Application of label-free immunohistochemical imaging method based on OIRD technology in tissue slice analysis

Abstract

The invention discloses an application of an unmarked immunohistochemical imaging method based on an OIRD (Oblique Incident Reflection Diffraction) technology in tissue slice analysis, which realizes unmarked IHC imaging of a tissue slice by introducing an OIRD technology and adopting an unmarked antibody. The method has high sensitivity (delta I signal resolution reaches mu V level) and high specificity (non-specific adsorption test delta I fluctuation lt; the device is compatible with any substrate, and realizes a real-time dynamic monitoring function under the spatial resolution (4-6 [mu] m). According to the technology, the experimental procedure is greatly simplified, the total detection cost is reduced, meanwhile, key dynamic information of antibody-antigen specific binding can be provided, and the technology has important significance on clinical application and basic pathological research.

Description

Technical Field

[0001] The present invention relates to the technical field of immunohistochemical detection, and particularly relates to the application of a label-free immunohistochemical imaging method based on OIRD technology in tissue section analysis. Background Art

[0002] Immunohistochemistry (IHC) is a widely used diagnostic technique that shows crucial applications in clinical pathological diagnosis and basic pathological research. Since the establishment of IHC, it has had a significant impact on tumor diagnosis, classification, and prognosis, and has deepened our understanding of various diseases. Generally, IHC realizes the localization and semi-quantitative detection imaging of specific biomarkers (antigens) by labeling (such as fluorescence, enzymes, etc.) antibodies, while the labeling of antibodies may affect the recognition activity of biomolecules, increase the experimental cost, and add additional operation steps. Therefore, label-free immunohistochemical imaging technology urgently needs to be developed.

[0003] Oblique-incidence reflectivity difference (OIRD) is an emerging method that detects the physicochemical change process occurring on the interface by measuring the change in the reflectivity difference of two polarized light components (i.e., s and p) of the reflected light incident on the interface, and has the advantages of label-free, real-time, and high-throughput in immunoassay. Compared with other label-free immunohistochemical imaging methods such as surface plasmon resonance imaging / microscopy and interferometric scatter microscopy, OIRD is compatible with any substrate and has a larger detection depth, so it has a wider applicability. It is of great significance to explore whether OIRD is suitable for label-free detection of tissue sections with complex interface structures. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the application of a label-free immunohistochemical imaging method based on OIRD technology in tissue section analysis.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] 1. The application of a label-free immunohistochemical imaging method based on OIRD technology in tissue section analysis.

[0007] In some embodiments of the present invention, the application specifically includes the following steps:

[0008] (1) Mount the glass slide carrying the tissue section on the OIRD sample platform, scan the entire chip surface, and obtain the original OIRD image after reaching stability in 0.01M PBS buffer solution;

[0009] (2) Introduce the unlabeled primary antibody solution into the reaction chamber for affinity binding, repeatedly collect the in-situ OIRD signal to monitor the binding degree. After the affinity binding is completed, scan and image the same area of the tissue section again to obtain the final image;

[0010] (3) Subtract the original image from the final image to obtain the OIRD differential image, and realize the detection of the local abundance and expression level of the target antigen.

[0011] In some embodiments of the present invention, the thickness of the tissue section is less than or equal to 8 microns.

[0012] In some embodiments of the present invention, the OIRD uses a He-Ne laser with a wavelength of 632.8 nm and a power of 3 mW as the light source.

[0013] In some embodiments of the present invention, the light source is obliquely incident on the surface of the tissue section at an incident angle of 60°.

[0014] In some embodiments of the present invention, in steps (1) and (2), the scanning is performed at a height above the 4 mm straight-line distance on the surface of the section.

[0015] The beneficial effects of the present invention are as follows: By introducing the oblique incidence reflectance difference (OIRD) technology and using unlabeled antibodies, the present invention realizes label-free IHC imaging of tissue sections, and has high sensitivity (the ΔI signal resolution reaches the μV level) and high specificity (the ΔI fluctuation of non-specific adsorption test < 0.6 μV), is compatible with any substrate, and at the same time realizes the real-time dynamic monitoring function at the spatial resolution of (4 - 6 μm). This technology greatly simplifies the experimental procedure and reduces the overall detection cost, and can also provide key kinetic information on the specific binding of antibody-antigen, which will be of great significance for clinical applications and basic pathology research. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to make the objectives, technical solutions, and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0017] Figure 1 It is a schematic diagram of the OIRD optical detection system.

