A method for preparing samples for transmission electron microscopy of samples requiring positioning based on a new embedding agent

Abstract

The application provides a sample preparation method for transmission electron microscopy of a sample to be positioned based on a novel embedding agent, and effectively improves the toughness of an embedding block, improves the quality of an ultrathin section of a sample, reduces the situation of section falling and section cracking of the ultrathin section, realizes the demand of semi-thin positioning section of the sample, guarantees the integrity of a target structure of the sample, effectively solves the problems of semi-thin positioning difficulty and non-ideal imaging effect of a sample such as a kidney tissue embedded by the novel embedding agent, and provides a solution for further application and popularization of the novel embedding agent 812R, and can be used for high-quality electron microscope sample preparation of human or mouse kidney tissue, so that ultramicrostructure observation and pathological diagnosis become possible.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical biological sample preparation, and relates to a method for preparing a transmission electron microscope sample based on a new embedding agent, in particular to a method for preparing a transmission electron microscope sample of a clinical kidney puncture sample. BACKGROUND

[0002] Transmission electron microscopy has been widely used to observe the ultrastructure of tissues and cells. In the preparation process of transmission electron microscope biological samples, the ultrathin section link is the premise of final structure observation and good results. However, due to the small area of random sections, it is easy to miss the effective target structure, causing waste of human, material and time costs. In order to accurately cut the target structure in the ultrathin section, a semi-thin section needs to be made for positioning before the ultrathin section.

[0003] Transmission electron microscopy (TEM) examination is crucial in kidney puncture pathological diagnosis, and semi-thin positioning of glomeruli is a key step, which is mainly reflected in the following aspects: 1. Improve detection efficiency and avoid missed detection. The number of glomeruli in kidney puncture samples is limited (usually only a few to a dozen), and the distribution is uneven. After semi-thin sectioning and staining, the tissue structure can be clearly observed under a light microscope, and the area containing glomeruli can be quickly positioned, avoiding missed detection caused by blind sectioning of TEM, and ensuring that ultrathin sectioning and electron microscope observation can be targeted at glomeruli. 2. Ensure the accuracy and relevance of observation. Glomeruli are the main lesion sites of kidney diseases (such as nephritis, nephrotic syndrome, etc.). By semi-thin positioning, typical glomeruli with lesions can be accurately selected for TEM analysis, avoiding missing key lesions due to random selection and improving the accuracy of diagnosis. 3. Save sample and experimental resources. Semi-thin positioning can clearly define the target area, reduce unnecessary ultrathin sectioning and electron microscope observation time, maximize the acquisition of effective information in limited samples, and save human, material and time costs. In summary, semi-thin positioning of glomeruli is the premise and basis of transmission electron microscopy diagnosis of kidney puncture samples, directly affecting the efficiency of subsequent TEM examination and the reliability of diagnostic results, and plays an irreplaceable role in the accurate diagnosis of kidney diseases.

[0004] SPI-PON 812 epoxy resin is one of the most widely used embedding resins for electron microscopy. With the discontinuation of SPI-PON 812 embedding agent (which can be referred to as 812), a substitute for the resin, SPI-PON 812R embedding agent (which can be referred to as 812R), has appeared. Although the manufacturer indicates that the section quality, staining effect and electron beam stability of SPI-PON 812R are not significantly different from those of SPI-PON 812, when preparing samples for samples such as kidney samples that require semi-thin section positioning, the embedding blocks prepared by embedding the samples with SPI-PON 812R based on the technical documents of the manufacturer are brittle and fragile, semi-thin sections are difficult to form, and structural positioning is not conducive. In addition, the efficiency of ultra-thin section fishing is low, the staining is prone to sheeting and cracking, and the imaging effect is poor. The kidney tissue samples embedded with the new embedding agent have problems such as difficulty in semi-thin positioning and unsatisfactory imaging effect, so it is necessary to develop a more reliable sample preparation method to improve the preparation effect of clinical kidney samples for semi-thin positioning and imaging observation and improve the accuracy of diagnosis. SUMMARY

