A method for processing a moss plant for cryosectioning

Abstract

The present application belongs to the technical field of section, and particularly relates to a moss freezing section processing method, which comprises the following steps: pretreatment: selecting a well-developed moss and placing it in clean water for soaking, peeling off the leaves of the moss and cutting the stems into segments; fixation treatment: placing the cut stem segments in FAA fixing solution; section processing: taking the stem segments out of the FAA fixing solution, fixing them on the cold table of a section machine and freezing them into ice blocks, and using a cold knife to trim the ice blocks until the surface of the ice blocks is flat and the stem segments are exposed; section processing: sectioning the stem segments, placing the sections on a glass slide and covering them with a cover glass; staining treatment: adding a staining solution to the sections for staining, and washing off the floating color after staining; and making a mounted section. The present method can maximize the integrity and clarity of the moss sections, the sectioning time is short, and the present method lays a foundation for the research of mosses.

Description

Technical Field

[0001] This invention belongs to the field of slicing technology and relates to a slicing method, specifically a method for processing frozen slices of bryophytes. Background Technology

[0002] Bryophytes are widely distributed and highly adaptable, capable of surviving in various environments. They are small plants with simple stem cross-sectional structures. Although some scholars have studied the cross-sectional structure of bryophyte stems, related reports are limited. The cross-sectional structures of stems differ among different bryophytes and can serve as one of the classification characteristics at the family, genus, and species levels. Therefore, a systematic study of the cross-sectional structure of bryophyte stems is both necessary and of great significance.

[0003] Cryosectioning is an experimental technique that involves rapidly freezing biological tissues at low temperatures until they reach a certain hardness before sectioning. While the process is quick, simple, and easy to perform, the freezing requirements make this technique unsuitable for all plant tissue materials, such as bryophytes. The main reason existing cryosectioning techniques are unsuitable for bryophytes is that bryophyte cells have cell walls and vacuoles, containing a large amount of water. During rapid freezing, this water easily forms ice crystals, leading to structural damage such as cell rupture, making the sections brittle. Furthermore, the water content varies significantly between different parts of a complete bryophyte, making it difficult to obtain relatively intact sections. Therefore, the pre-sectioning treatment of bryophyte tissues is crucial. Applying cryosectioning to bryophytes would lay the foundation for future bryophyte research. On the other hand, most plants are relatively large, and when fixed on a cold plate, only a section of the plant is selected, and only one section is presented in the slice. However, the stems of bryophytes are thin and soft, and the diameter is small, making it difficult to ensure that the section is suitable for observation. Therefore, multiple sections need to be prepared into slides, and then slides with complete structures need to be selected from them, which increases the difficulty of the work and results in low usability of the sections. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for processing frozen sections of bryophytes, which enables the obtaining of complete stem cross-sections of bryophytes through frozen sectioning. The method is simple and easy to operate, and multiple cross-sections can be obtained from the same section, effectively improving the utilization rate of the sections.

[0005] The specific technical solution adopted in this invention is as follows:

[0006] A method for processing frozen sections of mosses, the key of which includes the following steps:

[0007] S1. Pretreatment: Select well-developed bryophytes and soak them in clean water. After soaking, remove the leaves of the bryophytes and cut the stems of the bryophytes into stem segments.

[0008] S2. Fixation treatment: Prepare FAA fixative, stir evenly, and place the cut stem segments in FAA fixative for 24-48 hours;

[0009] S3. Trimming: Remove the stem segments from the FAA fixative, fix multiple stem segments at intervals on the cold stage of the microtome using a freezing tank and freeze them into frozen samples. Fix the cold stage with the frozen samples on the cold stage fixing slot and trim the frozen samples with a cold knife until the surface of the frozen samples is flat and the stem segments are exposed.

[0010] S4. Sectioning: Slice the stem segment, place the slice on a glass slide, and cover with a coverslip;

[0011] S5. Staining treatment: Add staining solution to the section for staining, and wash off the excess stain after staining for 1-3 minutes;

[0012] S6. Prepare temporary aqueous slides from the stained sections. The processing of frozen sections of bryophytes is now complete.

[0013] In step S1, the soaking time is 15-30 minutes, and the stem segment length is 0.8-1.2 cm.

