A method for preparing a transmission electron microscope sample of laver filaments

Abstract

The present invention provides a method for preparing an algae filament transmission electron microscope sample, which comprises the following steps: fixing the laver filament sample with a fixing solution I, rinsing with a rinsing solution after the fixing treatment is completed, fixing the rinsed filament with a fixing solution II for secondary fixation after the rinsing treatment is completed, rinsing with a rinsing solution, dehydrating the laver filament after the secondary rinsing treatment with an organic solvent after the rinsing treatment is completed, embedding the dehydrated laver filament with an embedding agent, tissue penetration, slicing, and staining to obtain a filament transmission electron microscope sample. The present invention improves the traditional fixing method, and fixes once with glutaraldehyde and Tritan X-100, thereby promoting the fixing solution to penetrate the cell wall, and not damaging the internal structure of the plant cell. Potassium permanganate is used for secondary fixation, which solves the problems of unclear boundaries of the membrane structure of algae samples in the past.

Description

Technical Field

[0001] The invention belongs to the technical field of transmission electron microscope sample preparation, and in particular relates to a method for preparing a transmission electron microscope sample of laver filaments. Background Art

[0002] Porphyra sensu lato belongs to the class Protoflorideac of the phylum Rhodophyta and the family Bangiaceae. It is widely distributed in the intertidal zones from the frigid to the subtropical zone. It is an edible red algae with a long history of cultivation in Northeast Asia and significant commercial value. In 2021, the cultivated area of ​​​​Porphyra in my country was 680,000 hectares, with an output of nearly 200,000 tons, creating an output value of over 10 billion yuan, and is an important pillar of my country's seaweed farming industry. The production process of Porphyra includes the indoor cultivation stage of conifers and the cultivation stage of thalli in the sea area. Free conifers are an important form of preservation of Porphyra germplasm, and can also be used to directly collect shell conifer seedlings, which are important production materials.

[0003] Transmission Electron Microscopy (TEM) imaging is achieved by emitting a high-speed electron beam from an electron gun, converging through 1-2 level condensers, and then evenly irradiating the sample's observation area. The incident electrons collide and do not collide with the sample. Since the sample is very thin, most of the electrons penetrate the sample, and their intensity distribution corresponds to the morphology, organization, and structure of the sample area being observed. The electrons projected out of the sample are magnified by the three-level magnetic lens of the objective lens, intermediate lens, and projection lens, and projected onto the fluorescent screen for observing the image. The fluorescent screen converts the electron intensity distribution into a light intensity distribution visible to the human eye, and displays an image corresponding to the sample's morphology, organization, and structure on the fluorescent screen for experimental needs. The resolution of TEM is as high as It is an important tool for observing biological ultrastructures and is widely used to analyze the characteristics of cell ultrastructures. Preparing high-quality TEM samples is a prerequisite for accurately observing cell microstructures. The sample preparation process of TEM ultrathin sections generally includes sampling, fixation, immersion, dehydration, embedding, infiltration, polymerization, sectioning and staining. Differences in the properties of biological samples will cause uneven sample preparation results and affect the final observation results.