[0005] The purpose of the present application is to solve the problems of brittle and fragile embedding blocks, difficulty in semi-thin positioning, and poor imaging effect in the preparation of transmission electron microscopy samples of kidney tissue and other samples that require positioning in the prior art. A transmission electron microscopy sample preparation method for samples that require positioning based on a new embedding agent is provided. The present application optimizes the stirring time of the 812R embedding agent mixture and combines the programmed temperature polymerization method to effectively improve the preparation effect of clinical kidney samples and solve the problems of difficulty in semi-thin positioning and poor imaging effect of kidney and other tissue samples embedded with the new 812R embedding agent for transmission electron microscopy.

[0006] The technical solutions adopted by the present application are as follows:

[0007] A transmission electron microscopy sample preparation method for samples that require positioning based on a new embedding agent, comprising:

[0008] Take the tissue to be prepared and put it into a fixed liquid pre-cooled in a 4℃ refrigerator and fix it overnight;

[0009] Rinse the sample, fix it with osmium acid, rinse it again, dye it, and dehydrate it;

[0010] Prepare an embedding mixture of the new embedding agent by mixing SPI-PON 812R 44.56 parts, NMA 17.83 parts, DDSA 35.65 parts and DMP-301.96 parts by volume; stir the obtained embedding mixture;

[0011] Add the obtained embedding mixture and acetone to the sample, and after embedding, perform programmed temperature polymerization;

[0012] The obtained embedding block is sliced to obtain a transmission electron microscope sample.

[0013] Further, the rinsing, osmium acid fixing and rinsing again are specifically: after rinsing with PBS, fixing at room temperature with osmium acid, discarding the osmium acid, rinsing with distilled water, and then staining at room temperature with uranyl acetate.

[0014] Further, the dehydration is specifically: sequentially performing 30% ethanol dehydration at room temperature for 15 minutes, 50% ethanol dehydration at room temperature for 15 minutes, 70% ethanol dehydration at room temperature for 15 minutes, 90% ethanol dehydration at room temperature for 15 minutes, 100% ethanol dehydration at room temperature for 20 minutes, and 100% acetone dehydration at room temperature for 20 minutes each time.

[0015] Further, it is characterized in that the SPI-PON 812R is 44.56 parts, the NMA is 17.83 parts, the DDSA is 35.65 parts, and the DMP-30 is 1.96 parts.

[0016] Further, the stirring time is 3-4 hours.

[0017] Further, the volume ratio of the embedding mixture to acetone is 1:1.

[0018] Further, after adding the embedding mixture and acetone into the sample, the sample is placed in a room temperature drying box, the embedding plate is filled with the embedding mixture without air bubbles, and then the obtained sample D is placed in the embedding plate.

[0019] Further, the programmed temperature polymerization is: placing in a 37 DEG C oven for 24 hours, placing in a 45 DEG C oven for 24 hours, and polymerizing in a 60 DEG C oven for 48 hours.

[0020] Further, the sample to be positioned is a kidney tissue sample, an ovary tissue sample or an endometrial tissue sample. Beneficial effects

[0021] The application improves the transmission electron microscope sample preparation method of the new embedding agent for clinical kidney puncture samples, effectively improves the toughness of the embedding block by studying the optimal formula of the 812R embedding mixture, optimizing the stirring time of the 812R embedding agent mixture, and combining the programmed temperature polymerization method, realizes the demand of sample semi-thin positioning section, improves the accuracy of sample observation, and can improve the quality of sample ultrathin section, reduce the situation of ultrathin section falling and cracking, ensure the integrity of the target structure of the sample, effectively solve the problems of new embedding agent embedding kidney tissue and other samples, such as semi-thin positioning difficulty and unsatisfactory imaging effect, provide a solution for further application and promotion of the new embedding agent 812R, and can be used for high-quality electron microscope sample preparation of human or mouse kidney tissue, so that the ultramicrostructure observation and pathological diagnosis become possible. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the method flow of the present invention;