[0014] In step S2, the stem segments are placed in FAA fixative for 24-48 hours and stored at -4°C.

[0015] In step S3, the temperature of the cold table is -20°C, the temperature of the cold blade is -15°C, and the freezing time of the stem segment on the cold table is 20 minutes.

[0016] In step S4, the slice thickness is 7-18 μm.

[0017] In step 5, safranin dye is used as the staining solution. Safranin dye is added to the slice for staining. After staining for 2 minutes, the excess dye is washed away with anhydrous ethanol.

[0018] The freezing tank includes an annular paraffin wall and a set of spaced freezing water tanks enclosed by the paraffin wall. In step S3, the process of making the frozen sample is as follows: the stem segment is placed in the freezing water tank and water is injected into the freezing water tank. After freezing, a frozen sample with paraffin coating is formed.

[0019] The slicer is equipped with an atomizing nozzle. The process of preparing the frozen sample is as follows: a water-permeable membrane is laid in the inner cavity of the freezing tank, with one end of the membrane positioned on the cooling stage. The atomizing nozzle sprays water mist onto the membrane. Then, stem segments are placed on the membrane, and the atomizing nozzle continues to spray water mist onto the segments. The membrane and other stem segments are alternately laid on top of the stem segments, with the atomizing nozzle spraying water mist onto both the membrane and each stem segment simultaneously. After laying, water is injected into the freezing tank, and the frozen sample is formed after freezing.

[0020] The freezing tank includes an ice column and a thin film fixed to one end of the ice column. The thin film forms a spiral cavity along the ice column. The process of making the frozen sample is as follows: N layers of stem segments are arranged along the ice column, N≥1, and the stem segments are arranged with gaps between them. The thin film wraps each layer of stem segments in sequence, and a cavity is formed between the tail end of the thin film and the wrapped stem segments. Water is injected into the cavity and the gaps between the stem segments, and frozen samples are formed after freezing.

[0021] The beneficial effects of this invention are:

[0022] This invention applies the frozen sectioning method to the preparation of bryophyte sections, effectively avoiding structural damage such as cell rupture caused by water crystal formation in bryophytes. The sections are less prone to breakage during slicing, maximizing the integrity and clarity of the bryophyte sections. This invention allows for simultaneous continuous slicing of multiple parts, providing microscopic structures of cross-sections of various layers of the bryophyte stem. A single cutter operation yields multiple cross-sections within the same section, which can be assembled into a single slide. Using a microscope, operators can select complete cross-sections and make more intuitive comparisons, significantly improving the usability of a single section and effectively reducing the number of slide preparation and selection steps, thus significantly improving work efficiency.

[0023] After fixation, no other pretreatment is required for cryosectioning, and no embedding agent is used. There is no residual contamination after slicing, no water washing is required, the steps are simple and convenient, and the preparation time is short, laying the foundation for the research of bryophytes. Attached Figure Description

[0024] Figure 1 A cross-sectional view of *Phyllostachys edulis* obtained using this invention;

[0025] Figure 2 This is a schematic diagram of the structure of the freezing tank in Example 1;

[0026] Figure 3 This is a schematic diagram of the structure of the freezing tank in Example 2;

[0027] Figure 4 This is a schematic diagram of the structure of the freezing tank in Example 3;

[0028] In the attached diagram, 1 is the cold platform, 2 is the freezing tank, 3 is the paraffin wall, 4 is the freezing water tank, 5 is the stem segment, 6 is the icicle, 7 is the water-permeable membrane, 8 is the plastic film, and 9 is the cavity. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments:

[0030] Example 1, as Figure 1-2 As shown, *Phyllostachys edulis* was selected as the test subject in this embodiment. A method for processing frozen sections of mosses includes the following steps:

[0031] S1. Select well-developed *Pterocarya stenoptera* and soak it in clean water for 20 minutes until it is completely moistened and plump. Remove the *Pterocarya stenoptera* and place it under a stereomicroscope to peel off its leaves. Keep the stem of the *Pterocarya stenoptera* and cut the stem into 1cm segments with a blade.