[0004] The cell membrane of the filamentous body of Porphyra is closely connected to the inner layer of the cell wall. A large number of chromatosomes are arranged close to the cell membrane. There are a large number of organelles such as mitochondria and endoplasmic reticulum between the chromatosomes. The various membrane structures of the filamentous body cells are stacked and squeezed, which brings challenges to the preparation and observation of cell ultrastructure. Although the commonly used aldehyde fixatives can terminate the biological activity of tissue cells and stabilize the organelles and various material components, the display effect on the cytoplasmic membrane is poor and cannot be stained. In addition, the salinity of seawater is generally between 28‰ and 35‰. The conventional fixation conditions and osmotic conditions of terrestrial plants are relatively low for marine algae, which is not conducive to maintaining the algae itself, especially the structural characteristics of the inner membrane system; resulting in different degrees of cavities, unclear chloroplast lamellar structures, and blurred organelle boundaries in the cells. Ultrastructure photos of such quality cannot truly reflect the state of the algae, resulting in unavailable data. Therefore, for algae materials such as Porphyra, the preparation scheme of general plants or animals cannot be directly adopted, and must be optimized. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing TEM samples of laver filaments, in view of the problems that the ultrastructure of cells is unclear, especially the boundaries of chloroplast lamellae and other organelles are blurred, and the sample preparation results are unstable and non-repeatable, etc., when preparing TEM samples of laver filaments at present. According to the characteristics of algae cells, the present invention optimizes the fixation method, fixative and embedding agent of laver filaments on the basis of the traditional sample preparation method, and adjusts the immersion, dehydration and embedding time of the sample, so that the ultrastructure of laver filaments can be presented completely and clearly, and the problems such as the large-scale degradation of chloroplasts and mitochondria in the previous sample preparation are solved, and the results are stable and highly repeatable.

[0006] The preparation method of the algae filament transmission electron microscope sample provided by the present invention comprises the following steps: fixing the laver filament sample with a fixing solution I, rinsing with a rinsing solution after the fixing treatment, fixing the rinsed filaments with a fixing solution II for a second time, rinsing with the rinsing solution, dehydrating the laver filaments after the second rinsing treatment with an organic solvent after the rinsing treatment, embedding the dehydrated laver filaments with an embedding agent, subjecting the embedded laver filaments to tissue penetration treatment, slicing, and staining treatment to obtain the filament transmission electron microscope sample.

[0007] The fixing solution I is a solution obtained by mixing glutaraldehyde (25% EM grade) with a mass concentration of 4%, Tritan X-100 with a mass concentration of 2% and sterilized seawater in a volume ratio of 16:2:82.

[0008] The sterilized seawater is sterilized seawater with a salinity of 22‰.

[0009] The rinsing solution is 0.1 mol / L PBS solution;

[0010] The fixing solution II is a potassium permanganate solution with a mass concentration of 1.5%;

[0011] The dehydration treatment is to first dehydrate with ethanol step by step, and then dehydrate with acetone;

[0012] Furthermore, the dehydration treatment is to first treat the filaments after the secondary rinsing treatment with 30% alcohol, then treat them with 50% alcohol, and then treat them with 70% and 90% alcohol, thereby completing the step-by-step dehydration treatment of the samples with different concentrations of alcohol;

[0013] The filaments that have been dehydrated step by step with different concentrations of alcohol are first treated with 90% acetone and then with anhydrous acetone to complete the step by step dehydration of the samples with different concentrations of acetone;

[0014] The embedding medium is a plant cell embedding medium (Spurr embedding medium);

[0015] The step-by-step tissue infiltration process comprises the following steps:

[0016] 1) Add Spurr embedding agent to the acetone dehydrated samples for infiltration treatment, and the mass ratio of Spurr embedding agent to acetone in the embedding system is 1:3;

[0017] 2) adding Spurr embedding agent to the embedding system of 1) and treating the system again, so that the mass ratio of Spurr embedding agent to acetone in the embedding system is 2:3;

[0018] 3) adding Spurr embedding agent to the embedding system of 2) and treating the system so that the mass ratio of Spurr embedding agent to acetone in the system is 1:1;

[0019] 4) Carefully aspirate all the liquid in the system, add Spurr embedding medium, evacuate the system and leave to complete the infiltration.

[0020] The staining agents are uranyl acetate (TED PELLA) and lead citrate (Merck KGaA).

[0021] The present invention can be applied to the preparation of most algae TEM samples, such as Porphyra thallus, Enteromorpha, Chlorella, etc.

