without any preceding warning symptoms (such as numbness, dizziness).
In case of doubt about the oxygen level in the air it is advisable to use
a gas analyzer with an oxygen level indication range of 0 to 25 %.
There is danger when the oxygen level drops below 18 %.
), please bear in mind the following:
2
8
Instructions for Use V1.2, RevB – 10/2012
2. Safety
If an unconscious person remains in such a low-oxygen environment,
death may occur.
In case of emergency immediately call a physician and an ambulance. If breathing stops, apply artificial respiration at once.
• For the reasons just explained, never place LN2 containers in closed
storage rooms. Due to defects because of improper handling (jolts,
mechanical damage) or simply by normal wear, the evaporation rate
of Dewar vessels may rise to several liters per day. In enclosed
spaces, this will cause a dangerous reduction of the oxygen level in
the air.
• Please remember that the LN22 system evaporates 2 to 3 liters of liq-
uid nitrogen per hour during normal operation. This quantity of liquid
corresponds to 2 to 3 m
area is kept well ventilated at all times and adhere to the precautionary measures described above.
• Be extremely careful when bringing objects at ambient temperature
into contact with LN
of gas will form around the object. This makes any significant transfer
of warmth impossible and so comparatively little LN2 will evaporate.
However, as soon as the object has cooled down sufficiently there
will often occur strong boiling and splashing of LN2 at a time when it
is no longer expected.
3
of GN2/h. Therefore, make sure your work
. During the first few moments an insulating layer
2
9
2. Safety
• There are clothing items and protective gear
with certain characteristics which you should
specifically avoid when working with LN2: Do
a
b
not wear closed safety goggles (a), boots (c),
shoes (e) and gloves (g) into which LN
splashes can enter, but from where they cannot escape. LN2 that has splashed into such
items will evaporate immediately inside the
item thus causing severe injuries, before the
item can be removed or taken off. Therefore,
always use safety goggles with side protec-
c
d
tion but which are open at the top and at the
bottom (b), so that LN2 splashes can escape
immediately. Wear boots only in combination
with long, loose-fitting (not tight!) pants that
fall over the boots (d) and completely cover
their upper opening. Only wear open slippers
(f) in the lab, no shoes or pumps that enclose
e
f
your feet entirely.
In combination with open slippers/sandals only
wear pants without cuffs, so that LN
will flow off freely. When pouring LN2 from one
container into another or when inserting the
Dewar pump into the Dewar, never wear safety
gloves! As an alternative to gloves you can use
an open flannel cloth (h) to protect your hands
gh
when touching cold components. Protective
gloves are useful when handling cold and dry
Fig. 1
objects, however they are not for work with LN2.
splashes
2
2
• If your skin is injured by splashing LN2, immediately rinse the affected skin area(s) thoroughly with lukewarm (body temperature!)
water. In case of serious injuries, immediately
see a dermatologist.
10
If LN2 splashes into your eyes, rinse the
eye immediately and thoroughly with
lukewarm water. If available in your lab,
use a special eye-rinsing device for that
purpose. After rinsing, immediately see
an ophthalmologist.
Instructions for Use V1.2, RevB – 10/2012
Loosely fitted stopper
Fig. 2
2. Safety
• Once every three months check the actual evaporation rate of your
Dewar vessels and compare the measuring results to the evaporation
rates specified by the manufacturer. The evaporation rate of an undamaged metal Dewar is far less than 1 liter per day. Defective
Dewars with evaporation rates higher than that are a safety risk and
should be taken out of service or repaired.
• Standard Dewar vessels, such as the metal Dewar of the LN22 system, are not pressure vessels, therefore make sure they are closed
only with a special Dewar topper (Fig. 2). These stoppers fit loosely
onto the Dewar neck and leave a sufficiently large gap to allow for
sudden evaporation of GN2. Check the stopper frequently to make
sure it will not be stuck to the Dewar neck by ice deposits (Fig. 3).
Never close a Dewar vessel with stoppers that you have crafted
yourself (Fig. 4). Through exposure to shock or vibration (e.g. transport), a gas-tight closed Dewar vessel can release GN2 in an explosion-like fashion and burst in the process. In that case the entire LN
content (35 liters = 35m3 GN2) will evaporate at once (that may e.g.
happen inside a vehicle during transport). When a Dewar moves, thus
causing a sudden evaporation of LN
per will rise due to the gas pressure, thus opening a wide enough gap
for the GN2 to escape (Fig. 5). The small quantities of LN2 which may
escape as well in such situations do not have to be regarded as a significant safety risk.