[0018] Figure 2 It is a schematic diagram of label-free IHC imaging based on OIRD.

[0019] Figure 3 It is the OIRD images of tumor sections collected in 0.01 M PBS at t = 0 (a) and t = 157 min (c) respectively; (b) The in-situ OIRD reactions collected at three positions of the tumor section, and 5 mg mL -1 BSA, 10 mg mL- 1 BSA, 10% human serum, and 100% human serum, with 0.01M PBS as the baseline; (d) OIRD differential image of the tumor section, obtained by subtracting the image in (a) from the image in (b).

[0020] Figure 4 (a, b) are OIRD images of the tumor section before reacting with the primary antibody in 0.01M PBS (a, t = 0) and after the reaction (b, t = 100 minutes); (c) is the OIRD differential image after the tumor section undergoes an immunoreaction with the primary antibody; (d) is the in-situ OIRD reaction collected at three positions on the tumor section (as shown in c) after adding the primary antibody, and 0.01M PBS is used for baseline collection; (e) is a schematic diagram of the fluorescence IHC imaging protocol after label-free OIRD detection; (f) is the fluorescence image of the tumor section in the same region as in (c) after reacting with a 1:1000 fluorescent secondary antibody for 15 minutes; (g) is a comparison of the average ΔI and fluorescence intensity of six 0.15mm × 0.15mm square regions in the two images of (c) and (f).

[0021] Figure 5 (a, b) are OIRD images of the section containing the tumor and adjacent tissues before reacting with the primary antibody (a, t = 0) and after the reaction (b, t = 145 min) collected in 0.01M PBS; (c) is the OIRD differential image after affinity binding with the primary antibody collected in 0.01M PBS; (d) is the in-situ OIRD reaction after adding the primary antibody collected at three positions on the section (as shown in c), and 0.01M PBS is used for baseline collection; fluorescence images (e, g) and microscopic images (f, h) of the tumor region I (e, f) and the adjacent normal tissue region II (g, h); the adjacent normal tissue region II includes tumor tissue and normal tissue, where 4 and 5 are normal tissues and 6 is tumor tissue in figure g; (i) is the ΔI and fluorescence intensity graphs of six 0.2mm × 0.2mm squares selected from the OIRD differential image and the fluorescence image as shown in c, e, and g.

[0022] Figure 6 (a) are OIRD differential images of sections with thicknesses of 4μm (a), 6μm (b), and 8μm (c) collected in 0.01M PBS after affinity binding of the primary antibody to the same tumor tissue; (d) are the corresponding OIRD signals of the three images; (e) is a representative in-situ OIRD response curve. Detailed implementation mode

[0023] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0024] The schematic diagram of the OIRD device of the present invention is as Figure 1 shown, which mainly consists of four parts: a light source, an optical path system, a sample stage, and a detection system. A He-Ne laser with a wavelength of 632.8 nm and a power of 3 mW is used as the light source. The incident light passes through a polarizer to provide its degree of polarization, and then passes through the modulation of an acousto-optic modulator. The polarization state of the incident light changes periodically between s and p at a frequency of 50 kHz. The laser light emerging from the acousto-optic modulator passes through a phase shifter, introducing an adjustable phase difference for background zeroing between the s and p polarized lights, and then is focused by a lens and obliquely incident on the surface of the tissue section on the sample stage at an incident angle of 60°. The laser light reflected from the sample surface is further subjected to background zeroing through a polarization analyzer, and finally is converted into a voltage signal by a silicon photodiode. Two lock-in amplifiers respectively collect the fundamental frequency and harmonic frequency components of the modulation frequency in the voltage signal and input them into a computer for recording and processing. The signals used in this method are all harmonic signals.

[0025] The schematic diagram of the label-free IHC imaging method based on OIRD is as Figure 2 shown. The glass slide carrying the tumor tissue section is mounted on the OIRD sample stage, and this platform can be scanned by the horizontal movement of the motor-driven stage. When performing OIRD measurement, first scan the entire chip surface at a spatial resolution of 4 μm to obtain the OIRD image after the OIRD signal (ΔI) reaches stability in 0.01 M PBS buffer. Then, introduce the label-free 1:50 primary antibody solution (such as CRTC2 Mouse Monoclonal Antibody) into the reaction chamber for affinity binding. During this process, the spatially resolved in-situ OIRD signals along a 4-mm straight line on the surface are repeatedly collected with a time resolution of 3.2 s. After affinity binding, image the same area of the tissue section again. By subtracting the original image from the final image, an OIRD differential image is obtained.

