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電顯 第二題
2. Cryobiology is a recently well-developed integrated field in biological and
physical sciences. Would you please mention principle method, application and
limitations of different cytotechniques, e. q. cryopreservation of sperm and/
or plant calli, freeze-drying, freeze-substitution, freeze-fracture, freeze-et
ching and cryoSEM.
Ans;
Cryopreservation of sperm
--method
1. Blood test to screen for infectious diseases such as HIV (the AIDS virus)
, hepatitis and syphilis.
2. Semen collection
3. The sperm will be mixed with a solution to reduce freezing damage and then
frozen in colour-coded, labeled plastic straws and stored in liquid nitrog
en at -196℃.
4. Thawed in 40℃ water bath.
--application
Homologous and donor insemination, cryopreservation prior to surgical infertil
ity treatment, intraoperative cryopreservation, postoperative cryopreservation
, cryopreservation prior to treatment for malignancies and nonmalignant diseas
es, as well as premortem and postmortem cryopreservation.
--limitation
There is concern about the transfer of genetically damaged sperm after cryopre
servation. There is no guarantee that the sperm will survive thawing. There is
also no guarantee that the thawed sperm will be fertile or that they will giv
e rise to a conception if used with technologies such as artificial inseminati
on, in vitro fertilization (IVF) and sperm microinjection (ICSI).
Cryopreservation of plant calli
--method
1. Calli samples were taken after subculture
2. Cryoprotectants were used: (1). 10% DM-SO + 0.5 mol/L sorbitol;
(2). 7.5% DMSO +5% glycerin +5% sucrose.
3. The specimens were cooled at a rate of l℃/min from 0℃, kept -40℃ for 2
h, put into liquid nitrogen(-196℃)
4. Thawed in 40℃ water bath.
--application
--limitation
Freeze-drying
--method
1. Deep-freeze
2. Sublimation: A vacuum is applied to pull all the water out in the form of
water vapor.
3. Slowly returned to room temperature.
--application
Preservation of materials, e. q. tissues for transplant, fresh flowers, fr
uits, and vegetables that maintain the natural shape and vibrant color of the
botanical
--limitation
Freeze-drying whole organs (or bodies) would be much more difficult and ti
me-consuming, though, so it's not clear that this approach will ever be a usef
ul alternative to cryonic suspension.
Freeze-substitution
--method
1. Cryoprotection and high-pressure freezing
2. Substituted frozen water with acetone containing some additives at -80℃
to -95℃
3. Warm up to r. t.
4. Embedding
--application
Preservation of cell structure, immunolabeling, and embedding specimens in
to resin
--limitation
Freeze-fracture
--method
1. Cryoprotection
2. Quick freeze in liquid nitrogen
3. The frozen specimen is rapidly transferred to a precooled stage (-150℃)
4. Chamber pumped to high vacumn
5. The stage is warmed to -100℃ and the tissue fractured by razor blade
6. Platinum is evaporated at 45。 relative to the specimen plane
7. Carbon is evaporated directly above
8. The chamber is brought to r. t. and pressure
9. The thawed tissue is digest in 5% sodium hypochloride
10. The replica is moved to an additional chlorine bleach change and then
through 2 or 3 water washes
11. The replica is put on a meshed grid and dried
12. The replica is examined under a TEM
--application
Examine membrane faces
--limitation
Freeze-etching
--method
1. Cryoprotected
2. Freeze
3. Fractured directly over the specimen
4. Warm up to -100℃ and remove the ice by sublimation at a rate of 2 to 3 nm
per second
--application
Examine actual membrane surface
--limitation
1. Poor freezing and accidental warming of tissue may lead to ice crystal
formation, causing deformation of tissue structure.
2. The replica is fragile and brittle and may fragment
CryoSEM
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