Leica CryoJane User manual

CryoJane®User Manual
Customer Service 1-800-237-2772
(In continental US only)
Customer Service 1-201-343-1313
(International)

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Instrumedics grants the purchaser of the CryoJane system
the rights to use the patented CryoJane (CFSA) materials
and process.
Patent # 4,545 831

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CRYOJANE TAPE-TRANSFER
STEP-BY-STEP
INSTALLATION INSTRUCTIONS
The following items are required for installation:
Absolute Alcohol 1600 Watt Hair Dryer Gauze Gloves
01 Remove release paper
from ECU
02 Place ECU on top left
corner of cryostat
03 Using absolute alcohol and gauze
clean chamber wall
04 Heat left wall for Mech &
Oil Bath placement
05 Place Oil Bath towards the
rear of the left chamber wall
06 Route Oil Bath & Mech Cables
underneath Mech
*07 Place Mech in Chamber.
Place Mech low in chamber
near the front wall.
(Placement near the freeze
bar is ideal in most cryostats)
08 Route cables in door bar 09 Place door bar with cables
onto front chamber ledge.
If required attach Door Bar Support Strip
underneath door bar

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10 Adhere Cable holders
along cryostat top outer wall
11 ECU Rear with Cable &
Power Connectors
12 Connect Mech, Oil Bath Cables to
ECU
& Power cord to the ECU and wall outlet
Turn the ECU on
13 Mech Cable
connected to ECU
14 External-Installation
Complete
Cryo-Vac Shown
15 Internal-Installation Complete
Cryo-Vac Shown
*The standard CryoJane Mech installation is shown in Fig. 07.
The Hacker/Bright OTF and Microm HM 560 cryostats require an S-Plate
Mech and separate mounting plate for installation.
S-Plate Mech for Hacker OTF and Microm HM 560 Cryostats
Mounting Plate locks onto S-Plate Mech.
Slide Nest Entrance
The entire assembly is adhered to the left cryostat wall as shown in Fig. 07.
Note: The S-Plate Mech must have enough clearance from the front wall of the
cryostat for slides to enter the Slide Nest.

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Table of Contents
Instrumedics Frozen Section Process Overview…………… 7
Freezing Process …………………………………………………………. 7
CryoJane Process ………………………………………………………… 7
CryoJane System Components …………………………………….. 8
CryoJane ECU ……………………………………………………………. 9
CryoJane Mech Components ………………………………………. 10
CryoJane Operating Procedure …………………………………… 11
Background ………………………………………………………………. 11
Preparation ……………………………………………………………….. 11
Use ……………………………………………………………………….. 12
Steps for a Single Section ………………………………………… 12
Steps for Multiple Sections ………………………………………. 18
Summary of the Instrumedics Frozen Sectioning Process. 19
Special Fixation … … … … … … … … … … … … … … … … … … … … … … 20
Aqueous Fixation …… ………………………………………………….. 20
“Anhydrous” Fixation …………………………………………………….. 21
CryoJane Maintenance ……………………………………………….. 22
ECU ……………………………………………………………………… 22

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Mech …………………………………………………………………….. 22
Troubleshooting Guide … … … … … … … … … … … … … … … 23
Troubleshooting the Mech and ECU …………………………………….. 23
CryoJane Process Troubleshooting ………………………………………. 25
Troubleshooting Finished Slides …………………………………………. 27
Oil Bath Accessories Kit Components ………………………….. 29
Oil Bath Accessory Kit Operating Procedure ………………. 30
Background ……………………………………………………………… 30
Preparation ………………………………………………………………. 30
Use ……………………………………………………………………… 31
Storing a Tissue Block ………………………………………….. 31
Sectioning a Stored Tissue Block ………………………………. 31
Oil Bath Accessory Kit Troubleshooting Guide ………….. 32

