A method for detecting cell pathological molecules based on LCTF optical tweezers and RET

Abstract

The present invention discloses a method for detecting cell pathological molecules based on LCTF optical tweezers and RET, including: S100: Pretreating and preparing a sample to be measured; S200: Making the donor protein molecules and the receptor protein molecules in the sample to be measured undergo resonance energy transfer, obtaining the real image signal generated by the fluorescence emitted by the receptor protein, thereby obtaining the position of the cell with the highest concentration of pathogenic molecules in the sample to be measured, and obtaining the cell by using optical tweezers; S300: Obtaining the spectral signal converted after filtering and coupling the scattering generated by the cell, synchronously analyzing the ultraviolet resonance Raman and fluorescence of the spectral signal to judge the content of the main pathogenic molecules in the cell with the highest concentration of pathogenic molecules, realizing the rapid screening and capture of pathological cells, and the rapid, accurate, trace and non-destructive detection of the pathological molecular tags of single cells.

Description

Technical Field

[0001] The present invention relates to the field of single-cell medical analyzers, and particularly to a method for detecting cell pathological molecules based on laser optical tweezers capture with a tunable liquid crystal filter and tunable resonance energy transfer, i.e., a method for detecting cell pathological molecules based on LCTF optical tweezers and RET. Background Art

[0002] In type II diabetic patients, hemoglobin in red blood cells binds to free glucose in the blood to form glycated hemoglobin, which can be used as the gold standard for long-term blood glucose detection in type II diabetic patients; the hemoglobin of patients with various anemias has characteristics such as abnormal structure or morphological changes in red blood cells; leukemia includes many subtypes. The conventional rapid detection method is to count white blood cells and cannot distinguish subtypes. The subtypes of leukemia need to be diagnosed through pathological indicators of molecular tags in white blood cells. Conventional pathological detection methods cannot quickly, accurately, minimally invasively, and nondestructively detect the molecular tags of single cells.

[0003] The means for obtaining pathological indicators of the above blood diseases need to combine refinement and rapidity, and merge microscale and non-destructiveness. Achieving refinement can rely on analysis techniques at the single-cell level. The main single-cell analysis techniques include microspectral chemical analysis, such as molecular vibration spectroscopy methods like Raman spectroscopy, resonance Raman, surface-enhanced Raman, coherent Raman, and infrared microspectroscopy; fluorescence-mediated microanalysis, such as those that require fluorescence staining, fluorescence microscopy, fluorescence lifetime imaging, fluorescence correlation spectroscopy, etc.; mass spectrometry and nuclear magnetic resonance, etc. Single-cell analysis often requires sample preparation in advance and combination with cell capture and trapping techniques to identify abnormal cells and perform accurate detection.

[0004] In some existing technical methods, the sample preparation process is relatively complex and requires a long analysis time; in addition, for cell detection samples of actual patients, there are a large number of cells, including both normal cells and abnormal cells. Moreover, some abnormal cells have morphological differences, while some do not have morphological differences but have changes in the characteristics of internal molecules. How to combine rapid and efficient discrimination between normal and abnormal cells and precise single-cell capture in microspectral and fluorescence-mediated analysis is a quite difficult problem. Summary of the Invention

[0005] In order to achieve rapid discrimination and capture of pathological cells and rapid, accurate, and comprehensive detection of cell pathological molecular tags, the present invention proposes a method for detecting cell pathological molecules based on LCTF optical tweezers and RET. By using LCTF laser optical tweezers in combination with RET hyperspectral technology, precise spatial and spectral information in a wider spectral range regarding patients, tissue samples, or different disease conditions can be provided, enabling rapid discrimination and capture of abnormal cells.