[0023] Figure 2 This is a schematic diagram of the experimental process in an embodiment of the present invention;

[0024] Figure 3 The repair effect of the kidney 812R embedded block obtained by method A;

[0025] Figure 4 Image of a 500nm semi-thin section of the kidney 812R embedded block obtained by method A;

[0026] Figure 5 Image of a 1μm semi-thin section of the kidney 812R embedded block obtained by method A;

[0027] Figure 6 Image of a 3μm semi-thin section of the kidney 812R embedded block obtained by method A;

[0028] Figure 7 Image of a 5μm semi-thin section of the kidney 812R embedded block obtained by method A;

[0029] Figure 8 The effect of 3μm semi-thin glomerular localization of the 812R kidney embedding block obtained by method A;

[0030] Figure 9 The effect of 5μm semi-thin 812R kidney embedding block for glomerular localization obtained by method A;

[0031] Figure 10 The image shows a microscopic ultrathin section of the kidney 812R embedded block obtained by method A.

[0032] Figure 11 The image shows a 90nm ultrathin section of the kidney 812R embedded block obtained by method A at low magnification (280 nm).

[0033] Figure 12 The image shows a 90nm ultrathin section of the kidney 812R embedded block obtained by method A at low magnification (2000).

[0034] Figure 13 The image shows a 90nm ultrathin section of the kidney 812R embedded block obtained by method A at low magnification (8500).

[0035] Figure 14 The image shows a 90nm ultrathin section of the kidney 812R embedded block obtained by method A at low magnification (2600).

[0036] Figure 15The low magnification 8500 effect diagram of the 90 nm ultrathin section of the kidney 812R embedding block obtained by the A group method;

[0037] Figure 16 The block effect of the kidney 812R embedding block obtained by the B group method;

[0038] Figure 17 The 500 nm semi-thin section effect diagram of the kidney 812R embedding block obtained by the B group method;

[0039] Figure 18 The 1 μm semi-thin section effect diagram of the kidney 812R embedding block obtained by the B group method;

[0040] Figure 19 The 3 μm semi-thin section effect diagram of the kidney 812R embedding block obtained by the B group method;

[0041] Figure 20 The 5 μm semi-thin section effect diagram of the kidney 812R embedding block obtained by the B group method;

[0042] Figure 21 The microscope ultrathin section effect diagram of the kidney 812R embedding block obtained by the B group method;

[0043] Figure 22 The low magnification 280 effect diagram of the 90 nm ultrathin section of the kidney 812R embedding block obtained by the B group method;

[0044] Figure 23 The low magnification 2000 effect diagram of the 90 nm ultrathin section of the kidney 812R embedding block obtained by the B group method;

[0045] Figure 24 The low magnification 8500 effect diagram of the 90 nm ultrathin section of the kidney 812R embedding block obtained by the B group method;

[0046] Figure 25 The low magnification 280, 2000, 8500 effect diagram of the 90 nm ultrathin section of the kidney 812R embedding block obtained by the B group method;

[0047] Figure 26 The low magnification 280, 2000, 8500 effect diagram of the 90 nm ultrathin section of the kidney 812R embedding block obtained by the B group method;

[0048] Figure 27 The block effect comparison of the kidney 812R embedding block before and after the optimization of the programmed temperature condition;

[0049] Figure 28 The low magnification 280, 2000, 8500 effect diagram of the 90 nm ultrathin section of the kidney 812R embedding block obtained by the three-stage gradient temperature polymerization method for 1 h;

[0050] Figure 29 The effect diagram of the 90 nm ultrathin section of the kidney 812R embedding block obtained by stirring for 2 h + three-stage gradient temperature polymerization at multiples of 280, 2000, and 8500;

[0051] Figure 30 The effect diagram of the 90 nm ultrathin section of the kidney 812R embedding block obtained by stirring for 5 h + three-stage gradient temperature polymerization at multiples of 280, 2000, and 8500;

[0052] Figure 31 The block repairing effect of the kidney 812R embedding block obtained by different stirring times + three-stage temperature polymerization. DETAILED DESCRIPTION

[0053] The technical solutions of the present application will be further described in detail below in combination with the drawings and specific embodiments. All the following described embodiments are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application. In addition, if not specifically stated, all reagents used in the following examples are commercially available or can be synthesized according to the methods described herein or known methods, and the reaction conditions not listed are also easily obtained by those skilled in the art. The percentages not specifically stated are all volume ratios.