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Instrumedics Frozen Section Process Overview
Freezing Process - Conventional Freezing vs. Snap Freezing
In the conventional freezing method, the tissue is typically frozen at about -30°C. During
this relatively slow freezing process, the ice crystals that form are usually large compared
to cellular dimensions. These crystals can cause considerable displacement and structural
damage to the tissue.
The Gentle Jane device is designed to snap-freeze the tissue in approximately 8 -10
seconds. The freezing agent can be liquid nitrogen, LN2chilled isopentane, dry ice or a
thermoelectric cooler (Peltier device). The temperature at which the tissue is frozen
should be -60°C or colder.
NOTE: Gentle Jane Snap-Freezing methods are described in a separate Snap-
Freezing User Manual.
CryoJane Process
The CryoJane process is built around three special methodologies:
1. The capture of an undistorted, thin, snap-frozen section on a special cold adhesive
tape.
2. The lamination of the captured section onto a cold glass microscope slide coated with
an ultraviolet light curable pressure sensitive adhesive.
3. The curing of the adhesive on the slide with an 8 millisecond ultraviolet flash, and the
subsequent removal of the adhesive tape, leaving a still frozen section firmly adhered
to the microscope slide.
The CryoJane system is designed to permit the facile application of these methodologies,
and can be adapted to virtually all model cryostats.

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CryoJane System Components
FIGURE 1. CryoJane System Components
1. Mechanism (Mech). See CRYOJANE MECH COMPONENTS for details.
2. Electronics Control Unit (ECU). See CRYOJANE ECU for details.
3. ½ inch diameter Mech Cable (connected to the Mech)
4. Door Channel
5. 3 Shelf Unit
6. Hand Roller
7. Adhesive Coated Slides
8. Tape Windows
9. Knife Facet Wipers
10. AC Power Cord (not shown)
OPTIONAL EQUIPMENT:
11. Protective Oil Accessories Kit
(see Oil Bath Accessories Kit sections in this manual)
12. Cryo-Vac-Away System

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(see Cryo-Vac-Away User Manual)
CryoJane ECU
Figure 2. CryoJane ECU -back
Figure 3. CryoJane ECU -front and right side
1. On/Off Switch
2. Pad LED
3. Power LED (or Bath LED if optional Oil Bath Accessory Kit is installed).
4. Flash/Pad Connector
5. Optional Oil Bath Connector
6. Fuses
7. AC Power Cord Connector
4 5
6
7
1
2
3

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CryoJane Mech Components
FIGURE 4. CryoJane Mech Components
1. Blue Pad
2. Stainless Steel Spring Clip
3. Black Knob
4. Flash Tray
5. Lid

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CryoJane Operating Procedure
Background
In conventional sectioning the operator usually guides the cut section over the knife using
a brush or an anti-roll device. Even with a skillful, steady hand, this step can be difficult,
especially if a 2 to 4 micron thick section is desired.
To mount the section, a room-temperature slide is touched to it. When contact is made
between the warm slide and the frozen section, instantaneous melting of the section
occurs, converting the ice crystals back into water. Surface tension causes the melted
section to adhere to the slide. When the ice melts, water fills the spaces formerly
occupied by the ice crystals, rehydrating the nucleoplasm and cytoplasm, causing
erythrocytes to lyse and soluble substances to be displaced. Flow of the melted solutions
can distort and displace fine structure. Often the melted section is then dried, during
which step, surface tension forces further distort, displace and collapse tissue structure.
Pathologists and clinicians have been trained to recognize normal and abnormal structure
against this background of degraded fine structure.
In contrast, the CryoJane process is designed specifically to avoid melting and air drying
and its associated artifacts. It uses a cold adhesive tape to support and capture the section
during the cutting step. The unthawed section on the tape is then transferred to a special
cold adhesive-coated slide. The section is not melted in the mounting step. The
adhesive coating on the slide is then polymerized, and the tape is peeled away, leaving the
section permanently anchored to the slide while still frozen.
Preparation
•A day’s supply of Adhesive Coated Slides and Tape Windows should be in the
cryostat, chilled and ready for use.
•The cryostat temperature should be -25°C to -30°C (depending on the cryostat
model).
•The Pad light (left LED) on the ECU should be green.