[0006] The present invention adopts the following technical solutions:

[0007] A cell pathological molecular detection method based on LCTF optical tweezers and RET, comprising: S100: Pretreatment and sample preparation of the sample to be tested; S200: Making the donor protein molecules and receptor protein molecules in the sample to be tested undergo resonance energy transfer, obtaining the real image signal generated by the fluorescence emitted by the receptor protein, thereby obtaining the position of the cell with the highest concentration of pathogenic molecules in the sample to be tested, and capturing the cell by optical tweezers; S300: Obtaining the spectral signal converted by filtering and coupling the scattering generated by the cell, and synchronously analyzing the ultraviolet resonance Raman and fluorescence in the spectral signal to judge the content of the main pathogenic molecules in the cell with the highest concentration of pathogenic molecules.

[0008] Preferably, the S200 includes: S201: Irradiating the sample to be tested with a single-frequency ultraviolet continuous laser with an output wavelength equal to the excitation wavelength of the donor fluorescent protein. After broadening and frequency selection, resonance energy transfer occurs between the donor protein molecules and the receptor protein molecules in the sample to be tested. The donor emits fluorescence to excite the receptor fluorescent protein molecules, and the receptor emits fluorescence after being excited.

[0009] Preferably, the S200 further includes: S202: The fluorescence emitted by the sample to be tested is filtered and then undergoes secondary imaging to form a secondary real image. Calculate the high-frequency energy of the image of the two-dimensional Fourier transform of the secondary real image, adjust the imaging parameters. When the high-frequency energy of the image is the largest, find the pixel point with the largest gray value on the secondary real image, and move the sample to be tested so that the pixel point with the largest gray value coincides with the main optical axis. This pixel point is the position of the cell with the highest concentration of pathogenic molecules.

[0010] Preferably, the S200 further includes: S203: Emitting a laser and focusing it on the cell with the highest concentration of pathogenic molecules in the sample to be tested, and using the optical tweezer effect to capture the cell with the highest concentration of pathogenic molecules.

[0011] Preferably, in the S202, control the first conjugate objective lens and the second conjugate objective lens located on both sides of the sample to be tested to perform mirror-symmetric sliding. When the high-frequency energy of the image obtained by Fourier transform analysis is the largest, achieve simultaneous focusing of the first conjugate objective lens and the second conjugate objective lens. After the focusing is completed, continue to analyze the secondary real image to find the pixel point with the largest gray value on the secondary real image.

[0012] Preferably, the 300 includes: S301: Driving the sample to be tested to move two-dimensionally. Due to the optical tweezer effect, the cell always remains in place and is extracted from the sample to be tested.

[0013] Preferably, the 300 further includes: S302: Converting the Rayleigh scattering formed by filtering and coupling the scattering generated by the cell into a spectral signal, and synchronously analyzing the ultraviolet resonance Raman and fluorescence in the spectral signal to judge the content of the main pathogenic molecules in the cell with the highest concentration of pathogenic molecules.

[0014] Preferably, the 300 further includes: S303: Scanning the frequency within a certain step in the spectral range. At each wavelength point, synchronously analyzing the ultraviolet resonance Raman and fluorescence at different wavelengths of the spectral signal to realize the analysis of the types and contents of other pathogenic molecules in pathological cells except the most main pathogenic molecule.

[0015] Preferably, the S100 includes: S101: According to the most main pathogenic molecule to be measured in the cell, determining its RET donor antigen molecule and selecting the donor fluorescent protein antibody; S102: According to its RET donor antigen molecule, determining the RET receptor antigen molecule and selecting the receptor fluorescent protein antibody; S103: Adding an appropriate amount of donor fluorescent protein and receptor fluorescent protein into the red blood cell suspension to be tested, adding a diluent, placing it in a stirrer for stirring, filtering and removing the excess fluorescent protein, and then smearing it into a monolayer cell to prepare a sample to be tested.

