Transmission electron microscope sample preparation method for sample to be positioned based on novel embedding medium

Abstract

The invention provides a transmission electron microscope sample preparation method of a sample to be positioned based on a novel embedding medium, which effectively improves the toughness of an embedding block, improves the quality of an ultrathin section of the sample and reduces the conditions of falling and cracking of the ultrathin section by researching the optimal formula of an SPI-PON 812R embedding mixture, optimizing the stirring time and combining with a programmed heating polymerization method. The requirement for semi-thin positioning and slicing of the sample is met, the integrity of the target structure of the sample is guaranteed, the problems that the semi-thin positioning of the kidney tissue and other samples embedded by the novel embedding medium is difficult, the imaging effect is not ideal and the like are effectively solved, a solution is provided for further application and popularization of the novel embedding medium 812R, and the application prospect is wide. The method can be used for preparing high-quality electron microscope samples of human or mouse kidney tissues and the like, so that ultrastructure observation and pathological diagnosis become possible.

Description

Technical Field

[0001] The invention belongs to the technical field of medical biological sample preparation, and relates to a transmission electron microscope sample preparation method for a sample requiring positioning based on a novel embedding agent, and particularly relates to a preparation method for a transmission electron microscope sample requiring positioning such as a clinical renal puncture sample. Background Art

[0002] Transmission electron microscopy (TEM) is widely used to observe the ultrastructure of tissues and cells. Ultrathin sectioning is a prerequisite for final structural observation and obtaining excellent results during the preparation of biological samples for TEM. However, the small size of random sections can easily lead to missed detection of valid target structures, resulting in a waste of manpower, material resources, and time. To ensure that ultrathin sections accurately target the target structure, semi-thin sections are performed before ultrathin sectioning to facilitate localization.

[0003] Transmission electron microscopy (TEM) examination is crucial for the pathological diagnosis of renal biopsy, and semi-thin localization of glomeruli is a key step. Its importance is primarily reflected in the following aspects: 1. Improving detection efficiency and avoiding missed detections. The number of glomeruli in renal biopsy samples is limited (usually only a few to a dozen) and their distribution is uneven. Semi-thin sections, after staining, allow for clear observation of tissue structure under a light microscope, quickly localizing areas containing glomeruli, avoiding missed detections caused by blind TEM sectioning, and ensuring targeted ultrathin sectioning and electron microscopic observation of glomeruli. 2. Ensuring accurate and targeted observations. Glomeruli are the primary lesion site for kidney diseases (e.g., nephritis, nephrotic syndrome, etc., often affect glomeruli). Semi-thin localization allows precise selection of glomeruli with typical lesions for TEM analysis, avoiding the possibility of missing key lesions due to random selection and improving diagnostic accuracy. 3. Saving specimens and experimental resources. Renal biopsy specimens are extremely rare and precious. Semi-thin positioning can clearly identify the target area, reduce unnecessary ultrathin section preparation and electron microscopy observation time, maximize the effective information obtained from limited samples, and save manpower, material resources, and time costs. In short, semi-thin positioning of glomeruli is the prerequisite and foundation for transmission electron microscopy diagnosis of renal biopsy specimens, directly affecting the efficiency of subsequent TEM examinations and the reliability of diagnostic results, and plays an irreplaceable role in the accurate diagnosis of kidney disease.

[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 medium (abbreviated as 812), a replacement, SPI-PON 812R resin embedding medium (abbreviated as 812R), has emerged. Although the manufacturer states that SPI-PON 812R offers no significant differences in section quality, staining efficacy, and electron beam stability compared to SPI-PON 812, when preparing samples requiring semi-thin sectioning and positioning, such as kidneys, according to the manufacturer's technical documentation, embedding blocks prepared with SPI-PON 812R are brittle and fragile, making semi-thin sections difficult to slice and hindering structural localization. Furthermore, ultra-thin sectioning is inefficient, and the slides are prone to falling and splitting during staining, resulting in poor imaging. Kidney tissue samples embedded in the new embedding agent have problems such as difficulty in semi-thin positioning and unsatisfactory imaging effects. Therefore, for this new embedding agent, how to effectively and fully utilize it in the preparation of samples that need to be positioned? It is necessary to develop a more reliable sample preparation method to improve the preparation effect of clinical kidney samples, facilitate semi-thin positioning and imaging observation, and improve diagnostic accuracy. Summary of the Invention