[0054] SPI-PON 812R is an upgraded version of the original SPI-PON 812, and has high continuity in technical parameters and operation processes. Therefore, the SPI-PON 812R formula is studied by the transmission electron microscope sample preparation method of the original SPI-PON 812 (i.e. the traditional method). It is found that the best formula is SPI-PON 812R 44.56 parts by volume, NMA 17.83 parts, DDSA 35.65 parts, and DMP-30 1.96 parts, and the sample picture quality is best under this formula, but the embedding block is brittle, which affects the positioning of the section, and the operation process needs to be further optimized.

[0055] This embodiment takes mouse kidney as an example to provide a new embedding agent transmission electron microscope sample preparation method for clinical kidney biopsy samples, such as Figure 1 、 Figure 2 , the specific process is as follows:

[0056] 1. Fresh ICR mice were sacrificed, and the kidney cortex was immediately taken out with a size of 1*1*1 cubic millimeter, and was put into 4℃ refrigerator pre-cooled 2.5% glutaraldehyde fixing solution, and was fixed overnight.

[0057] 2. The sample was rinsed with 0.1M PBS (pH=7.4) for 3 times, 10 minutes / time.

[0058] 3. 1wt% osmium acid room temperature fixed samples for 1 hour. Then discard osmium acid, distilled water rinse the sample 3 times, 10 minutes / time.

[0059] 4. 2wt% uranyl acetate room temperature staining for 30 minutes.

[0060] 5. 30% ethanol room temperature dehydration for 15 minutes, 50% ethanol room temperature dehydration for 15 minutes, 70% ethanol room temperature dehydration for 15 minutes, 90% ethanol room temperature dehydration for 15 minutes, 100% ethanol room temperature dehydration for 20 minutes, 100% acetone room temperature dehydration for 2 times, 20 minutes / time.

[0061] 6. SPI-PON 812R 44.56 parts, NMA 17.83 parts, DDSA 35.65 parts, DMP-30 1.96 parts by volume in advance to configure the embedding agent mixture;

[0062] Stir the embedding agent mixture for different lengths of time (1-5 hours), such as:

[0063] a: stir for 4 hours;

[0064] b: stir for 1 hour.

[0065] 7. Add the embedding agent mixture prepared in advance to the mixture with a volume ratio of 1:1 with 100% acetone, and place it in a room temperature drying oven for 2 hours.

[0066] 8. Fill the embedding plate with pure embedding agent and place the sample in the embedding plate.

[0067] 9. The embedding plate is subjected to programmed temperature treatment, and the treatment scheme is:

[0068] Three-stage temperature rise: 37°C oven for 24 hours, 45°C for 24 hours, and 60°C for 48 hours.

[0069] Two-stage temperature rise: 37°C oven for 24 hours and 65°C oven for 48 hours.

[0070] A series of experiments were conducted to study the influence of stirring time and programmed temperature rise scheme on the quality of the sample to be positioned. Among them: group A is stirring for 4 hours + three-stage temperature rise scheme, group B is stirring for 1 hour + two-stage temperature rise scheme (i.e. traditional scheme process).

[0071] 10. Leica UC7 ultrathin microtome block, the block effect diagram of group A is shown in Figure 3 .

[0072] 11. Semi-thin section, 500nm, 1μm, 3μm, 5μm semi-thin sections were made on the embedding block respectively, and the semi-thin section effect diagram is shown in Figure 4 、 5, 6, 7. The 3 μm, 5 μm semi-thin sections were stained with toluidine blue and observed under microscope. The pictures after staining are shown in Figure 8 , 9 .