[0016] Compared with the prior art, the present invention has the following advantages: The present invention provides a method for detecting cell pathological molecules based on LCTF optical tweezers and RET. The RET subsystem and the LCTF subsystem are mutually coordinated. Through the analysis of the LCTF receptor emission wavelength image, according to the image pixel intensity (i.e., the image gray value), the pathological molecule and the cell where the pathological molecule is located are judged. An adjustable energy optical tweezer is used to capture pathological cells. Through the frequency scanning output in the RET subsystem, the spectral signal is synchronously analyzed for ultraviolet resonance Raman and fluorescence. Through the above technical route, the rapid identification and capture of pathological blood cells are realized, and the rapid, accurate and comprehensive detection of the pathological molecule tags of single cells is carried out. By using the tunable liquid crystal filter LCTF laser optical tweezer combined with the RET hyperspectral technology, it has the advantages of non-contact, label-free, non-destructive, etc. of the optical tweezer, and also has the characteristics of spectrum-image integration of hyperspectral imaging, and can provide more accurate spatial and spectral information in a wider spectral range about patients, tissue samples or different disease conditions. It can not only reflect the external quality characteristics such as the size, shape and defects of the sample, but also reflect the differences in its internal physical structure and chemical composition, and can quickly identify and capture abnormal cells. The resonance energy transfer RET spectrum can accurately label and detect specific pathological molecules, and the ultraviolet tunable resonance Raman spectrum can detect other abnormal molecular characteristics. The complementarity of the two spectra can improve the detection accuracy of specific molecules while achieving full coverage of other pathological molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic structural diagram of an analyzer according to an embodiment of the present invention.

[0018] Figure 2 It is a flowchart of the method for detecting cell pathological molecules of the present invention.

[0019] As shown in the figure, the load device 1; the RET subsystem 2, the ultraviolet continuous laser 21, the photonic crystal fiber 22, the RET frequency selector 23, the pump beam expander 24; the LCTF subsystem 3, the first conjugate objective lens 31, the LCTF tube lens 32, the LCTF lens 33, the area array CCD 34, the LCTF filter 35; the optical tweezer system 4, the optical tweezer laser 41, the energy regulator 42, the optical tweezer beam expander 43; the spectral detection subsystem 5, the RET conjugate cut-off filter 51, the coupling mirror 52, the fiber optic spectrometer 53; the main controller 6; the perforated ultraviolet total reflector 7; the second conjugate objective lens 8; the focusing device 9, the linear guide 91, the first moving motor 92, the second moving motor 93, the motor controller 94. Detailed implementation manners

[0020] To make the objectives, technical solutions and advantages of the embodiments of this article clearer, the following will clearly and completely describe the technical solutions in the embodiments of this article with reference to the accompanying drawings in the embodiments of this article. Obviously, the described embodiments are some, but not all, of the embodiments of this article. Based on the embodiments in this article, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of this article. It should be noted that, without conflict, the embodiments in this article and the features in the embodiments may be combined with each other arbitrarily.

[0021] Unless the context clearly requires otherwise, the words "including", "comprising" and similar words throughout the specification and claims shall be interpreted in an inclusive sense rather than an exclusive or exhaustive sense; that is, the meaning of "including but not limited to".

[0022] In the description of the disclosure of the present invention, it should be understood that the terms "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance. In addition, in the description of the disclosure of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0023] An embodiment of the present invention provides a method for detecting cell pathological molecules based on LCTF optical tweezers and RET. By using tunable liquid crystal filter LCTF laser optical tweezers combined with RET hyperspectral technology, it has the advantages of non-contact, label-free, non-destructive, etc. of optical tweezers, and the characteristic of spectrum-image integration of hyperspectral imaging. It can provide more accurate spatial and spectral information in a wider spectral range about patients, tissue samples or different disease conditions. It can not only reflect external quality characteristics such as the size, shape, and defects of the sample, but also reflect the differences in its internal physical structure and chemical composition, and can quickly identify and capture abnormal cells. The resonance energy transfer RET spectrum can accurately label and detect specific pathological molecules, and the ultraviolet tunable resonance Raman spectrum can detect other abnormal molecular characteristics. The complementarity of the two spectra can improve the detection accuracy of specific molecules while achieving full coverage of other pathological molecules.