[0005] The present invention aims to address the existing problems in the preparation of transmission electron microscopy samples requiring localization, such as kidney tissue, resulting in brittle and fragile embedding blocks, difficulty in semi-thin-thin positioning, and poor imaging. By providing a method for preparing samples for transmission electron microscopy using a novel embedding agent, the present invention optimizes the stirring time of the 812R embedding agent mixture and combines it with a temperature-programmed polymerization method to effectively improve the preparation of clinical kidney samples, addressing the difficulties in semi-thin-thin positioning and poor imaging of transmission electron microscopy samples of kidney tissues using the novel 812R embedding agent.

[0006] The technical solutions adopted in the present invention are as follows: A method for preparing a sample for transmission electron microscopy based on a novel embedding agent for positioning a sample, comprising: Take the tissue to be prepared and put it into the fixative solution pre-cooled in a 4℃ refrigerator and fix it overnight; The samples were rinsed, fixed with osmate, rinsed again, stained, and dehydrated in sequence; An embedding mixture of the novel embedding agent was prepared by respectively comprising 44.56 parts of SPI-PON 812R, 17.83 parts of NMA, 35.65 parts of DDSA, and 1.96 parts of DMP-30 by volume; the resulting embedding mixture was stirred; The obtained embedding mixture and acetone were added to the sample, and after embedding, temperature-programmed polymerization was performed; The embedded block was sliced ​​to obtain a transmission electron microscope sample.

[0007] Furthermore, the rinsing, osmium acid fixation, and re-rinsing specifically include: rinsing with PBS and then fixing with osmium acid at room temperature, discarding osmium acid, rinsing with distilled water, and then staining with uranyl acetate at room temperature.

[0008] Furthermore, the dehydration is specifically carried out in sequence: dehydration with 30% ethanol by volume for 15 minutes at room temperature, dehydration with 50% ethanol by volume for 15 minutes at room temperature, dehydration with 70% ethanol by volume for 15 minutes at room temperature, dehydration with 90% ethanol by volume for 15 minutes at room temperature, dehydration with 100% ethanol by volume for 20 minutes at room temperature, and dehydration with 100% acetone by volume for 2 times, each time for 20 minutes.

[0009] Furthermore, it is characterized by 44.56 parts of SPI-PON 812R, 17.83 parts of NMA, 35.65 parts of DDSA, and 1.96 parts of DMP-30. Furthermore, the stirring time is 3 to 4 hours.

[0010] Furthermore, the volume ratio of the embedding mixture to acetone is 1:1.

[0011] Furthermore, embedding mixture and acetone were added to the sample and then placed in a drying oven at room temperature. The embedding plate was filled with embedding mixture until there were no bubbles. Then, the obtained sample D was placed in the embedding plate.

[0012] Furthermore, the programmed temperature polymerization is as follows: placing in an oven at 37°C for 24 hours, placing in an oven at 45°C for 24 hours, and polymerizing in an oven at 60°C for 48 hours.

[0013] Furthermore, the sample to be located is a kidney tissue sample, an ovarian tissue sample, or an endometrial tissue sample. Beneficial effects

