In-vivo in-situ integrated analysis method for micro-plastic organisms based on phthalocyanine blue labeling and Raman imaging
By using phthalocyanine blue labeling and Raman imaging, the incompatibility between microplastic detection technology and paraffin pathology section procedures has been resolved, enabling accurate qualitative, localization, and quantitative analysis of microplastics and providing a low-cost, high-efficiency detection solution.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-20
- Publication Date
- 2026-04-03
AI Technical Summary
Existing microplastic detection technologies are difficult to integrate with paraffin pathology slide procedures. Fluorescent labeling methods are prone to signal leakage, while other methods are costly or require expensive equipment. Furthermore, they are difficult to achieve accurate qualitative, quantitative, and seamless integration of microplastics with histopathology.
Phthalocyanine blue labeling and Raman imaging were used to prepare paraffin sections after microplastic particles were enriched in vivo by phthalocyanine blue staining. In-situ qualitative and quantitative analysis of microplastics was performed using Raman spectroscopy and imaging technology, and spatial distribution images were generated by combining peak clipping method.
It achieves compatibility between microplastic detection technology and paraffin pathology sectioning procedures, provides chemical composition-specific information, ensures the reliability and accuracy of detection, has submicron resolution, is low in cost, and is applicable to various types of plastics.
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Figure CN121783948A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of environmental toxicology and analytical chemistry, specifically relating to an in-situ integrated method for the analysis of microplastics in vivo based on phthalocyanine blue labeling and Raman imaging. Background Technology
[0002] The bioaccumulation and toxic effects of microplastics are at the forefront of current research in environmental science. In related toxicity studies, paraffin-embedded tissue pathology sections have been widely used in the toxicological assessment of microplastics because they can clearly show the structure of tissue cells and facilitate the observation and diagnosis of pathological changes.
[0003] However, existing mainstream microplastic tracing technologies are difficult to effectively integrate with standard paraffin sectioning procedures. The most commonly used fluorescent labeling methods (such as Nile Red staining) require treatment with organic solvents (such as ethanol) and high-temperature baking during paraffin preparation, during which the fluorescence signal is highly susceptible to leakage or quenching, leading to detection failure. Furthermore, there is a risk of fluorescent dyes leaking from the plastic matrix, and the biological tissue itself may also emit fluorescence, causing background interference and false positives. Other methods, such as stable polymer tracing (e.g., PTFE), are only applicable to a single plastic type and have poor universality; techniques such as time-of-flight secondary ion mass spectrometry (TOF-SIMS) are destructive to samples and require expensive equipment; while rare earth element or isotope labeling methods are costly and difficult to widely apply.
[0004] Therefore, there is an urgent need in this field to develop a new labeling and detection technology that should meet the following key requirements: (1) the label has extremely high chemical and physical stability and can withstand the entire process of standard paraffin sectioning without failure; (2) it can provide specific information on chemical components to achieve accurate qualitative and quantitative identification and localization of microplastics; (3) it can be seamlessly integrated with histopathological observation to facilitate direct correlation with toxic effects; (4) it has quantitative analysis capabilities; and (5) it is low in cost and easy to promote. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an in-situ integrated analysis method for microplastics in vivo based on phthalocyanine blue labeling and Raman imaging.
[0006] The specific technical solution adopted in this invention is as follows:
[0007] This invention provides an in-situ integrated method for the analysis of microplastics in biological organisms based on phthalocyanine blue labeling and Raman imaging. The specific steps are as follows:
[0008] S1: Expose the organism to be studied and analyzed to phthalocyanine blue-stained microplastic particles until the phthalocyanine blue-labeled microplastic particles are enriched in the organism.
[0009] S2: Obtain the exposed biological tissue, prepare paraffin sections and perform histopathological staining;
[0010] S3: Raman spectroscopy and imaging analysis of samples after histopathological staining: First, locate the tissue region under an optical microscope, and obtain the spectrum of the region using Raman spectroscopy spot scan mode. Identify the target region by recognizing the characteristic Raman peaks of phthalocyanine blue and microplastics. Perform Raman surface scanning on the target region, and extract the intensity information of the characteristic Raman peaks of phthalocyanine blue or microplastics using the peak clipping method to generate spatial distribution images of the corresponding components in biological tissues, and perform in-situ qualitative analysis of microplastics in biological tissues.