[0032] S2. Fixation: In a clean bench, measure 450ml of 50% alcohol, 25ml of 38% formaldehyde, and 25ml of glacial acetic acid into beakers, and stir thoroughly with a glass rod to prepare FAA fixative. Place the prepared fixative in a wide-mouth bottle. Place the cut stem segments 5 into the FAA fixative and store the fixative at -4℃ for 24 hours. This fixation effectively prevents damage to the bryophyte samples due to temperature differences during slide preparation on a cold stage 1 at even lower temperatures, thus maintaining sample integrity.

[0033] S3. Slice Preparation: Set the temperature of the microtome's cooling stage 1 to -20℃ and the cold blade temperature to -15℃. After the temperature drops to the set temperature, remove the stem segment 5 from the FAA fixative, fix it on the cooling stage 1, and freeze it into a frozen sample. The cooling stage 1 is equipped with a freezing tank 2, which includes an annular paraffin wall 3 and a set of spaced-apart freezing water tanks 4 formed by the paraffin wall 3. The preparation process of the freezing tank 2 is as follows: Using a set of molds with different inner cavity sizes, the largest mold is fixed on the cooling stage 1 as an outer ring, and the remaining smaller molds are placed inside the outer ring and fixed to the cooling stage 1 as inner rings. The inner ring and the outer ring are spaced apart, and adjacent inner rings are spaced apart. Paraffin wax is poured between the inner ring and the outer ring. After the paraffin wax solidifies, the mold is removed, forming a paraffin wall 3 with freezing water tanks 4. The specific process for preparing the frozen sample is as follows: the stem segment 5 is placed into the freezing water tank 4 formed by the inner ring of the paraffin, and water is injected into the freezing water tank 4. The stem segment 5 is suspended in the water and frozen to obtain a good condition; the cold stage 1 freezes the water in the freezing water tank 4 to fix the stem segment 5 and form a frozen sample with paraffin coating; after freezing and fixing for 20 minutes, the cold stage 1 with the frozen sample is fixed on the cold stage fixing groove, and the frozen sample is trimmed with a cold knife until the surface of the frozen sample is flat and the stem segment 5 is exposed.

[0034] S4. Sectioning: Section the stem segment 5 into several 10μm thick sections, ensuring that there are 5 complete sections; place the complete sections on a glass slide and cover with a coverslip;

[0035] S5. Staining treatment: Add safranin stain to the section, stain for 2 minutes, and then wash away the excess stain with anhydrous ethanol.

[0036] S6. Prepare temporary aqueous slides from the stained sections, and photograph the complete slides under a microscope for record-keeping.

[0037] Example 2, as Figure 3As shown, this embodiment is basically the same as embodiment 1, except that: the slicer is equipped with an atomizing nozzle, and the inner cavity of the freezing tank is rectangular. The process of preparing the frozen sample is as follows: the end of the freezing tank 2 is fixed to the cold stage 1, which can be placed horizontally or vertically (in this embodiment, the cold stage 1 is placed vertically). The water-permeable membrane 7 is laid in the inner cavity of the freezing tank 2 and flattened. One end of the water-permeable membrane 7 is located on the cold stage 1 and in contact with the cold stage 1. The atomizing nozzle sprays water mist onto the water-permeable membrane 7, and the water-permeable membrane 7 is quickly frozen and fixed with the help of the cold stage 1. Then, the stem segment 5 is placed on the water-permeable membrane 7, and the atomizing nozzle continues to spray water onto the stem segment 5. The water mist around the permeable membrane 7 and stem segments 5 quickly condenses, fixing the stem segments 5 onto the permeable membrane 7. The permeable membrane 7 and other stem segments 5 are alternately laid on top of the fixed stem segments 5. Simultaneously, atomizing nozzles spray water mist onto the permeable membrane 7 and each stem segment 5. The water mist evenly penetrates the permeable membrane 7, freezing and fixing the water mist around the permeable membrane 7 and stem segments 5. At the same time, the frozen permeable membrane 7 provides support for the subsequently placed stem segments 5, using less water and achieving a faster freezing speed. In this embodiment, six stem segments 5 are used. After laying, the cooling table 1 is rotated so that the opening of the freezing tank 2 faces upwards, and clean water is injected into the freezing tank 2. The cooling table 1 freezes and fixes the clean water, stem segments 5, and permeable membrane 7 in the freezing tank 2, forming a frozen sample with the stem segments 5 arranged in layers. The permeable membrane 7 is a plastic film with mesh openings. Water mist passes through the permeable membrane 7, freezing and fixing the stem segments 5 placed on its upper and lower sides. The stem segment 5 is placed on a water-permeable film 7 and frozen without direct contact with the cold table 1, so that it is less likely to break during slicing.