[0014] The present invention improves the method for preparing transmission electron microscopy samples with a new embedding agent for clinical renal puncture samples. By studying the optimal formula of the 812R embedding mixture, optimizing the stirring time of the 812R embedding agent mixture, and combining it with a programmed temperature polymerization method, the toughness of the embedding block is effectively improved, the demand for semi-thin positioning sectioning of the sample is met, and the accuracy of sample observation is improved. At the same time, the quality of ultra-thin sectioning of the sample can be improved, the occurrence of slice dropout and cracking in the ultra-thin section is reduced, and the integrity of the target structure of the sample is ensured. The present invention effectively solves the problems of difficult semi-thin positioning of samples such as kidney tissue embedded in the new embedding agent and unsatisfactory imaging effects, and provides a solution for the further application and promotion of the new embedding agent 812R. The present invention can be used for the preparation of high-quality electron microscopy samples such as human or mouse kidney tissue, making ultrastructural observation and pathological diagnosis possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the process of the present invention; Figure 2 Schematic diagram of the experimental process in an embodiment of the present invention; Figure 3 This is the trimming effect of the kidney 812R embedded block obtained by the method of group A; Figure 4 This is the effect of 500nm semi-thin section of kidney 812R embedded block obtained by group A method; Figure 5 This is the 1μm semi-thin section of the kidney 812R embedded block obtained by the method of group A; Figure 6 This is the effect of 3μm semi-thin section of kidney 812R embedded block obtained by the method of group A; Figure 7 This is the result of 5μm semi-thin section of kidney 812R embedded block obtained by the method of group A; Figure 8 The 3μm semi-thin 812R embedded block of kidney obtained by the method of group A shows the effect of locating glomeruli; Figure 9 The 5μm semi-thin 812R embedded block of kidney obtained by the method of group A shows the effect of locating glomeruli; Figure 10 This is the image of the ultrathin section of the kidney 812R embedded block obtained by the method of group A under a microscope; Figure 11 This is the effect picture of 90nm ultra-thin section of kidney 812R embedded block obtained by group A method at low magnification 280; Figure 12 This is the effect picture of 90nm ultra-thin section of kidney 812R embedded block obtained by group A method at low magnification 2000; Figure 13 This is the effect picture of 90nm ultra-thin section of kidney 812R embedded block obtained by group A method at low magnification 8500; Figure 14 This is the effect picture of 90nm ultra-thin section of kidney 812R embedded block obtained by group A method at low magnification 2600; Figure 15 This is the effect picture of 90nm ultra-thin section of kidney 812R embedded block obtained by group A method at low magnification 8500; Figure 16 This is the trimming effect of the kidney 812R embedded block obtained by the method of group B; Figure 17 This is the result of 500nm semi-thin section of kidney 812R embedded block obtained by group B method; Figure 18 This is the 1μm semi-thin section of the kidney 812R embedded block obtained by the method of group B; Figure 19 This is the effect of 3μm semi-thin section of kidney 812R embedded block obtained by the method of group B; Figure 20 This is the result of 5μm semi-thin section of kidney 812R embedded block obtained by the method of group B; Figure 21 This is the image of the ultrathin section of the kidney 812R embedded block obtained by the method of group B under a microscope; Figure 22 This is the effect picture of 90nm ultra-thin section of kidney 812R embedded block obtained by group B method at low magnification 280; Figure 23 This is the effect picture of 90nm ultra-thin section of kidney 812R embedded block obtained by group B method at low magnification 2000; Figure 24 This is the effect picture of 90nm ultra-thin section of kidney 812R embedded block obtained by group B method at low magnification 8500; Figure 25 The effect of 90nm ultrathin section of 812R (Formula 1) kidney embedded block at magnifications of 280, 2000, and 8500. Figure 26 The effect of 90nm ultrathin sections of 812R (Formula 2) kidney embedded blocks at magnifications of 280, 2000, and 8500. Figure 27 To compare the trimming effects of kidney 812R embedded blocks before and after optimization of programmed temperature conditions; Figure 28 The effect of 90nm ultrathin sections of kidney 812R embedded blocks obtained by stirring for 1h and three-stage gradient temperature polymerization method at magnifications of 280, 2000, and 8500; Figure 29 The effect of 90nm ultrathin sections of kidney 812R embedded blocks obtained by stirring for 2h and three-stage gradient temperature polymerization method at magnifications of 280, 2000, and 8500; Figure 30 The effect of 90nm ultrathin sections of kidney 812R embedded blocks obtained by stirring for 5h and three-stage gradient temperature polymerization method at magnifications of 280, 2000, and 8500; Figure 31 The repair effect of kidney 812R embedded blocks obtained by different stirring times + three-stage temperature rising polymerization method. DETAILED DESCRIPTION

[0016] The technical scheme of the present invention is further described in detail below in conjunction with the accompanying drawings and specific examples. All the following described embodiments are exemplary and are intended to be used to explain the present invention, but are not to be construed as limiting the present invention. In addition, unless otherwise expressly stated, all reagents used in the following examples are commercially available or can be synthesized according to this document or known methods. For reaction conditions not listed, they are also readily available to those skilled in the art. Percentages not otherwise specified refer to volume ratios.