[0011] Preferably, the method for preparing the phthalocyanine blue stained microplastic particles is as follows:
[0012] S11: Dissolve the target plastic in an organic solvent, add phthalocyanine blue dye and mix thoroughly; after the organic solvent evaporates, obtain phthalocyanine blue dyed plastic; wherein the mass of phthalocyanine blue dye added is 0.5%~5% of the target plastic.
[0013] S12: The phthalocyanine blue dyed plastic is placed in liquid nitrogen to embrittle it and then mechanically crushed until the particle size is submicron, to obtain phthalocyanine blue dyed microplastic particles.
[0014] Preferably, the target plastic is one or more of polyvinyl chloride, polystyrene, or polymethyl methacrylate; the particle size of the phthalocyanine blue-stained microplastic particles ranges from 0.1 to 100 μm.
[0015] Preferably, the histopathological staining in step S2 is performed using the hematoxylin-eosin staining method.
[0016] Preferably, in step S3, the Raman spectroscopy spot scanning mode uses a 633 nm laser, a 600 nm grating, and a 50x objective lens for spectral acquisition; the spectral scanning range is 500–3000 cm⁻¹. -1 .
[0017] Preferably, the laser power of the laser is set to 40~60 mW and the single-point integration time is 10~30 seconds.
[0018] Preferably, the characteristic Raman peak of the phthalocyanine blue is 686 cm⁻¹. -1 742 cm -1 Or 1528 cm -1 At least one of them.
[0019] Preferably, the peak-sandwiching method is as follows: select the wavenumber range of the characteristic Raman peak in the Raman spectrum, calculate the integral intensity of the spectral signal of each pixel within this range, and generate a spatial distribution map of the component accordingly.
[0020] Preferably, the step size of the Raman surface scanning in step S3 is set to 1~5 μm, and the scanning area is not less than 10 μm×10 μm.
[0021] As a preferred method, the obtained biological tissue was subjected to quantitative analysis of microplastic content, specifically as follows: the biological tissue enriched with phthalocyanine blue-stained microplastics was acid-digested until the microplastics and phthalocyanine blue dye were completely decomposed; then, the concentration of copper ions in the digestion solution was determined by inductively coupled plasma atomic emission spectrometry; based on the measured copper ion concentration, the copper mass fraction in the phthalocyanine blue dye, and the proportion of phthalocyanine blue dye added, the mass of microplastics enriched in the biological tissue was calculated.
[0022] Compared with the prior art, the present invention has the following advantages:
[0023] (1) The core advantage of this invention lies in solving the incompatibility problem between microplastic detection technology and the gold standard paraffin pathology section process. The phthalocyanine blue dye used in this invention has extremely high chemical and physical stability, and can withstand a series of tissue processing processes such as ethanol dehydration, paraffin embedding and high temperature baking of biological tissues, and does not experience signal attenuation or leakage. This ensures that the marker signal can still exist stably on the final HE-stained sections, thus ensuring the reliability and success rate of the detection results.
[0024] (2) The method provided by this invention achieves seamless integration of morphological observation and chemical composition analysis. Researchers can first assess tissue pathological damage (such as cell necrosis, inflammatory infiltration, etc.) using conventional optical microscopy on the same histopathological section, and then accurately locate the spatial distribution of microplastics in situ using confocal Raman imaging. This method can directly and intuitively correlate the distribution location of microplastics with the lesion area of the tissue, providing a powerful technical means for elucidating the toxic mechanism of microplastics.
[0025] (3) In the method provided by this invention, confocal Raman technology can provide submicron spatial resolution and can detect plastic particles of hundreds of nanometers. The strong Raman characteristic peak of phthalocyanine blue is significantly distinguishable from the Raman background signal of biological tissue matrix, which can effectively avoid background interference from biological tissue and ensure the specificity of detection. The combination of the two realizes high-precision and high-reliability localization and analysis of microplastics, especially submicron particles, in biological tissue.
[0026] (4) The method provided by this invention is highly universal and inexpensive. Phthalocyanine blue dyed microplastic particles can be prepared using a variety of common plastics (PVC, PS, PMMA, etc.). In addition, phthalocyanine blue is a common industrial dye that is inexpensive and easy to obtain, which greatly reduces research costs and application barriers. Attached Figure Description
[0027] Figure 1 The overall flowchart of the in-situ integrated analysis method for microplastics in vivo based on phthalocyanine blue labeling and Raman imaging provided by the present invention;
[0028] Figure 2 The stability results of the phthalocyanine blue-dyed PVC microplastics prepared in Example 1 are shown in the figure.