[0022] In the prior art, the preparation of marine algae electron microscope samples is relatively rare and immature. Since the cell wall of marine algae is relatively thick, it will hinder the penetration of chemicals such as fixative, rinsing solution or embedding agent. In addition, the algae sample itself is relatively sensitive to osmotic pressure, which brings great difficulties to sample preparation. Compared with the prior art, the present invention has the following technical effects:

[0023] (1) The present invention provides a method for preparing TEM samples of laver filaments, which can clearly and completely present the ultrastructure of algae, and the operation is standardized, making the method stable and repeatable.

[0024] (2) The present invention improves the traditional fixation method. Glutaraldehyde and Tritan X-100 are used together without damaging the cell structure; thereby promoting the fixative to penetrate the cell wall without damaging the plant cell structure. This method can quickly fix the sample and solves the problem of insufficient fixation of algae samples in the past.

[0025] (3) The present invention improves the traditional secondary fixation method. Analytical pure potassium permanganate powder is used to prepare a 1.5% potassium permanganate solution, which is filtered twice through a 0.45 μm filter membrane before use. Potassium permanganate can effectively deposit on the surface of the biomass membrane, forming a good contrast, which is conducive to the observation of the membrane structure of organelles such as chloroplasts and mitochondria in the later stage.

[0026] (4) The present invention uses Spurr low viscosity epoxy resin embedding agent (Spurr embedding agent) to replace the Epon 812 epoxy resin embedding agent in the conventional method. Spurr embedding agent has low viscosity and is easy to penetrate, and is more suitable for embedding algae cells.

[0027] (5) The present invention optimizes the processing time of each step according to the characteristics of algae. The use of a rotary oscillator in some processes is conducive to giving full play to the role of each step, increasing the effectiveness of sample preparation, and providing a basis for the clear presentation of samples.

[0028] (6) The method of the present invention is stable and repeatable in preparing samples of algae filaments. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Comparison of the results of TEM of Porphyra filaments prepared by different methods, where a, c: TEM results of Porphyra filaments prepared by conventional methods; b, d: TEM results of Porphyra filaments prepared by improved methods. Note: C: chromatin; P: pyrenoid; SW: cell wall; Pm: cell membrane. DETAILED DESCRIPTION

[0030] The existing steps for preparing the transmission electron microscopy sample of Porphyra filaments are as follows:

[0031] 1) Take a 1.5 ml sterile centrifuge tube and add the treated laver filaments. Rinse with 3% glutaraldehyde for 3 times.

[0032] 2) After completion, add 4% glutaraldehyde to immerse the free filaments of Porphyra and fix them at 4°C in the dark for 24 hours.

[0033] 3) Wash with 1M PBS three times, 15 min each time, and fix with 1% osmium acid in a dark place in a refrigerator for 1 h to 2 h.

[0034] 4) After completion, wash three times with 0.1 mol / L PBS.

[0035] 5) Dehydration: 30% alcohol (15 min) → 50% alcohol (15 min) → 70% alcohol (15 min) → 90% alcohol (15 min) → 90% acetone (freshly prepared for use, 15 min) → anhydrous acetone (15 min), repeat 4 times → aspirate the acetone for the fourth time, leaving one drop for the last time.

[0036] 6) Infiltration: add 1-2 drops of embedding agent, embedding agent: acetone = 1:1, cover the lid and mix well; add another 1-2 drops of embedding agent, embedding agent: acetone = 2:1, cover the lid, suck out the old embedding agent, add pure embedding agent, and leave the lid open overnight.

[0037] 7) After the infiltration is completed, polymerize at above 37℃ (12h) and 60℃ (36h).

[0038] 8) Ultra-thin sections were double-stained with uranyl acetate and lead citrate and observed on a microscope (Hitachi H-7650).

[0039] The cell wall and cell membrane structures of the samples prepared by conventional methods were blurred, the intracellular chromatin membrane layer structure was unclear, and the protein nucleus and other organelle structures were diffuse.