[0017] As an upgraded version of the original SPI-PON 812, the SPI-PON 812R shares a high degree of continuity in technical parameters and operational procedures. Therefore, the SPI-PON 812R formulation was investigated using the original SPI-PON 812 transmission electron microscopy sample preparation method (i.e., the traditional method). The optimal formulation was found to be 44.56 parts SPI-PON 812R, 17.83 parts NMA, 35.65 parts DDSA, and 1.96 parts DMP-30, by volume. This formulation yielded the best sample image quality, but the embedding blocks were brittle, hindering section positioning, necessitating further optimization of the operational procedures.

[0018] This example uses mouse kidney as an example to provide a new embedding medium transmission electron microscopy sample preparation method for clinical renal puncture samples. Figure 1 、 Figure 2 The specific process is as follows: 1. Fresh ICR mice were sacrificed and a 1*1*1 mm3 section of kidney cortex was immediately removed and fixed in 2.5% glutaraldehyde solution pre-cooled in a 4°C refrigerator overnight.

[0019] 2. Rinse the sample three times with 0.1M PBS (pH=7.4), 10 minutes each time.

[0020] 3. Fix the sample in 1 wt% osmium hydroxide at room temperature for 1 hour. Discard the osmium hydroxide and rinse the sample with distilled water three times, 10 minutes each time.

[0021] 4. Stain with 2wt% uranyl acetate at room temperature for 30 minutes.

[0022] 5. Dehydrate in 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 2 times, 20 minutes each time.

[0023] 6. Prepare the embedding agent mixture in advance by volume: 44.56 parts SPI-PON 812R, 17.83 parts NMA, 35.65 parts DDSA, and 1.96 parts DMP-30; The embedding agent mixture is stirred for different lengths of time (1 to 5 hours), such as: a: Stir for 4 hours; b: Stir for 1 hour.

[0024] 7. Add the pre-prepared embedding agent mixture and 100% acetone in a 1:1 volume ratio and place in a drying oven at room temperature for 2 hours.

[0025] 8. Fill the embedding plate with pure embedding medium and place the sample into the embedding plate.

[0026] 9. The embedding plate is subjected to programmed temperature treatment. The treatment options are: Three-stage heating: place in a 37°C oven for 24 hours, 45°C for 24 hours, and 60°C for 48 hours.

[0027] Second stage heating: place in 37℃ oven for 24 hours, and polymerize in 65℃ oven for 48 hours.

[0028] A series of experiments were conducted to investigate the effects of stirring time and programmed temperature ramping on the quality of the sample being positioned. Group A used a 4-hour stirring schedule followed by a three-stage temperature ramp, while Group B used a 1-hour stirring schedule followed by a two-stage temperature ramp (the traditional process).

[0029] 10. Leica UC7 ultra-thin section machine repair block, group A repair block effect diagram see Figure 3 .

[0030] 11. Semi-thin sectioning: 500nm, 1μm, 3μm, and 5μm semi-thin sections were performed on the embedded block. The semi-thin sectioning effect is shown in the figure. Figure 4 、 5 , 6, 7. 3μm and 5μm semi-thin sections were stained with toluidine blue and observed under a microscope. Figure 8 、 9 .

[0031] 12. Ultrathin sectioning: Cut the sample into 90nm ultrathin sections. Figure 10 Then use a 200-mesh copper mesh without film to hydrophilize the film and pick it up.

[0032] 13. Double staining of sections: stain with 4% uranyl acetate for 45 minutes, rinse, stain with 1% lead nitrate for 5 minutes, rinse, and air dry.

