AU2021104690A4 - Method for preparing section for rapidly observing microstructure of frozen aquatic products - Google Patents
Method for preparing section for rapidly observing microstructure of frozen aquatic products Download PDFInfo
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- AU2021104690A4 AU2021104690A4 AU2021104690A AU2021104690A AU2021104690A4 AU 2021104690 A4 AU2021104690 A4 AU 2021104690A4 AU 2021104690 A AU2021104690 A AU 2021104690A AU 2021104690 A AU2021104690 A AU 2021104690A AU 2021104690 A4 AU2021104690 A4 AU 2021104690A4
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- sample
- section
- frozen
- tissue
- adhesive film
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- 238000000034 method Methods 0.000 title claims abstract description 19
- 239000005030 aluminium foil Substances 0.000 claims abstract description 15
- 239000002313 adhesive film Substances 0.000 claims abstract description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000000853 adhesive Substances 0.000 claims abstract description 7
- 230000001070 adhesive effect Effects 0.000 claims abstract description 7
- 239000011888 foil Substances 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 239000007864 aqueous solution Substances 0.000 claims abstract description 5
- 238000005520 cutting process Methods 0.000 claims abstract description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 5
- 238000009966 trimming Methods 0.000 claims abstract description 5
- 238000010186 staining Methods 0.000 claims abstract description 3
- -1 OCT compound Chemical class 0.000 claims description 10
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 2
- 235000011089 carbon dioxide Nutrition 0.000 claims description 2
- 239000013078 crystal Substances 0.000 abstract description 13
- 238000002360 preparation method Methods 0.000 abstract description 4
- 101100012902 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FIG2 gene Proteins 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 abstract 2
- 241000238557 Decapoda Species 0.000 description 9
- 238000010586 diagram Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 3
- 230000003187 abdominal effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 238000005096 rolling process Methods 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000004018 waxing Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
- G01N2001/2833—Collecting samples on a sticky, tacky, adhesive surface
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
- G01N33/12—Meat; fish
Abstract
OF THE DISCLOSURE
The present disclosure discloses a method for preparing a section for rapidly
observing a microstructure of frozen aquatic products. The method specifically
comprises the following steps: preparing a cylindrical foil mold, and pouring an OCT
compound as an embedding medium into the aluminium foil mold; cutting a sample into
cuboid, and putting the sample in the foil mold to embed the sample; putting the
aluminium foil packed with the sample into liquid nitrogen to freeze the OCT
compound; attaching the sample after being embedded and frozen to a chuck of a
microtome-cryostat for trimming; adhering a cryogenic adhesive film which is
pre-cooled to below -20°C to the sample after being trimmed for sectioning; fixing a
sample section obtained above with an ethanol aqueous solution which is below -20°C
and in volume percentage of 70%- 80% for 2-5min, and then staining with a
hematoxylin-eosin stain; and putting an adhesive side of the cryogenic adhesive film
adhered with the sample section after being stained with a hematoxylin-eosin stain to a
microscope slide for direct mounting. In the present disclosure, a frozen section of a
sample is adhered through a cryogenic adhesive film, achieving a rapid preparation of a
section, and preserving the frozen morphology and ice crystals state in the tissue during
the sectioning, so that the contour of the ice crystals in the tissue is clear with minimal
changes.
ABSTRACT DRAWING - Fig 1
17920686_1 (GHMatters) P116864.AU
1/1
FIGI
FIG2
Description
1/1
FIG2
[01] The present disclosure relates to the field of microscopic tissue section, specifically relates to a method for preparing a section for rapidly observing a
microstructure of frozen aquatic products.
[02] Observation of the microstructure of biological tissues is often achieved by microscope. As the most commonly-used equipment for the observation of the
microstructure, the optical microscope can better present the original morphology of the
tissue under the precondition that the prepared section has high quality. Therefore, the
quality of the tissue section is a decisive factor for thefinal observation effect. The
commonly-used section preparation methods include the paraffin section method or the
frozen section method.
[03] As the most traditional section preparation method, the paraffin section method
requires a long time for fixation, clearing and gradient waxing before sectioning. After
sectioning, dewaxing is required before the sample being stained. Samples in the frozen
section method can be sectioned just by being embedded, which greatly reduces the
pre-processing time, and the sample is closer to status of the tissue in a original frozen
state. However, since there are ice crystals in the frozen section, sectioning often cause
ice crystals crushing and tissue fragmentation. When the tissue is directly embedded to
section and adsorbed on a microscope slide in the unfixed state, the tissue is rapidly
melted because of temperature difference. The tissue changes accordingly in the
transition process from solid to liquid and the observed tissue changes greatly due to the
melting of the ice crystals. If the sample is fixed in advance, and then goes through the
frozen section, the tissue will be severely dehydrated due to the influence of the fixing
solution, which impacts the observation of the original state of the tissue.