[0038] Example 3, as Figure 4As shown, this embodiment is basically the same as Embodiment 1, except that the freezing tank includes an ice column 6 and a plastic film 8 fixed to one end of the ice column 6. The plastic film 8 forms a spiral cavity along the ice column 6. The process of making the frozen sample is as follows: N layers of stem segments 5 (N≥1) are set along the ice column 6; a gap may be set between the lower end of the stem segment 5 and the cold table 1, and the layers of stem segments 5 are set with gaps. The plastic film 8 wraps each stem segment 5 in sequence, and a cavity 9 is formed between the tail end of the plastic film 8 and the wrapped stem segment 5. The lower end of the plastic film 8 is in contact with the cold table 1; water is injected into the cavity 9 and the gap between the stem segments 5, and frozen to form a frozen sample. In this embodiment, N=2, that is, two layers of stem segments 5 are set along the ice column 6, each layer has multiple stem segments 5, and adjacent stem segments 5 are set with gaps. The front end of the plastic film 8 is frozen and fixed to the ice column 6. The specific operation is as follows: The first layer of stem segments 5 is placed against the ice pillars 6. For each stem segment 5 placed, the end of the plastic film 8 is pulled to wrap it around the ice pillar 6. After the first layer of stem segments 5 is placed, the second layer of stem segments 5 is placed against the plastic film 8 already wrapped around the first layer, with the end of the plastic film 8 continuing to wrap the second layer of stem segments 5. Then, the plastic film 8 continues to wrap, forming a cavity 9 outside the second layer of stem segments. Water is injected into the cavity 9 and the gaps between each stem segment 5, freezing all stem segments 5 within the ice block. The gaps between each stem segment and between the two layers of stem segments are small, the water volume is small, and freezing is done in sections. Simultaneously, the ice pillars 6 cool the water and stem segments for freezing, greatly improving the freezing speed. The freezing and fixing time after injecting water can be shortened by 3-8 minutes compared to Example 1.

[0039] Examples 1-3 all implemented the setting of having two or more stem segments 5 in the same frozen sample. The stem segments 5 were selected from the same part of multiple bryophyte samples. A single slice can obtain the stem cross-section of multiple bryophyte samples at the same layer, which is convenient for observation and comparison. Moreover, the frozen stem segments 5 can maintain a good condition, and the probability of the slice containing a complete cross-section structure is significantly improved. The preparation of the slides is effectively reduced, the process is simple, and the utilization rate of the slides is high.

[0040] In Examples 2 and 3, ice pillars 6 and permeable membranes 7 were used as supports, which conveniently, quickly and effectively ensured that the small and soft stem segments 5 of bryophytes remained relatively vertical when making frozen samples, reducing the degree of bending and helping to obtain complete and effective stem cross sections. This effectively reduced the number of slices and the number of slides, and significantly reduced the workload of slicing and subsequent steps.

[0041] In the slice provided in Example 3, the cross-sections of stem segment 5 are arranged in a regular, layered, ring-like pattern using a thin film. This facilitates easy marking and identification of each cross-section during microscopic observation, avoiding repeated selection of different cross-sections, making the process clear and less prone to confusion.

[0042] The stem cross-sections of bryophytes obtained by this method have high integrity and clarity. It eliminates the need for frequent and repeated steps such as material selection, processing, freezing, slicing, and mounting to select usable section structures, greatly shortening the experimental time, ensuring the efficiency of section preparation, and improving the clarity of the section microstructure plates.