[0073] 12. Ultra-thin sections. The sample was cut into 90 nm ultra-thin sections, as shown in Figure 10 , and then the sections were hydrophilic treated with 200 mesh non-film copper screen and collected.

[0074] 13. Section double staining: 4% uranyl acetate staining for 45 minutes, then rinsing; 1% lead nitrate staining for 5 minutes, then rinsing, and air-drying.

[0075] 14. Observation and photography under Talos120C transmission electron microscope. The electron micrographs of LM280x are shown in Figure 11 , the electron micrographs of SA2000x of renal tubules are shown in Figure 12 , the electron micrographs of SA8500x of renal tubules are shown in Figure 13 , the electron micrographs of SA2600x of glomerulus are shown in Figure 14 , and the electron micrographs of SA8500x of glomerulus are shown in Figure 15 .

[0076] As can be seen from Figure 3 , no debris occurs during the trimming of the embedding block (see Figure 16 ), indicating that the toughness of the embedding block is improved. From the effects of semi-thin sections Figure 4 , 5 , 6 and 7, compared with the effects of semi-thin sections Figure 17 , 18 , 19 and 20 of the traditional method, the semi-thin sections obtained by this method are all intact without damage, and the tissue structures of the 3 μm, 5 μm semi-thin sections after staining are clear, and the glomerulus in the kidney tissue can be directly observed (see Figure 8 and 9 , respectively), proving that the quality of the embedding block can meet the requirements of semi-thin positioning of transmission electron microscope sample preparation for clinical kidney biopsy samples. The ultra-thin sections in Figure 10 and 11 are undamaged, indicating that the ultra-thin sections have better strength (the traditional method ultra-thin sections are shown in 21, 22, 23 and 24), which is conducive to protecting the structural integrity of the sample and facilitating observation. From Figure 12 , 13 , 14 and 15, the clear ultrastructure of the glomerulus and renal tubule in the kidney tissue can be observed, indicating that this sample preparation method can meet the observation requirements of transmission electron microscope.

[0077] After a series of experimental studies, it is found that the formula of the embedding agent mixture, the stirring time of the embedding agent mixture and the temperature rising polymerization procedure have great influence on the quality of the final embedding block and the quality of the transmission electron microscope sample. Among them: according to formula one (SPI-PON 812R 41.72 parts, NMA 10.43 parts, DDSA 45.89 parts, DMP-30 1.96, formula two (SPI-PON 812R 48.05 parts, NMA 28.83 parts, DDSA 21.62 parts, DMP-30 1.49) respectively in the traditional method process, the transmission electron microscope images of the samples prepared are shown in Figure 25 and Figure 26 It can be seen that the sample block obtained by formula one under the traditional method is brittle, the slice is wrinkled, the image contrast is poor, and the effect is not ideal, and it is not as good as that of formula three (SPI-PON 812R 44.56 parts, NMA 17.83 parts, DDSA 35.65 parts, DMP-30 1.96 parts), and the effect of formula three is shown in Figure 22 , Figure 23 and Figure 24 The slice is wrinkle-free and the image contrast is clear; but the sample of formula three has the problems of brittle embedding block and easy to break affecting the positioning of the slice. Therefore, on this basis, the traditional method process is optimized with the best formula (formula three), and it is found that only optimizing the temperature rising procedure can improve the brittleness of the embedding block, as shown in Figure 27 Before optimization, 1 μm semi-thin section cannot be obtained, but after optimization, 1 μm semi-thin section can be obtained. However, formula three only program temperature optimization still cannot meet the positioning requirements of 3-5 μm semi-thin section of kidney puncture sample (the thinner the semi-thin section, the more difficult to see the morphology of the glomerulus during positioning, generally a slightly thicker thickness such as 3 or 5 μm is used, and compared with 1 μm semi-thin section, 3-5 μm semi-thin section is more difficult to obtain). Therefore, on the basis of program temperature optimization, the stirring time (1 h / 2 h / 3 h / 4 h / 5 h) of the 812R embedding agent mixture is explored, and the samples obtained by using three-stage temperature rising treatment are shown in Figure 28 , Figure 29 , Figure 30 The difference between them is that the stirring time of the embedding agent mixture is 1 h, 2 h and 5 h respectively, and it can be seen from Figure 31 that the sample blocks obtained by stirring for 1 h and 2 h are brittle and broken, and cannot meet the positioning requirements of 3-5 μm semi-thin section; although the sample block obtained by the stirring scheme of 5 h can be used for 3 μm semi-thin section, the ultrathin section is broken, the image dyeing is deep under high magnification, and the contrast is poor Figure 30 , which still cannot meet the sample positioning and imaging requirements. That is, the stirring time also has important influence on the sample preparation quality, and it is not the longer the better, and the preferred stirring time is 3-4 h.