[0029] Figure 3 The images show the Raman spectra of PVC microplastics, paraffin, muscle tissue stained with phthalocyanine blue at different particle sizes and under different laser intensities in Example 1.
[0030] Figure 4 The image shows the Raman scan results of phthalocyanine blue stained PVC particles in the tadpole intestine in Example 2. a1 is the localization map of microplastics in the tadpole intestine tissue; b1 is the Raman spectrum of this sample; c1, d1, and e1 are the Raman spectra of the sample at wavenumber 643 cm⁻¹. -1 686 cm -1 and 742 cm -1 Imaging images under certain conditions;
[0031] Figure 5 The image shows the Raman scan results of phthalocyanine blue stained PVC particles in tadpole muscle in Example 2. a1 is the localization map of microplastics in the tadpole muscle tissue; b1 is the Raman spectrum of this sample; c1, d1, and e1 are the Raman spectra of the sample at wavenumber 643 cm⁻¹. -1 686 cm -1 and 742 cm -1 Imaging images under certain conditions;
[0032] Figure 6 The images show Raman scans of phthalocyanine blue stained PVC particles in different organs and tissues of tadpoles during the peak metamorphosis period in Example 2. A1 to E1 represent the localization of microplastics in the liver, hind limbs, forelimbs, blood, and brain tissues of the tadpoles, respectively; A2 to E2 correspond to samples stained at a wavenumber of 643 cm⁻¹. -1 Imaging images under the following conditions; a3~e3 correspond to samples at wavenumber 686 cm⁻¹, respectively. -1 Imaging images under certain conditions;
[0033] Figure 7 The images show Raman scan results of the distribution of microplastics and their association with lesions in the intestinal tissue of tadpoles during the peak metamorphosis period in Example 2. Image a shows the intestinal lesions, and images b and c show the lesions at a wavenumber of 643 cm⁻¹. -1 686 cm -1 The image under the condition; d represents the sample at wavenumbers of 500–1600 cm⁻¹. -1 The results of the point scan between the points;
[0034] Figure 8The figure shows the quantitative analysis results of microplastics in Example 2, where a is the recovery rate and b is the mass of microplastics in the intestines of tadpoles in the phthalocyanine blue stained PVC particle exposure group and the control group with different particle sizes. Detailed Implementation
[0035] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.
[0036] In the examples, common polyvinyl chloride (PVC) was used as the plastic, and tadpoles of the black-spotted lateral fold frog were selected as the experimental material to provide a detailed description of the invention. The experimental materials in the examples are only one aspect of the invention and are not intended to limit it.
[0037] The overall flowchart of the method provided by this invention is as follows: Figure 1 As shown.
[0038] Example 1
[0039] This embodiment provides a method for preparing phthalocyanine blue dyed microplastics, and verifies the performance of phthalocyanine blue dyed microplastics with different particle sizes, as detailed below:
[0040] (1) Preparation of phthalocyanine blue stained microplastics
[0041] Dissolve 10 g of polyvinyl chloride (PVC, extrusion grade) in 50 mL of tetrahydrofuran and let stand in a fume hood until completely dissolved. Transfer the solution to a 500 mL beaker, add 0.2 g of phthalocyanine blue (CuPc) dye (2% of the plastic mass), place the beaker in a magnetic rotor, and heat with magnetic stirring at 60 °C until the solvent is completely evaporated to obtain uniformly colored phthalocyanine blue dyed PVC plastic.
[0042] The phthalocyanine blue-stained PVC plastic was embrittled in liquid nitrogen and then crushed using a ball mill. This liquid nitrogen embrittlement-mechanical crushing process was repeated several times to obtain particles with smaller diameters. The crushed mixture was ultrasonically dispersed in anhydrous ethanol for 30 minutes to remove small fragments adhering to the surface. Finally, it was subjected to gradient filtration through stainless steel sieves with different pore sizes (e.g., 100 μm, 50 μm, 20 μm, and 5 μm) and polycarbonate filter membranes (e.g., 1 μm and 0.2 μm) to obtain phthalocyanine blue-stained microplastic particles with diameters of 0.2–5 μm, 5–20 μm, 20–50 μm, and 50–100 μm, respectively. These phthalocyanine blue-stained microplastic particles of different diameters were dispersed in anhydrous ethanol to prepare a mother liquor for later use.
