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.
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. This document is protected under copyright laws. Any copyrights of this document are re­tained by Leica Biosystems Nussloch GmbH. Any reproduction of text and illustrations (or of any parts thereof) by means of print, photocopy, microfiche, web cam or other methods – includ­ing any electronic systems and media – requires express prior permission in writing by Leica Biosystems Nussloch GmbH.
© Leica Biosystems Nussloch GmbH
Published by: Leica Biosystems Nussloch GmbH
Heidelberger Strasse 17 - 19 D-69226 Nussloch Germany Telephone: +49 (0) 62 24 143-0 Fax: +49 (0) 6224 143-2 68 Internet: http://www.LeicaBiosystems.com
Leica LN22
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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
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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
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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.
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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!
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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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