[04] Thus, to observe the morphology of the frozen tissue, especially the
morphology of the ice crystals, a frozen section method that maintains the original
17920686_1 (GHMatters) P116864.AU I moisture state of the tissue as much as possible is needed, to solve the disadvantage that the tissue of the frozen section is fragile and deformable.
[05] In view of the shortcomings of the prior art, the present disclosure provides a method for preparing a frozen section for rapidly observing a state of ice crystals in a
frozen tissue. The method may assist rapid observation of the morphology and structure
in the tissue while maintaining the original morphology of the tissue, and may be more
conducive to observing the relevant information about the formation of the ice crystals
in the tissue.
[06] To achieve the above object, the technical solution adopted by the present
disclosure is a method for preparing a frozen section for rapidly observing a state of
ice crystals in a frozen tissue, which specifically includes the following steps:
[07] (1) preparing a cylindrical aluminium foil mold with a flat bottom, and pouring an OCT compound (SAKURA Finetek) as an embedding medium into the foil mold;
[08] (2) cutting a sample into cuboid on a cutting board with a periphery being covered with dry ice, and putting the sample in the aluminium foil mold to embed the
sample;
[09] (3) putting the aluminium foil packed with the sample into liquid nitrogen to
freeze the OCT compound, and moving it into a refrigerator at -80°C for storage for
later use after being frozen;
[10] (4) adhering the sample after being embedded and frozen to a chuck of a
microtome-cryostat where a thickness of a section is adjusted to 40 m, using a sample
fast-forward button to move the sample closer to a slicer, adjusting a plane to be cut,
and then using a slow-forward button to start trimming;
[11] (5) adhering a cryogenic adhesive film which is pre-cooled to below -20°C to
the sample after being trimmed for sectioning, wherein the force for sectioning is
uniform;
[12] (6) fixing a sample section obtained in step (5) with an ethanol aqueous solution
which is below -20°C and in a volume percentage of 70%-80% for 2-5min, and then
17920686_1 (GHMatters) P116864.AU 2 staining with a hematoxylin-eosin stain; and
[13] (7) putting an adhesive side of the cryogenic adhesive film adhered with the sample section after being stained with the hematoxylin-eosin stain to a microscope
slide, for direct mounting; observing the obtained tissue section under a microscope,
then recording and photographing; observing the morphology and structure in the tissue
and the ice crystals to obtain a clear image of the tissue section.
[14] Further, the cryogenic adhesive film is a product with model of Cryofilm type IIC (10) from Leica Camera Japan Co., Ltd..
[15] Due to the application of the above technical solution, the present disclosure has
the following advantages compared with the prior art: The morphology of the section
prepared by the method is better preserved, the morphology in the tissue of the tissue
section in the frozen state may be observed more clearly, and the formation state of the
ice crystals may be studied more accurately. The tissue change caused by using an
optically transparent film that is still adhesive at low temperature to adhere to the tissue
for sectioning is smaller than that caused by using a microscope slide to absorb the
tissue by temperature difference. The sample adhered to the cryogenic adhesive film
may be fixed and stained in batches and then directly mounted by a cover slip, to
perform the microscopic examination without waiting for drying after being mounted,
which greatly shortens the section preparation time and maintains the original
morphology of the tissue.
[16] FIG. 1 is an observation diagram of a tissue section absorbed to a microscope
slide prepared in Example 1; and
[17] FIG. 2 is an observation diagram of a tissue section adhered to a cryogenic
adhesive film prepared in Example 2.
[18] The specific examples of the present disclosure are described in further details
below in conjunction with the drawings.
[19] The examples were implemented with the tissue of frozen shrimp as the raw
17920686_1 (GHMatters) P116864.AU 3 material.
[20] Example 1
[21] A cylindrical aluminium foil mold with a flat bottom was prepared, and an OCT compound as an embedding medium was poured into the aluminium foil mold; a cuboid shrimp tissue with a volume of 5 mm x 3 mm x 3 mm was cut from the first abdominal segment of the pretreated shrimp, and the side with a size of 3 mm x 3 mm that was near to the back of the shrimp was put toward the bottom of the aluminium foil mold, then the aluminium foil packed with the shrimp tissue as a sample was put into a thermostatic cup filled with liquid nitrogen, and then was moved into a -80°C refrigerator after the OCT compound was frozen; the OCT compound was used to fix the sample on the chuck of the microtome-cryostat after the aluminium foil was removed, and the section thickness on the microtome-cryostat was adjusted to 40 m, the sample fast-forward button was used to move the sample closer to the slicer, the plane to be cut was adjusted, the slow-forward button was used to start trimming, then anti-rolling plate was put down to start sectioning after the sample was trimmed, and the force for sectioning was uniform; the obtained section was absorbed to the adhesive microscope slide, and was fixed with an ethanol aqueous solution which was below -20°C and in a volume percentage of 70%- 8 0% for 2-5min, and then was stained with a hematoxylin-eosin stain; the stained section was transparentized with xylene to remove ethanol, and the neutral gum was dropped as the cover slip to mount the section. An microscopic examination was carried out after the section was dried. FIG. 1 is an observation diagram of the tissue section absorbed to the microscope slide.