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•The right LED should be red if it is labeled Power and green if it is labeled Oil Bath.
Use
Once a tissue specimen has been frozen and mounted in the microtome chuck, follow the
procedure outlined below.
Steps for a Single Section:
1. If the Cryo-Vac-Away is installed,
position the collection nozzle at the
knife edge and step on the foot switch
to automatically remove debris during
the trimming sequence. If the Cryo-
Vac-Away is not installed, trim the
block to the desired depth to obtain a
full block face and remove the debris
from the knife. Set the micrometer for
the desired section thickness and cut
two or three sections to be sure a full
section will be cut. Figure 5. Block trimming and debris removal.
2. Take a pre-cooled, pouched, Adhesive
Coated Slide, and while holding it
inside the cryostat chamber, remove
the glass slide from the pouch.
Figure 6. Adhesive coated slide in pouch.
.
Nozzle

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3. Holding the Slide at the frosted end,
rapidly peel off the protective mylar
film which covers the adhesive and
discard it.
Figure 7. Peeling mylar from adhesive coated
slide.
4. Place the Coated Slide, adhesive-side-
up, on the Blue Pad of the Mech by
slipping the frosted end of the slide
under the stainless steel Spring Clip.
Figure 8. Adhesive Coated Slide on Blue Pad.
5. Inside the cryostat, pick up a pink Tape
and peel away the protective film
covering the adhesive window. Discard
the protective film.
Figure 9. Exposing the Tape window.

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6. Hold the Tape up by the narrow tab
with the adhesive side towards the
block face. Position the Tape in front of
the trimmed block so that the bottom
edge of the exposed adhesive is aligned
with the bottom edge of the block.
Place the Tape on the block face
making sure that the bottom wider tab
of the Tape overhangs the knife. NO
PORTION OF THE ADHESIVE
WINDOW SHOULD OVERHANG
THE BLOCK FACE.
TO AVOID MELTING THE
SURFACE OF THE TISSUE DO
NOT TOUCH THE PORTION OF
THE TAPE THAT COVERS THE
BLOCK FACE.
Figure 10. Placing Tape on block face.
7. Using the cold Hand Roller, laminate
the Tape to the face of the block by
moving the Roller up and down one to
two times and then left to right one to
two times. Some pressure is required
for good adhesion. When finished,
place the Hand Roller back to its
original position inside the cryostat.
Figure 11. Laminating the Tape to the block face.

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8. Turn the flywheel slowly and evenly to
cut a section. If a sharp knife is used,
the section captured on the Tape will be
flat, intact, uncompressed and as thin as
two microns. No need for a brush or
an anti-roll device!
Figure 12. Cutting the section.
9. Pick up the Tape at either tab and
transfer it to the Slide on the Blue Pad
making sure to keep it down inside the
cryostat to avoid melting the section.
Figure 13. Handling the cut section.
10. Position the Tape, section-side-
down,
on the Adhesive Coated Slide. For
multiple sections refer to the Steps for
Multiple Sections.

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Figure 14. Positioning the Tape on the Slide.
11. Use the cold Hand Roller to laminate
the Tape to the Slide. Apply even
pressure on the Roller and roll it over
the section under the Tape two or three
times
Figure 15. Laminating the section to the Slide.
12. Holding the frosted end of the Slide,
remove it from the Blue Pad and
transfer it into the Flash Tray. Gently
push the Slide in as far as it will go,
against the stop. (To check, lift the
Lid.) Close before actuating the flash.
Figure 16. Placing the slide into the Flash Tray.
13. Push and release the Black Knob to
actuate the UV flash. The UV cures the
adhesive on the Slide into a polymer,
anchoring the frozen section to the
Slide.
Figure 17. Curing the adhesive on the Slide.

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14. Holding the frosted end, immediately
remove the cured Slide from the Flash
Tray. DO NOT TAKE THE SLIDE
OUT OF THE CRYOSTAT AT THIS
TIME AS THAT WILL CAUSE THE
SECTION TO MELT.
Figure 18. Removing the cured Slide.
15. Keeping the Slide deep inside the
cryostat chamber, carefully remove the
pink Tape to expose the frozen section
on the Slide.
Figure 19. Removing Tape from cured Slide.
16. Peel off the Tape diagonally and
downward to minimize the tension on
the section and to insure complete
transfer. Do not touch the section with
your fingers. Once removed, discard
the Tape.
The slide can now be:

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a) fixed in the Instrumedics Aqueous
Fixative,
b) freeze-dried or freeze-substituted in
the cryostat before “anhydrous”
fixation,
Note: The section should be immersed
in the fixative inside the cryostat.
Fixation should then continue at room
temperature.
c) melted or air-dried and fixed with
the fixative of choice for your
protocol.
Figure 20. Technique for removing Tape from
Slide.
Steps for Multiple Sections:
17. Multiple sections can be positioned on
o
ne Slide by placing the Tapes
perpendicular to the Slide. For small
tissue specimens, the Tape can be cut
into narrower strips. Follow Steps 5 - 9.
18. To cure the adhesive on the slide, lift
the Lid of the Mech and seat the Slide
on the Flash Tray. The Tape tabs will
overhang the Flash Tray.
Figure 21. Processing multiple sections.

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19. Close the Lid and push and release the
Black Knob to actuate the UV flash.
Then lift the Lid, remove the Slide and
carefully peel away each strip of Tape
as described in Steps 15 and 16.
Figure 22. Curing multiple sections.
Summary of the Instrumedics Frozen Sectioning Process
FIGURE 23. To minimize ice-crystal
size, tissue is snap-frozen with a
chilled heat extractor.
Snap-freezing and embedding.
A blockholder is placed on the Gentle Jane snap-
freezing device.
CryoGel or other embedding media is dispensed onto the blockholder
and a tissue specimen is positioned on top. The chilled heat extractor is
placed in its holder and released. When the heat extractor contacts the
specimen, the tissue and embedding medium is snap-frozen into a flat
block. The plane of the flat block face minimizes trimming. When
specimen shape or orientation are essential, the tissue is snap-frozen
using the Instrumedics CryoGel / Rubber Mold method.
FIGURE 24. A cold adhesive tape is
used to support the section as it is
being cut.
Cutting
After the block is trimmed, a cold adhesive tape is adhered to the block
face. The tape supports and captures the section as it is being cut,
eliminating the need for a brush or anti-roll device.
FIGURE 25. The still frozen section,
Transfer to Slide
A cold adhesive-coated slide is placed on a temperature-
controlled pad.
The adhesive tape is placed section-side-down on the adhesive-coated
slide, and is laminated to the adhesive layer using a cold roller.

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adhered to the tape is rolled onto the
cold adhesive-coated slide.
FIGURE 26. An ultraviolet flash
converts the adhesive coating into a
hard solvent-resistant plastic.
Curing the Adhesive Coating
A flash of ultraviolet light passes through the slide to polymerize the
adhesive layer on the slide into a hard, solvent-resistant plastic, tightly
anchoring the section. to the slide.
FIGURE 27. The tape is removed.
Several options are available
Removal of Tape
The tape is peeled away leaving the still frozen section tightly bonded to
the plastic layer.
The slide can then be:
a) fixed in the Instrumedics Aqueous Fixative,
b) freeze-dried or freeze-substituted in the cryostat before “anhydrous”
fixation,
c) melted or air-dried and fixed with the fixative of your choice.
Special Fixation Protocols
Aqueous Fixative:THIS FIXATION SHOULD BE USED FOR ALL
HISTOLOGICAL STAINING OF CRYOJANE PREPARED SECTIONS.
This fixative melts and fixes the tissue simultaneously (direct controlled “melting”). Ice
crystal artifacts (holes) are masked. Nuclear morphology is usually excellent. Some cell
components are lost and most of the red blood cells are lysed.
1. PREPARATION OF AQUEOUS BUFFER
Dissolve Buffer-Salt Mix in 180 ml of water.
Buffer-Salt Mix is available from Instrumedics (Cat. #AB)
Store in refrigerator. Usable for at least 6 months.
2. PREPARATION OF AQUEOUS FIXATIVE
Add 20 ml of 25% glutaraldehyde to 30 ml of Aqueous Buffer.
Mix well.
Use at room temperature.
Discard after 1 week. (If using 50% glut. add 10 ml glut. and 10 ml water.)
CAUTION! Contains Cacodylic Acid.
Avoid contact with fingers or skin.
Do not breathe dust from vial.
3. PROCEDURE