[0026] Example 1. Nonspecific study of tissue sections.

[0027] Tumor tissue sections of mouse breast cancer (with a thickness of about 4 μm) were used to demonstrate label-free immunohistochemistry based on OIRD. After obtaining a stable baseline in 0.01 M PBS, as Figure 3 , as shown in a, the OIRD image of the section was collected at t = 0. The image shows that the OIRD signal fluctuates in a 2 mm × 2 mm area, indicating that the spatial dielectric constant of the tissue section is inhomogeneous, probably due to changes in cell density and / or local physicochemical environment. Through the use of in-situ OIRD detection, the nonspecific protein adsorption on the tissue section was studied for the first time. Figure 3 , b shows that after exposure to different nonspecific protein solutions, three different positions on the section (such as Figure 3The representative in-situ OIRD signals collected (as shown in a) showed no obvious upward trend or net increase in ΔI, indicating that after blocking, the section had excellent resistance to non-specific protein adsorption. After the non-specific adsorption test (t = 157 min), the same area of the tumor section was imaged again, as Figure 3 , c shown. Compared with the initial image ( Figure 3 , a), no obvious differences were found. The differential image obtained by subtracting the initial image ( Figure 3 , a) from the image collected at t = 157 min ( Figure 3 , c) is shown in Figure 3 , d, and the average value of the whole surface is 0.09 ± 0.6 μV, indicating that the section performed excellently in non-specific protein adsorption.

[0028] Case 2. Label-free IHC imaging based on OIRD technology.

[0029] First, OIRD imaging was used to study the differences in the expression levels of the target protein CRT (calreticulin) in different regions within a tumor using mouse breast cancer tissue sections containing only tumors. OIRD images of the sections were collected in 0.01 M PBS ( Figure 4 , a). After adding the primary antibody for immunoreaction, the sections were imaged again in 0.01 M PBS ( Figure 4 , b). The OIRD differential image ( Figure 4 , c) showed different OIRD signal intensities in the whole area, with a peak of about 15 μV and a valley as low as about 2 μV. The in-situ OIRD responses collected from three different positions A, B, and C on the section ( Figure 4 , d) showed that the ΔI values at sites A, B, and C were approximately 12, 15, and 3 μV respectively. The different ΔI values at the three sites indicated that compared with A and C, there was more CRT antigen presentation at site B, which was related to the different expression levels of the CRT antigen in different regions.

[0030] To verify the effectiveness of OIRD-based IHC imaging, as Figure 4 , e shown, after the immunoreaction monitored by OIRD, a fluorescently labeled 1:1000 secondary antibody (Alexa Fluor 488 labeled goat anti-rabbit lgG(H+L)) was further added to the sections. From the obtained fluorescence image ( Figure 4 , f), it could be observed that the spatially resolved fluorescence intensity in the whole area showed similar fluctuations to those detected by OIRD. By quantitatively analyzing the ΔI values and fluorescence intensities of six different 0.15 mm × 0.15 mm square regions ( Figure 4, g) It can be obtained that the ΔI value and the fluorescence intensity show exactly the same changing trend. The high consistency between the two images proves that the OIRD differential image can, like the fluorescence immunohistochemistry image, effectively reflect the local abundance and expression level of the biomarker of interest on the tissue section.