[0043] (2) Stability verification
[0044] To verify the stability of phthalocyanine blue dye in the paraffin sectioning process, the following experiment was conducted: phthalocyanine blue-stained PVC microplastics were immersed in 70%, 95%, and 100% ethanol for 72 hours, respectively, and the paraffin embedding and baking process was simulated by high-temperature treatment at 65℃. After treatment, the supernatant was collected by centrifugation, and the absorbance value at the maximum absorption peak (~650nm) was measured using a UV-Vis spectrophotometer. The results are as follows: Figure 2 As shown.
[0045] The results showed that no significant phthalocyanine blue characteristic absorption peak was detected in the supernatant of any of the ethanol-treated groups, and the dye leaching rate was less than 1%. Meanwhile, the Raman signal intensity of the microplastics after immersion did not change significantly compared with that before immersion (p>0.05). These results demonstrate that phthalocyanine blue labeling exhibits excellent stability under organic solvents and high-temperature conditions, and can fully adapt to the standard paraffin section preparation process.
[0046] (3) Raman spectroscopy characterization
[0047] Raman spectroscopy characterization was performed on the phthalocyanine blue-stained microplastics of different particle sizes prepared in this embodiment, and the results are as follows: Figure 3 As shown in the figure. The results show that the prepared microplastics also possess the characteristic peaks of PVC (~643 cm⁻¹). -1 ~703 cm -1 ~2917 cm -1 The strong characteristic peaks of phthalocyanine blue (~686 cm⁻¹) and phthalocyanine blue. -1 ~742 cm -1 ~1528 cm -1 As the particle size decreases to the submicron level, the characteristic peak intensity of PVC decreases significantly, while the characteristic peak intensity of phthalocyanine blue remains stable and clearly distinguishable. This indicates that when detecting submicron-sized microplastics, the characteristic peak of phthalocyanine blue dye (especially at 686 cm⁻¹) remains relatively constant. -1 and 742 cm -1 This can serve as a more reliable identification criterion, effectively avoiding confusion with characteristic peaks of paraffin or biological tissues (such as 1341 cm⁻¹). -1 1446 cm -1 and 2917 cm -1 The overlapping interference between them.
[0048] In this embodiment, phthalocyanine blue-dyed microplastic particles with a particle size of 50-100 μm and phthalocyanine blue dye were characterized by Raman spectroscopy using 100% laser power and 10% laser power (approximately 50 mV), respectively. The results are as follows: Figure 3As shown in the figure, the results indicate that all four particle sizes of phthalocyanine blue-stained PVC microplastics exhibited strong Raman signals with distinct characteristic peaks. The characteristic peaks of muscle tissue and paraffin in the tissue-stained section substrate did not overlap with those of phthalocyanine blue dye and PVC microplastics, thus having no interference with this embodiment. Furthermore, even at 10% laser intensity, phthalocyanine blue and PVC microplastics still exhibited strong Raman signals; therefore, this embodiment selected a 10% laser intensity (approximately 50 mV) for Raman spectroscopy characterization.
[0049] Example 2
[0050] (1) Microplastic exposure experiment of tadpoles
[0051] This study used Gosner tadpoles at stage 26 / 27, provided by the Amphibian Laboratory of Lishui University, as the research subjects. Healthy individuals of similar size were selected for microplastic exposure experiments. Phthalocyanine blue-stained microplastic particles with particle sizes of 0.2–5 μm, 5–20 μm, 20–50 μm, and 50–100 μm, prepared in Example 1, were used. The microplastic concentration for each exposure group was 10. 6 The exposure solution was set up at 100 tadpoles / L, with a control group without microplastic exposure. Each group had 3 replicates, with 30 tadpoles in each replicate tank. The feeding amount was controlled at 2% of the tadpoles' body weight, and the exposure solution was changed every three days until the tadpoles completed metamorphosis.
[0052] (2) Preparation of paraffin sections of tadpole tissues and organs
[0053] During the experiment, the growth of tadpoles was observed daily. After periodic anesthesia with low-concentration MS-222, the morphological characteristics of the tadpoles (such as body length, tail length, and hind limb length) were observed and photographed under a microscope to determine their developmental stage. At different developmental stages (premetamorphosis, premetamorphosis, peak metamorphosis, and metamorphosis completion), 15 tadpoles or froglets were randomly collected, euthanized, and fixed in formalin solution for subsequent paraffin pathological section examination.
[0054] Formalin-fixed specimens were removed and dehydrated using a gradient of ethanol solutions (70%–100%). Tadpole samples from the pre-metamorphosis and early metamorphosis stages were embedded in paraffin with the abdomen facing downwards. Due to the large sample size, tadpoles from the peak metamorphosis stage were cut in half and embedded in paraffin with one half parallel and the other perpendicular to allow for tissue observation from different angles. Next, sections with a thickness of 5 μm were prepared using a Leica rotary microtome and stained with hematoxylin and eosin (H&E) solution.