, an appropriately designed stop-
2
2
Fig. 3
Ice deposits
Tightly fitted stopper
with seal
Fig. 4
GN
Fig. 5
2
GN
2
11
2. Safety
• Never leave LN2 in open containers where it can exchange with the
room atmosphere. The boiling point of LN2 (-196°C) is lower than that
of liquid oxygen (-183°C). If a large LN2-surface is exposed to the air,
oxygen from the air is absorbed by the LN2 while nitrogen is released
into the air. LN2 with a high content of liquid oxygen has a bluish color.
In that case, caution is advised, since concentrated liquid oxygen is a
fire hazard.
• Make sure that your Dewar vessel is filled with no other substances
than LN2. If at the filling location also other gases are refilled, install a
warning sign there that clearly states: “Liquid nitrogen only”. Check
the color of the cryogen! Bluish color indicates a high oxygen con-
tent (see previous paragraph). If LN2 is stored during a prolonged
period of time, liquid oxygen will accumulate in the LN2 vessel, since
liquid oxygen has a higher boiling point (-183°C) than liquid LN2
(-196°C).
• Never fill a Dewar vessel up to the neck with LN2 (Fig. 6). In case of a
boiling delay, there is danger that the rapidly expanding gas bubbles
will force the LN
in the neck upwards causing it to splash (Fig. 7). For
2
this reason, always make sure that the LN2 surface level is below the
neck of the vessel (Fig. 8). Also bear in mind, that in most cases the
length ‘l’ of the outer insulation differs from the length ‘L’ of the actual
Dewar neck.
LN2 level
LN
2
Fig. 6 Fig. 7
12
GN
LN2 level
2
Fig. 8
Instructions for Use V1.2, RevB – 10/2012
Warnings – Installation/Connection to mains
The room where the instrument is installed must either be well ventilated or be large enough to
avoid a dangerous decrease of the oxygen level in the air.
Please read chapter 2 ‘Safety instructions’ carefully!
Pay careful attention to chapter 3 - ‘Technical data’.
The voltage selector is preset to 230V in the factory.
Before connecting the instrument to the mains power, please check whether the factory setting
is identical with the local mains power in your lab!
The mains cable inlet is sealed with an adhesive tape that indicates the factory setting the instrument is delivered with.
Severe damage may be caused to the instrument when operating it at an incorrect voltage setting.
Do not change the voltage selector setting with the instrument being connected to the mains!
The Dewar must be externally grounded!
The instrument may only be connected to a grounded mains power outlet socket. To connect
the instrument only use one of the mains cables that are supplied together with the instrument!
2. Safety
Warnings – Maintenance
Only authorized and qualified service personnel may access the internal components of the instrument for service and repair!
Before opening the instrument, switch off the mains switch and pull the mains plug!
Before exchanging the fuses, switch off the mains switch and pull the mains plug!
When exchanging fuses, only replacement fuses with the same specification as the original
ones may be used. Refer to chapter 3 ‘Technical data’ for the correct specifications.
Before replacing the lamp, switch off the mains switch and pull the mains plug!
Leica LN22
13
2. Safety
Warnings – working at the instrument
During operation, metal surfaces and components inside the cryochamber can reach temperatures from as low as -196°C up to as hot as +110°C!
Touching these extremely cold or hot parts can cause severe injury!
For that reason, for any type of manipulations inside the cryochamber you may use only the
special tools which come as part of the standard delivery and the use of which is described in
detail in the present manual.
Transfer of frozen specimens may also only be done by means of the corresponding special
tools.
Always attach the specimen holder before inserting the knife holder!
The light has to be on as long as the knife is inside the cryochamber!
Exercise extreme caution when handling knives! The cutting edges are extremely sharp and
can cause severe injury!
Never try to catch a falling knife!
The knife may only be inserted into the knife holder shortly before sectioning and must be removed from the knife holder directly afterwards!
Cleaning
14
To insert the knife holder into the cryochamber, always use the corresponding manipulator
(part of standard delivery)!
The knife edge is exposed! Risk of injury!
Do not use acetone or xylene to clean the lacquered surfaces of the instrument.
Instructions for Use V1.2, RevB – 10/2012
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