[0078] In summary, the quality of the embedding block is the best when the optimal formula is adopted, the stirring time is 3-4 hours, and the three-stage temperature programming scheme is adopted, which can meet the positioning requirement of semi-thin section and obtain high-quality transmission electron microscope images.

[0079] The above shows that the transmission electron microscope sample preparation method of the novel embedding agent for clinical kidney puncture samples of the application provides a new experience method for high-quality electron microscope sample preparation of biological samples needing positioning.

[0080] The above-described embodiments are only some of the preferred schemes of the application, and are not intended to limit the application. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the application. Therefore, any technical scheme obtained by equivalent replacement or equivalent transformation falls within the protection scope of the application.

Claims

1. A method for preparing a sample for transmission electron microscopy of a sample to be positioned based on a novel embedding agent, characterized in that, The method comprises the following steps: Taking the tissue to be prepared, and putting into the pre-cooled fixing liquid at 4 DEG C for fixing overnight; Rinsing, osmium acid fixing, rinsing again, dyeing and dehydrating the sample in sequence; A new embedding mixture of embedding agent is prepared by mixing SPI-PON 812R, NMA, DDSA and DMP-30 in a volume ratio of 44.56:17.83:35.65:1.96 respectively; and the obtained embedding mixture is stirred for 3-4 hours; After adding the obtained embedding mixture and acetone into the sample, the sample is subjected to programmed temperature polymerization, i.e., being placed in an oven at 37 DEG C for 24 hours, being placed in an oven at 45 DEG C for 24 hours, and being polymerized in an oven at 60 DEG C for 48 hours; The obtained embedding block is sliced to obtain a transmission electron microscope sample.

2. The method for preparing a sample for transmission electron microscopy of a new type of embedding agent-based positioning sample according to claim 1, characterized in that, The rinsing, osmium acid fixing and rinsing again are specifically as follows: rinsing with PBS, fixing with osmium acid at room temperature, rinsing with distilled water after discarding the osmium acid, and dyeing with uranyl acetate at room temperature.

3. The method for preparing a sample for transmission electron microscopy of a new type of embedding agent-based positioning sample according to claim 1, characterized in that, The dehydrating is specifically as follows: sequentially dehydrating with 30% ethanol at room temperature for 15 minutes, 50% ethanol at room temperature for 15 minutes, 70% ethanol at room temperature for 15 minutes, 90% ethanol at room temperature for 15 minutes, 100% ethanol at room temperature for 20 minutes, and 100% acetone at room temperature for 20 minutes twice.

4. The method for preparing a sample for transmission electron microscopy of a new type of embedding agent based positioning sample according to claim 1, characterized in that, The volume ratio of the embedding mixture to acetone is 1:

1.

5. The novel-embedding-agent-based method for preparing a localized sample for transmission electron microscopy according to claim 1, wherein, After adding the embedding mixture and acetone into the sample, the sample is placed in a room temperature drying oven, the embedding plate is filled with the embedding mixture without air bubbles, and then the obtained sample is placed in the embedding plate.

6. The novel-embedding-agent-based method for preparing a localized sample for transmission electron microscopy according to claim 1, wherein, The sample to be positioned is a kidney tissue sample, an ovary tissue sample or an endometrial tissue sample.

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