[0040] The present invention aims at the technical deficiencies in the prior art, such as unclear chloroplast lamella structure, high failure rate, difficulty in clearly presenting the ultrastructure of cells, instability and non-repeatability of sample preparation results, etc., when preparing TEM samples of laver filaments, and provides a method for preparing transmission electron microscope samples of laver filaments. The present invention optimizes the fixation method of filaments, the selection of fixative and embedding agent based on the traditional sample preparation method through multiple experimental tests, combined with practical experience, and according to the characteristics of algae, improves the time of immersion, dehydration and embedding, increases the skills in some sample preparation processes, and can make the ultrastructure of laver filaments completely and clearly present, with stable results and strong repeatability, and solves the technical problems such as large-scale degradation of cytoplasmic membrane in previous sample preparation.

[0041] Example 1: Preparation of Porphyra filamentous body transmission electron microscopy samples

[0042] Select the filaments required for the experiment → fix the samples with fixative I → rinse the samples with rinse solution → fix the filaments for a second time with fixative II → rinse the filaments for a second time with rinse solution → dehydrate the filaments with an organic solvent → infiltrate the filaments with an embedding agent → embed and polymerize the filaments → slice → stain → obtain the filament TEM sample → observe and inspect on the machine.

[0043] In the above technical solution, the preparation method specifically comprises the following steps:

[0044] (1) Sampling:

[0045] Select healthy filaments for the experiment and check the state of the algae under a light microscope to ensure that the sample structure is complete, well-developed, healthy in color, and the volume of the algae ball is about 1mm 3 ;

[0046] (2) Fixation:

[0047] Transfer the filaments selected in step (1) to a corner of a glass slide, carefully absorb excess culture medium with filter paper, rinse the algae with fixative I into a centrifuge tube containing 1 mL of fixative I, carefully shake by hand, and vacuum filter for 30 minutes to allow the fixative I to fully penetrate the sample. Then transfer the sample to a 4°C refrigerator to avoid light and fix for 24 hours, then replace with fresh fixative I to ensure that the fixative is in full contact with the sample;

[0048] (3) Rinse:

[0049] The sample fixed in step (2) is first rinsed with a rinse solution for 3 times, and then rinsed with pure water once, for a total of 4 times. During each rinse, the sample is placed in the rinse solution and rinsed by shaking in a rotary mixer. Before each immersion, the rinse solution of the previous step should be dried as much as possible. The shaking rinsing is performed at room temperature;

[0050] (4) Secondary fixation:

[0051] Aspirate the rinse solution from the sample rinsed in step (3), add 20 μL of fixative II, and then transfer the sample to a 4°C refrigerator for secondary fixation; the secondary fixation must be protected from light and the processing time is 1 hour;

[0052] (5) Secondary rinsing:

[0053] The fixative II in the sample obtained after the secondary fixation in step (4) is sucked dry, and the sample is immediately rinsed twice with a rinse solution, and then rinsed once with pure water; before each rinsing, the residual liquid in the previous step must be sucked dry, and then the rinse solution is added, and the sample is shaken and rinsed in a rotary mixer;

[0054] (6) Dehydration:

[0055] The rinse liquid in the sample obtained after the secondary rinsing treatment in step (5) is sucked dry, and the sample is first dehydrated step by step using alcohols of different concentrations from low to high, and then the sample is further dehydrated using acetone to replace the free water in the sample;

[0056] (7) Penetration:

[0057] The sample obtained after dehydration treatment in step (6) is subjected to gradual tissue penetration treatment using embedding agents of different concentrations;

[0058] (8) Embedding:

[0059] The sample obtained after infiltration in step (7) is embedded in a centrifuge tube with a pure embedding agent;

[0060] (9) Polymerization:

[0061] The sample obtained after embedding in step (8) was polymerized at 70° C. for 24 h;

[0062] (10) Trimming and slicing:

[0063] The sample obtained after the polymerization treatment in step (9) is taken out from the centrifuge tube and continuously ultra-thin sliced ​​on a microtome. The ultra-thin slice thickness is 70 nm, and a 150-mesh copper mesh of aromatic film is used for scooping the slices.