[0055] (3) Raman spectroscopy and imaging analysis process
[0056] Raman imaging was performed on the stained tadpole tissue samples. The specific procedure is as follows:
[0057] Raman spot scanning mode: First, the suspected plastic particle area was located under an optical microscope. A 633 nm laser, along with a 600 nm grating and a 50x objective lens, was used for spectral acquisition. To reduce optical damage to the sample and obtain a spectrum with a good signal-to-noise ratio, the laser power was optimized to 10% (approximately 50 mV), the integration time was 20 seconds, and three scans were performed. The scanning range was 500–3000 cm⁻¹. -1 Under these parameters, phthalocyanine blue markers exhibit strong and stable characteristic Raman peaks, while the characteristic peaks of the PVC microplastic matrix are relatively weak. Therefore, the characteristic peaks of phthalocyanine blue are used as the primary basis for the detection and confirmation of microplastics in the analysis.
[0058] Raman surface scan mode: To visualize the spatial distribution of microplastics, a surface scan (mapping) is performed on a region of interest (e.g., 15 μm × 15 μm). Set the step size to 1–5 μm (adjust according to the target particle size). Select a stride of 500–3000 cm⁻¹. -1 The wavenumber range was determined to cover all characteristic peaks of PVC and phthalocyanine blue. This was achieved through the "peak clipping method" (setting a specific wavenumber range, such as 686 cm⁻¹). -1 ± 2 cm -1 Generate an intensity distribution map of the characteristic peak. If the shape of the high-signal region in the distribution map matches the particle morphology observed under an optical microscope, it can be confirmed as the target microplastic. Results are as follows: Figures 3-5 As shown.
[0059] Figure 4 and Figure 5 The images show the Raman scan results of phthalocyanine blue stained PVC particles in the intestinal cavity and muscle of tadpoles.
[0060] according to Figure 6 Phthalocyanine blue-stained PVC microplastics were successfully located in various tissues, including the tadpole's forelimbs, liver, blood, and even brain, and particles smaller than 4 μm could be clearly imaged. For submicron-sized particles, the Raman signal may be weaker, but by optimizing scanning parameters (such as increasing the integration time) and focusing on analyzing the characteristic peak with the highest signal-to-noise ratio (e.g., 686 cm⁻¹), the Raman signal can be improved. -1 Even with these limitations, effective detection can still be achieved. This confirms the effectiveness of the method of the present invention for detecting submicron-sized microplastics.
[0061] (4) Correlation study between tadpole tissue pathology and microplastics
[0062] Histopathological observation of stained tadpole intestinal tissue sections under a light microscope revealed pathological changes such as villus damage and excessive proliferation of mucus cells in the intestine. Figure 7 As shown in (a) of the diagram.
[0063] The suspected particles were scanned in situ using a confocal Raman spectrometer (LabRAM HR Evolution, Horiba) for spot scanning and area scanning within the same field of view (at 686 cm⁻¹). -1 Characteristic peak imaging successfully acquired the spectral and distribution maps of microplastics in intestinal tissue. For example... Figure 7 As shown, the area of microplastic enrichment with the strongest Raman signal (red area) highly overlaps with the area of most severe tissue damage in the H&E image. This directly and strongly demonstrates that the accumulation of microplastics at this site is the direct cause of the observed intestinal pathological damage.
[0064] (5) Quantitative analysis of microplastics
[0065] Weigh an appropriate amount of frozen intestinal tissue, transfer it to a digestion tube, add sufficient concentrated nitric acid, and heat at 85°C for 1 hour until the solution is clear, ensuring complete decomposition of microplastics and phthalocyanine blue dye. After cooling, determine the copper ion concentration in the digestion solution using ICP-OES (iCAP7000, Thermo Fisher Scientific).
[0066] The formula for calculating microplastic content is as follows:
[0067] Microplastic mass (μg) = [Measured Cu mass (μg) / Cu mass fraction in phthalocyanine blue dye] / Dye addition ratio
[0068] The accuracy and precision of this quantitative method were evaluated using a spiked recovery experiment. The procedure involved precisely adding a known mass of stained microplastic standard to a blank tadpole tissue matrix that was known to be free of the target microplastics, followed by the complete digestion and ICP-OES assay described above.