[22] Example 2
[23] A cylindrical aluminium foil mold with a flat bottom was prepared, and an OCT compound as an embedding medium was poured into the foil mold; a cuboid shrimp tissue with a volume of 5 mm x 3 mm x 3 mm was cut from the first abdominal segment of the pretreated shrimp, and the side with a size of 3 mm x 3 mm that was near to the back of the shrimp was put toward the bottom of the aluminium foil mold, then the aluminium foil packed with the shrimp tissue as a sample was put into a thermostatic cup filled with liquid nitrogen, and then was moved into a -80°C
17920686_1 (GHMatters) P116864.AU 4 refrigerator after the OCT compound was frozen; the OCT compound was used to fix the sample on the chuck of the microtome-cryostat after the aluminium foil was removed, and the section thickness on the microtome-cryostat was adjusted to 40 m, the sample fast-forward button was used to move the sample closer to the slicer, the plane to be cut was adjusted, the slow-forward button was used to start trimming, then anti-rolling plate was put down to start sectioning after the sample was trimmed, and the force for sectioning was uniform; the obtained section was absorbed to the adhesive microscope slide, and was fixed with an ethanol aqueous solution which was below
-20 °C and in a volume percentage of 70%-80% for 2-5min, and then was stained with
a hematoxylin-eosin stain; the stained section adhered to the cryogenic adhesive film
was directly pasted to the microscope slide to mount for the microscopic examination.
FIG. 2 is an observation diagram of the tissue section adhered to the cryogenic adhesive
film.
[24] Judging from the process of Example 1 and Example 2, Example 2 saved the
time for clearing, mounting, and drying by using the cryogenic adhesive film; and the
observed diagram of the tissue showed that the ice crystals of the tissue in Example 2
were more rounded, and the original morphology of the ice crystals in the tissue was
better maintained.
[25] It is to be understood that, if any prior art publication is referred to herein, such
reference does not constitute an admission that the publication forms a part of the
common general knowledge in the art, in Australia or any other country.
[26] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary
implication, the word "comprise" or variations such as "comprises" or "comprising" is
used in an inclusive sense, i.e. to specify the presence of the stated features but not to
preclude the presence or addition of further features in various embodiments of the
invention.
17920686_1 (GHMatters) P116864.AU
Claims (2)
1. A method for preparing a section for rapidly observing a microstructure of
frozen aquatic products, wherein, the method comprises:
(1) preparing a cylindrical foil mold, and pouring an OCT compound as an
embedding medium into the foil mold;
(2) cutting a sample into cuboid on a cutting board with a periphery being covered
with dry ice, and putting the sample into the foil mold to embed the sample;
(3) putting the aluminium foil packed with the sample into liquid nitrogen, to
freeze the OCT compound;
(4) attaching the sample after being embedded and frozen to a chuck of a
microtome-cryostat for trimming;
(5) adhering a cryogenic adhesive film which is pre-cooled to below -20°C to the
sample after being trimmed for sectioning;
(6) fixing a sample section obtained in step (5) with an ethanol aqueous solution
which is below -20°C and in a volume percentage of 70%-80% for 2-5min, and then
staining with a hematoxylin-eosin stain; and
(7) covering an adhesive side of the cryogenic adhesive film adhered with the
sample section after being stained with the hematoxylin-eosin stain to a microscope
slide, for direct mounting.
2. The method for preparing a section for rapidly observing a microstructure of
frozen aquatic products according to claim 1, wherein, the cryogenic adhesive film is a
product with model of Cryofilm type IIC (10) from Leica Camera Japan Co., Ltd.
17920686_1 (GHMatters) P116864.AU 6
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021104690A AU2021104690A4 (en) | 2021-07-29 | 2021-07-29 | Method for preparing section for rapidly observing microstructure of frozen aquatic products |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021104690A AU2021104690A4 (en) | 2021-07-29 | 2021-07-29 | Method for preparing section for rapidly observing microstructure of frozen aquatic products |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2021104690A4 true AU2021104690A4 (en) | 2021-09-30 |
Family
ID=77857710
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2021104690A Active AU2021104690A4 (en) | 2021-07-29 | 2021-07-29 | Method for preparing section for rapidly observing microstructure of frozen aquatic products |
Country Status (1)
| Country | Link |
|---|---|
| AU (1) | AU2021104690A4 (en) |
-
2021
- 2021-07-29 AU AU2021104690A patent/AU2021104690A4/en active Active
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| FGI | Letters patent sealed or granted (innovation patent) |