[0064] (10) Dyeing treatment:

[0065] The ultra-thin sections obtained after sectioning in step (9) are stained with a dye. After staining, the obtained samples are observed on a machine to check the sample preparation effect.

[0066] In the above technical solution, in step (2), the fixing solution I is a solution obtained by mixing glutaraldehyde (Glutaraldehyde 25% EM grade) with a mass concentration of 4%, Tritan X-100 with a mass concentration of 2% and sterilized seawater with a salinity of 28‰, and the volume ratio of the three is 16:2:82.

[0067] In the above technical scheme, in step (3), the rinsing liquid is a 0.1 mol / L PB solution; each time the sample is rinsed, the sample is placed in a rotary oscillator, 800 μL of sterilized seawater or rinsing liquid is added to the centrifuge tube and then the sample is shaken and rinsed, and the time for each shaking and rinsing is 15 minutes; when the rotary oscillator is used for shaking and rinsing, the speed is 40 r / min.

[0068] In the above technical solution, in step (4), the stationary liquid II is potassium permanganate with a mass concentration of 1.5% (filtered with a 0.45 μm filter membrane before use).

[0069] In the above technical solution, in step (5), the rinsing liquid is a 0.1 mol / L PB solution. During each rinsing, the sample is placed in a rotary mixer, and 800 μL of rinsing liquid is added to the centrifuge tube for rinsing. The time for each shaking and rinsing is 15 minutes; when the rotary mixer is used for shaking and rinsing, the speed is 40 r / min.

[0070] In the above technical solution, in step (6), the dehydration treatment comprises the following steps:

[0071] ① The rinse liquid in the sample obtained after the secondary rinsing treatment in step (5) is sucked dry, and 800 μL of 30% alcohol is added to a centrifuge tube, and the mixture is treated on a rotary mixer at room temperature for 15 minutes, and then the mixture is sucked dry; then 800 μL of 50% alcohol is added to the centrifuge tube, and the mixture is treated on a shaker at room temperature for 15 minutes, and then the mixture is sucked dry; then 800 μL of 70% alcohol is added to the centrifuge tube, and the mixture is treated on a rotary mixer at room temperature for 15 minutes, and then the mixture is sucked dry; finally, 800 μL of 90% alcohol is added to the centrifuge tube, and the mixture is treated on a shaker at room temperature for 15 minutes, and then the liquid is sucked off, leaving the last drop, thereby completing the stepwise dehydration treatment of the sample with different concentrations of alcohol;

[0072] ② After being dehydrated step by step with different concentrations of alcohol, transfer the filaments to a 1 mL centrifuge tube and continue to dehydrate with acetone (do not damage the sample during transfer): add 400 μL of 90% acetone to the centrifuge tube, treat it on a shaker at room temperature for 15 minutes, and then absorb it dry; then add 400 μL of anhydrous acetone to the centrifuge tube, treat it on a shaker at room temperature for 10 minutes, and then absorb it dry; repeat the anhydrous acetone dehydration step three times, and finally add 30 μL of anhydrous acetone to complete the step-by-step dehydration of the sample with acetone of different concentrations;

[0073] In this step, alcohol and acetone of different concentrations are prepared freshly.

[0074] In the above technical solution, in step (7), the stepwise tissue infiltration treatment comprises the following steps:

[0075] ① Add 10 μL of Spurr embedding agent to the sample to which 30 μL of anhydrous acetone was added in step (6) so that the mass ratio of Spurr embedding agent to acetone in the system is 1:3, cover the lid, and perform the infiltration treatment for 1 hour;

[0076] ② Add 10 μL of Spurr embedding agent to the system in step ①, so that the mass ratio of Spurr embedding agent to acetone in the system is 2:3, cover the lid, and perform infiltration treatment for 1 hour;

[0077] ③ Add 10 μL of Spurr embedding agent to the system in step ②, so that the mass ratio of Spurr embedding agent to acetone in the system is 1:1, cover the lid, and perform infiltration treatment for 1 hour;

[0078] ④ Carefully aspirate all the liquid in step ③, add 100 μL Spurr embedding medium, evacuate for 30 minutes, and leave the lid open overnight to complete the infiltration.