[0033] 14. Observe and photograph under a Talos120C transmission electron microscope. The electron microscope images of LM280× are shown in Figure 11 , SA2000× electron microscopic image of the lower renal tubules is shown in Figure 12 , SA8500× electron microscopy of the lower renal tubules Figure 13 , the electron microscopic image of the glomerulus under SA2600× is shown in Figure 14 , the electron microscopic image of the glomerulus under SA8500× is shown in Figure 15 .

[0034] from Figure 3 It can be seen that no debris occurs during the trimming of the embedded block as in the traditional method (see Figure 16 ), indicating that the toughness of the embedded block has been improved. Figure 4 、 5, 6 and 7, compared with the semi-thin section effect of the traditional method Figure 17 、 18 , 19 and 20, the semi-thin sections obtained by this method were intact and undamaged, and the tissue structure of 3μm and 5μm semi-thin sections was clear after staining, and the glomeruli in the kidney tissue could be directly observed (see Figure 2). Figure 8 and 9 ), proving that the quality of the embedding block can meet the requirements of semi-thin positioning of clinical renal puncture samples for transmission electron microscopy. Figure 10 and 11 The ultrathin sections were not damaged, indicating that the ultrathin sections had better strength (see 21, 22, 23, and 24 for ultrathin sections using traditional methods), which is beneficial for protecting the structural integrity of the samples and facilitating observation. Figure 12 、 13 , 14 and 15, clear ultrastructures of glomeruli and renal tubules in kidney tissue can be observed, indicating that this sample preparation method can meet the needs of transmission electron microscopy observation.

[0035] After a series of experimental studies, it was found that the formula of the embedding agent mixture, the stirring time of the embedding agent mixture, and the temperature polymerization procedure have a significant impact on the quality of the final embedding block and the quality of the transmission electron microscopy sample. Among them, the transmission electron microscopy images of the samples prepared by the traditional method process according to the following formula: SPI-PON 812R 41.72 parts, NMA 10.43 parts, DDSA 45.89 parts, DMP-30 1.96 parts; and formula 2 (SPI-PON 812R 48.05 parts, NMA 28.83 parts, DDSA 21.62 parts, DMP-30 1.49 parts) are shown as follows: Figure 25 and Figure 26 As shown in the figure, it can be seen that the sample blocks obtained by the traditional method are brittle, the slices are wrinkled, and the image contrast is poor. The results are not ideal and are not as good as those obtained by the recipe 3 (SPI-PON 812R 44.56 parts, NMA 17.83 parts, DDSA 35.65 parts, DMP-30 1.96 parts). The effect of recipe 3 is as follows Figure 22 、 Figure 23 and Figure 24 The slices were wrinkle-free and the image contrast was clear; however, the sample with recipe 3 had the problem of brittle and fragile embedding blocks that affected the positioning of the slices. Therefore, based on this, the traditional method process was optimized with the best recipe (recipe 3). It was found that only optimizing the heating program could improve the brittleness of the embedding blocks, such as Figure 27Before optimization, 1μm semi-thin sections could not be obtained, but after optimization, 1μm semi-thin sections could be obtained. However, the programmed temperature optimization of formula three still could not meet the 3-5μm semi-thin section positioning requirements for renal puncture samples (the thinner the semi-thin section, the more difficult it is to see the morphology of the glomeruli during positioning. Generally, a slightly thicker thickness, such as 3 or 5μm, is used. When the sample toughness is not good, 3-5μm semi-thin sections are more difficult to obtain than 1μm semi-thin sections). Therefore, based on the programmed temperature optimization, the stirring time (1h / 2h / 3h / 4h / 5h) of the 812R embedding agent mixture was explored, such as Figure 28 、 Figure 29 、 Figure 30 The samples were obtained by three-stage programmed temperature treatment, the difference being that the stirring time of the embedding agent mixture was 1h, 2h, and 5h respectively. Figure 31 It can be seen that the sample blocks obtained by stirring for 1 hour and 2 hours are brittle and broken, and cannot meet the positioning requirements of 3-5μm semi-thin sections. Compared with the traditional solution, the sample blocks obtained by stirring for 5 hours can be sliced ​​into 3μm semi-thin sections, but the ultra-thin sections are broken, the images under high-power microscope are deeply stained, and the contrast is poor ( Figure 30 ), still cannot meet the requirements of sample positioning and imaging. That is, stirring time also has a significant impact on sample preparation quality, and longer is not necessarily better. It is preferably stirred for 3-4 hours.