[0069] Calculations showed that the mass of microplastics in the intestines of tadpoles exposed to microplastics of different particle sizes was as follows: Figure 8 As shown in (b) in the diagram. Figure 8 As shown in (a), the average spiked recovery rate of this method was 84.7 ± 4.6%. This recovery rate is within the generally acceptable range for complex matrix analysis of environmental biological samples. The results indicate that although there is some matrix effect or pretreatment loss, this method has good reproducibility and accuracy, and can be reliably used for relative quantification and comparative studies of microplastic accumulation among different biological tissue samples.
[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.
Claims
1. A method for in-situ integrated analysis of microplastics in biological organisms based on phthalocyanine blue labeling and Raman imaging, characterized in that, The specific steps are as follows: S1: Expose the organism to be studied and analyzed to phthalocyanine blue-stained microplastic particles until the phthalocyanine blue-labeled microplastic particles are enriched in the organism. S2: Obtain the exposed biological tissue, prepare paraffin sections and perform histopathological staining; S3: Raman spectroscopy and imaging analysis of samples after histopathological staining: First, locate the tissue region under an optical microscope, and obtain the spectrum of the region using Raman spectroscopy spot scan mode. Identify the target region by recognizing the characteristic Raman peaks of phthalocyanine blue and microplastics. Perform Raman surface scanning on the target region, and extract the intensity information of the characteristic Raman peaks of phthalocyanine blue or microplastics using the peak clipping method to generate spatial distribution images of the corresponding components in biological tissues, and perform in-situ qualitative analysis of microplastics in biological tissues.
2. The in-situ integrated analysis method for microplastics in vivo according to claim 1, characterized in that, The method for preparing the phthalocyanine blue stained microplastic particles is as follows: S11: Dissolve the target plastic in an organic solvent, add phthalocyanine blue dye and mix thoroughly; after the organic solvent evaporates, obtain phthalocyanine blue dyed plastic; wherein the mass of phthalocyanine blue dye added is 0.5%~5% of the target plastic. S12: The phthalocyanine blue dyed plastic is placed in liquid nitrogen to embrittle it and then mechanically crushed until the particle size is submicron, to obtain phthalocyanine blue dyed microplastic particles.
3. The in-situ integrated analysis method for microplastics in vivo according to claim 1, characterized in that, The target plastic is one or more of polyvinyl chloride, polystyrene, or polymethyl methacrylate; the particle size of the phthalocyanine blue-stained microplastic particles ranges from 0.1 to 100 μm.
4. The in-situ integrated analysis method for microplastics in vivo according to claim 1, characterized in that, The histopathological staining in step S2 uses the hematoxylin-eosin staining method.
5. The in-situ integrated analysis method for microplastics in vivo according to claim 1, characterized in that, In step S3, the Raman spectroscopy spot scanning mode uses a 633 nm laser, a 600 nm grating, and a 50x objective lens for spectral acquisition; the spectral scanning range is 500–3000 cm⁻¹. -1 .
6. The in-situ integrated analysis method for microplastics in vivo according to claim 1, characterized in that, The laser power of the laser is set to 40~60 mW, and the single-point integration time is 10~30 seconds.
7. The in-situ integrated analysis method for microplastics in vivo according to claim 1, characterized in that, The characteristic Raman peak of the phthalocyanine blue is 686 cm⁻¹. -1 742 cm -1 Or 1528 cm -1 At least one of them.
8. The in-situ integrated analysis method for microplastics in vivo according to claim 1, characterized in that, The peak-squeezing method is as follows: Select the wavenumber range of the characteristic Raman peak in the Raman spectrum, calculate the integral intensity of the spectral signal of each pixel within this range, and generate a spatial distribution map of the component accordingly.
9. The in-situ integrated analysis method for microplastics in vivo according to claim 1, characterized in that, In step S3, the step size of the Raman surface scanning is set to 1~5 μm, and the scanning area is not less than 10 μm×10 μm.
10. The in-situ integrated analysis method for microplastics in vivo according to claim 1, characterized in that, The microplastic content of the obtained biological tissue was quantitatively analyzed as follows: the biological tissue enriched with phthalocyanine blue-stained microplastics was acid digested until the microplastics and phthalocyanine blue dye were completely decomposed; then, the copper ion concentration in the digestion solution was determined by inductively coupled plasma atomic emission spectrometry; based on the measured copper ion concentration, the copper mass fraction in phthalocyanine blue dye, and the proportion of phthalocyanine blue dye added, the mass of microplastics enriched in the biological tissue was calculated.