[0079] In the above technical solution, in step (10), the dyes are uranyl acetate (TED PELLA) and lead citrate (Merck KGaA), and the uranyl acetate staining is first performed and then the lead citrate staining is performed according to the traditional two-color staining method.

[0080] In the above technical solution, the rotary oscillator is continuous and slow to ensure sufficient treatment.

[0081] The cell wall of the algae filaments treated by this method is clear. Under a high-power electron microscope, the cell membrane is a three-layer structure of two dark and one bright. The middle layer has a low electron density and is a transparent layer, while the inner and outer layers are high electron density layers. The double-layer membrane of the intracellular chromatin body is clearly visible, the lamella system is complete, and the protein nucleus structure is intact ( Figure 1 ). This overcomes the technical problems of the existing methods in which the sample cell wall and cell membrane structure are blurred, the intracellular chromatin membrane layer structure is unclear, and the protein nucleus and other organelle structures are diffuse.

Claims

1. A method for preparing a transmission electron microscope sample of laver filaments, characterized in that: The method comprises the following steps: fixing the laver filament sample with a fixing solution I, rinsing with a rinsing solution after the fixing treatment, fixing the rinsed filaments with a fixing solution II for a second time after the rinsing treatment, rinsing with the rinsing solution, dehydrating the capsules after the second rinsing treatment with an organic solvent after the rinsing treatment, embedding the dehydrated capsules with an embedding agent, and performing tissue penetration treatment, sectioning, and staining on the embedded capsules to obtain a filament transmission electron microscope sample; The fixing solution I is prepared by mixing 4% glutaraldehyde, 2% Tritan X-100 and sterile seawater, wherein the volume ratio of 4% glutaraldehyde, 2% Tritan X-100 and sterile seawater is 16:2:82; The rinsing solution is 0.1 mol / L PBS solution; The stationary solution II is a potassium permanganate solution with a mass concentration of 1.5%, and the 1.5% potassium permanganate solution is prepared by using analytically pure potassium permanganate powder and filtering it twice through a 0.45 μm filter membrane; The sterilized seawater is sterilized seawater with a salinity of 22‰; The rinsing solution is 0.1 mol / L PBS solution; The embedding medium is Spurr embedding medium.

2. The preparation method according to claim 1, characterized in that The dehydration treatment is to first use ethanol for step-by-step dehydration and then use acetone for dehydration.

3. The preparation method according to claim 2, characterized in that: The dehydration treatment is to first treat the filamentous cysts after the secondary rinsing treatment with 30% alcohol, then treat them with 50% alcohol, and then treat them with 70% and 90% alcohol to complete the step-by-step dehydration treatment of the sample with alcohol of different concentrations; then the filaments after the step-by-step dehydration treatment with alcohol of different concentrations are first treated with 90% acetone, and then treated with anhydrous acetone to complete the step-by-step dehydration of the sample with acetone of different concentrations.

4. The preparation method according to claim 1, characterized in that: The tissue infiltration treatment comprises the following steps: 1) Add Spurr embedding agent to the acetone dehydrated samples for infiltration treatment, and the mass ratio of Spurr embedding agent to acetone in the embedding system is 1:3; 2) adding Spurr embedding agent to the embedding system of 1) and treating the system again, so that the mass ratio of Spurr embedding agent to acetone in the embedding system is 2:3; 3) adding Spurr embedding agent to the embedding system of 2) and treating the system so that the mass ratio of Spurr embedding agent to acetone in the system is 1:1; 4) Carefully aspirate all the liquid in the system, add Spurr embedding medium, evacuate the system and leave to complete the infiltration.

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