[0036] In summary, the quality of the embedding block was optimal when the optimal formula, combined with stirring for 3-4 h and a three-stage programmed temperature scheme, was used, which could not only meet the positioning requirements of semi-thin sections but also obtain high-quality transmission electron microscopy images.

[0037] The above results indicate that the novel embedding medium transmission electron microscopy sample preparation method of the present invention for clinical renal puncture samples provides a new empirical method for preparing high-quality electron microscopy samples of biological samples that require positioning.

[0038] The embodiments described above are merely some preferred embodiments of the present invention and are not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent substitution or equivalent transformation falls within the scope of protection of the present invention.

Claims

1. A method for preparing samples for transmission electron microscopy based on a novel embedding agent, characterized in that: include: Take the tissue to be prepared and put it into the fixative solution pre-cooled at 4℃ and fix it overnight; The samples were rinsed, fixed with osmate, rinsed again, stained, and dehydrated in sequence; An embedding mixture of the novel embedding agent was prepared by mixing 44.56 parts of SPI-PON 812R, 17.83 parts of NMA, 35.65 parts of DDSA, and 1.96 parts of DMP-30 by volume; the resulting embedding mixture was stirred; The obtained embedding mixture and acetone were added to the sample, and after embedding, temperature-programmed polymerization was performed; The embedded block was sliced ​​to obtain a transmission electron microscope sample.

2. The method for preparing a sample for transmission electron microscopy based on a novel embedding medium according to claim 1, characterized in that: The rinsing, osmium acid fixation, and re-rinsing specifically include: rinsing with PBS and then fixing with osmium acid at room temperature, discarding the osmium acid, rinsing with distilled water, and then staining with uranyl acetate at room temperature.

3. The method for preparing a sample for transmission electron microscopy based on a novel embedding medium according to claim 1, characterized in that: The dehydration is specifically carried out in sequence: dehydration with 30% ethanol by volume for 15 minutes at room temperature, dehydration with 50% ethanol by volume for 15 minutes at room temperature, dehydration with 70% ethanol by volume for 15 minutes at room temperature, dehydration with 90% ethanol by volume for 15 minutes at room temperature, dehydration with 100% ethanol by volume for 20 minutes at room temperature, and dehydration with 100% acetone by volume for 2 times, each time for 20 minutes.

4. The method for preparing samples for transmission electron microscopy based on a novel embedding medium according to claim 1, wherein: SPI-PON 812R 44.56 parts, NMA 17.83 parts, DDSA 35.65 parts, DMP-30 1.96 parts.

5. The method for preparing samples for transmission electron microscopy based on a novel embedding medium according to claim 1, characterized in that: The stirring time is 3 to 4 hours.

6. The method for preparing a sample for transmission electron microscopy based on a novel embedding medium for positioning of a sample according to claim 1, characterized in that: The volume ratio of the embedding mixture to acetone was 1:

1.

7. The method for preparing samples for transmission electron microscopy based on a novel embedding agent according to claim 1, characterized in that: After adding embedding mixture and acetone to the sample, place it in a drying oven at room temperature. Fill the embedding plate with embedding mixture until there are no bubbles. Then place the obtained sample D into the embedding plate.

8. The method for preparing a sample for transmission electron microscopy based on a novel embedding medium for positioning according to claim 1, characterized in that: The programmed temperature polymerization is as follows: placing in an oven at 37° C. for 24 hours, placing in an oven at 45° C. for 24 hours, and polymerizing in an oven at 60° C. for 48 hours.

9. The method for preparing a sample for transmission electron microscopy based on a novel embedding medium according to claim 1, characterized in that: The samples to be located are kidney tissue samples, ovarian tissue samples, and endometrial tissue samples.

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