[0031] Secondly, sections containing tumors and adjacent non-tumor tissues were used to study the difference in the expression level of the target protein CRT between tumors and adjacent non-tumor tissues using OIRD imaging. Similar to the above detection steps, OIRD images of 4 mm × 4 mm before and after the reaction were obtained respectively, as Figure 5 , shown in a and b. Figure 5 , c shows the OIRD differential image obtained after incubating with anti-CRT monoclonal antibody for 140 minutes. The figure shows that the ΔI on the entire surface is highly different, representing different CRT expression levels. Specifically, compared with the adjacent non-tumor tissues, the tumor region at the bottom of the section (mainly distributed in Figure 4 , the upper region in c) shows a higher ΔI value, indicating that the expression level of CRT protein in tumor cells is higher than that in normal cells. Figure 5 , d shows the in-situ OIRD curves simultaneously collected at three points A, B, and C in Figure 5 , c. After adding the antibody, the OIRD signal continued to rise during the 140-minute incubation process, representing the binding of the antibody to the tissue. The net ΔI values of the three regions are very consistent with the values in the Figure 4 , c differential image. Figure 5 , for two representative regions in c, namely region Ⅰ and region Ⅱ, microscopy and fluorescence imaging were further performed using the same protocol as Figure 4 , c. According to the corresponding microscopy images in Figure 5 , e and g, region Ⅰ is pure tumor tissue, while region Ⅱ is adjacent non-tumor tissue. There are obvious differences in cell morphology and tissue structure between the two regions. Figure 5 , the fluorescence images in e and g show clear surface patterns, which are exactly the same as the OIRD differential image in Figure 5 , c. In the OIRD differential images and fluorescence images of each region, all surface features including ridges and grooves are very consistent, which confirms the feasibility of label-free IHC imaging based on OIRD. To further explore the consistency between OIRD and fluorescence images, the ΔI and fluorescence intensity at six positions (each position occupies an area of 0.2 mm × 0.2 mm) on the section were quantitatively calculated from Figure 5 , c (ΔI) and e, g (fluorescence intensity), and plotted as Figure 5, i. Both the quantitative ΔI and fluorescence intensity showed that the expression level of CRT protein in cells in the pure tumor regions (positions 1, 2, 3, and 6) was higher than that in the adjacent non-tumor regions (positions 4 and 5). Notably, the trends of ΔI and fluorescence signals in the six regions were exactly the same, showing a high degree of consistency. This indicates that, similar to the gold-standard fluorescence IHC imaging, this OIRD-based label-free IHC imaging technique can reflect the spatially resolved abundance of biomarkers of interest on tissue sections with high sensitivity and specificity.

[0032] Case 3. Influence of section thickness on OIRD signals.

[0033] According to previous studies, the thickness of the section may be a key factor affecting the sensitivity of OIRD-based label-free IHC imaging. By serially sectioning tumor tissues, three sections with thicknesses of 4, 6, and 8 μm were obtained to study the influence of section thickness on the OIRD signals induced by antibody binding. The differential images of tissue sections with a thickness of 4 μm ( Figure 6 , a), 6 μm ( Figure 6 , b), and 8 μm ( Figure 6 , c) showed that all three surfaces exhibited considerable ΔI signals across the entire surface, indicating that they were all CRT-positive. At the same time, the average ΔI intensity decreased with increasing thickness, as shown in Figure 6 , d. The ΔI signal reached its maximum at a section thickness of 4 μm. Figure 6 , e. The representative in-situ OIRD signals also showed that the signals gradually weakened with increasing section thickness. This comparison indicates that the OIRD label-free IHC imaging has the highest sensitivity at a thickness of 4 μm, and thicker sections result in a decrease in sensitivity.

[0034] The above-described embodiments are merely preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Application of a label-free immunohistochemical imaging method based on OIRD technology in tissue section analysis.

2. The use according to claim 1, characterized in that: The specific steps include: (1) Mount the slide containing the tissue section on the OIRD sample platform, scan the entire chip surface, and obtain the OIRD original image after stabilization in 0.01 M PBS buffer; (2) introducing the unlabeled primary antibody solution into the reaction chamber for affinity binding, repeatedly collecting in situ OIRD signals to monitor the degree of binding, and after the affinity binding is completed, scanning and imaging the same area of ​​the tissue section again to obtain the final image; (3) The original image is subtracted from the final image to obtain the OIRD difference image, which enables the detection of the local abundance and expression level of the target antigen.

3. The use according to claim 2, characterized in that: The thickness of the tissue section is less than or equal to 8 microns.

4. The use according to claim 2, characterized in that: The OIRD uses a He-Ne laser with a wavelength of 632.8 nm and a power of 3 mW as a light source.

5. The use according to claim 4, characterized in that: The light source is incident obliquely onto the surface of the tissue slice at an incident angle of 60°.

6. The use according to claim 4, characterized in that: In steps (1) and (2), the scanning is performed at a straight line distance of 4 mm above the slice surface.

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