Claims

1. A method for processing frozen sections of bryophytes, characterized in that, Includes the following steps: S1. Pretreatment: Select well-developed bryophytes and soak them in clean water. After soaking, remove the leaves of the bryophytes and cut the stems of the bryophytes into stem segments. S2. Fixation treatment: Prepare FAA fixative, stir evenly, and place the cut stem segments in FAA fixative for 24-48 hours; S3. Trimming: Remove the stem segments from the FAA fixative, fix multiple stem segments at intervals on the cold stage of the microtome using a freezing tank and freeze them into frozen samples. Fix the cold stage with the frozen samples on the cold stage fixing slot and trim the frozen samples with a cold knife until the surface of the frozen samples is flat and the stem segments are exposed. S4. Sectioning: Slice the stem segment, place the slice on a glass slide, and cover with a coverslip. S5. Staining treatment: Add staining solution to the section for staining, and wash off the excess stain after staining for 1-3 minutes; S6. Prepare temporary aqueous slides from the stained sections. The processing of frozen sections of bryophytes is now complete. The freezing tank includes an annular paraffin wall and a set of spaced-apart freezing water tanks enclosed by the paraffin wall, or the freezing tank includes ice columns and a plastic film fixed to one end of the ice columns. When the freezing tank includes an annular paraffin wall and a set of spaced-apart freezing water tanks enclosed by the paraffin wall, the frozen sample preparation process in step S3 is as follows: stem segments are placed in the freezing water tanks, and water is injected into the freezing water tanks. After freezing, a frozen sample with a paraffin coating is formed. Alternatively, if the slicer is equipped with an atomizing nozzle, the frozen sample preparation process is as follows: a water-permeable membrane is laid in the inner cavity of the freezing tank, with one end of the water-permeable membrane in contact with the cold stage. Atomizing nozzles spray water mist onto a water-permeable membrane, which is then frozen and fixed using a cooling platform. Stem segments are then placed on the membrane, and the atomizing nozzles continue to spray water mist onto the stem segments. The water mist condenses on the membrane and around the stem segments, fixing them in place. Water-permeable membrane and other stem segments are alternately laid over the fixed stem segments, with atomizing nozzles spraying water mist onto both the membrane and each stem segment simultaneously. After laying, water is injected into the freezing tank, and the samples are frozen to form a frozen sample. When the freezing tank includes an ice column and a plastic film fixed to one end of the ice column, the plastic film forms a spiral cavity along the ice column, and the lower end of the plastic film contacts the cold stage. The process of making the frozen sample is as follows: two layers of stem segments are set along the ice column, each layer has multiple stem segments, and there is a gap between the lower end of the stem segment and the cold stage. Adjacent stem segments are spaced apart. The front end of the plastic film is frozen and fixed to the ice column. The first layer of stem segments is set against the ice column. Each time a stem segment is placed, the tail end of the plastic film is pulled to wrap it around the ice column. After the first layer of stem segments is placed, the second layer of stem segments is placed against the plastic film that has wrapped the first layer of stem segments. The tail end of the plastic film continues to wrap the second layer of stem segments. Then the plastic film continues to wrap, forming a cavity outside the second layer of stem segments. Water is injected into the cavity and the gaps between the stem segments to freeze and wrap all the stem segments in ice. Water is injected into the cavity and the gaps between the stem segments, and after freezing, a frozen sample is formed.

2. The method for processing frozen sections of bryophytes according to claim 1, characterized in that: In step S1, the soaking time is 15-30 minutes, and the stem segment length is 0.8-1.2 cm.

3. The method for processing frozen sections of bryophytes according to claim 1, characterized in that: In step S2, the stem segments are placed in FAA fixative for 24-48 hours and stored at -4°C.

4. The method for processing frozen sections of bryophytes according to claim 1, characterized in that: In step S3, the temperature of the cold table is -20°C, the temperature of the cold blade is -15°C, and the freezing time of the stem segment on the cold table is 20 minutes.

5. The method for processing frozen sections of bryophytes according to claim 1, characterized in that: In step S4, the slice thickness is 7-18 μm.

6. The method for processing frozen sections of bryophytes according to claim 1, characterized in that: In step S5, safranin dye is used as the staining solution. Safranin dye is added to the slice for staining. After staining for 2 minutes, the excess dye is washed away with anhydrous ethanol.

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