[0024] Example 1

[0025] A method for detecting cell pathological molecules based on LCTF optical tweezers and RET, comprising:

[0026] S100: Pretreatment and sample preparation of the sample to be tested;

[0027] Specifically, the S100 includes:

[0028] S101: Determine the donor antigen molecule of its RET according to the most important pathogenic molecule to be measured in the cell, select the donor fluorescent protein antibody;

[0029] S102: Determine the receptor antigen molecule of RET according to its donor antigen molecule, and select the receptor fluorescent protein antibody;

[0030] S103: Add an appropriate amount of donor fluorescent protein and receptor fluorescent protein to the red blood cell suspension to be tested, add a diluent, place it on a stirrer and stir, after filtering and removing the excess fluorescent protein, smear it as a single-layer cell, and prepare a sample to be a sample to be tested.

[0031] S200: Make the donor protein molecule and the receptor protein molecule in the sample to be tested undergo resonance energy transfer, obtain the real image signal generated by the fluorescence emitted by the receptor protein, thereby obtaining the position of the cell with the highest concentration of pathogenic molecules in the sample to be tested, and obtain the cell through optical tweezers;

[0032] Specifically, the S200 includes:

[0033] S201: Output a single-frequency ultraviolet continuous laser equal to the excitation wavelength of the donor fluorescent protein, irradiate it to the sample to be tested after broadening and frequency selection, the donor protein molecule and the receptor protein molecule in the sample to be tested undergo resonance energy transfer, the donor emits fluorescence to excite the receptor fluorescent protein molecule, and the receptor emits fluorescence after being excited;

[0034] S202: After filtering the fluorescence emitted by the sample to be tested, form a secondary real image through secondary imaging, calculate the high-frequency energy of the two-dimensional Fourier transform of the secondary real image, adjust the imaging parameters, when the high-frequency energy of the image is the largest, find the pixel point with the largest gray value on the secondary real image, move the sample to be tested so that the pixel point with the largest gray value coincides with the main optical axis, and this pixel point is the position of the cell with the highest concentration of pathogenic molecules;

[0035] Among them, control the first conjugate objective lens and the second conjugate objective lens located on both sides of the sample to be tested to perform mirror-symmetric sliding. When the high-frequency energy of the image obtained by Fourier transform analysis is the largest, realize the simultaneous focusing of the first conjugate objective lens and the second conjugate objective lens. After the focusing is completed, continue to analyze the secondary real image and find the pixel point with the largest gray value on the secondary real image.

[0036] S203: Emit a laser and focus it on the cell with the highest concentration of pathogenic molecules in the sample to be measured, and capture the cell with the highest concentration of pathogenic molecules using the optical tweezers effect.

[0037] S300: Obtain the spectral signal converted from the scattered light generated by the cell after filtering and coupling, and perform synchronous analysis of ultraviolet resonance Raman and fluorescence on the spectral signal to determine the content of the main pathogenic molecule in the cell with the highest concentration of pathogenic molecules.

[0038] Specifically, step 300 includes:

[0039] S301: Drive the sample to be measured to move two-dimensionally. Due to the optical tweezers effect, the cell always remains in place and is extracted from the sample to be measured.

[0040] S302: Convert the Rayleigh scattering formed by filtering and coupling the scattered light generated by the cell into a spectral signal, and perform synchronous analysis of ultraviolet resonance Raman and fluorescence on the spectral signal to determine the content of the main pathogenic molecule in the cell with the highest concentration of pathogenic molecules.

[0041] S303: Perform frequency sweeping at a certain step size within the spectral range. At each wavelength point, perform synchronous analysis of ultraviolet resonance Raman and fluorescence at different wavelengths on the spectral signal to realize the analysis of the types and contents of other pathogenic molecules in the pathological cells except the main pathogenic molecule.

[0042] Embodiment 2

[0043] As Figure 1 shown, in this embodiment, an analyzer is included. The analyzer includes a sample stage device 1, a RET subsystem 2, an LCTF subsystem 3, an optical tweezers system 4, a spectral detection subsystem 5, a main controller 6, a perforated ultraviolet total reflection mirror 7, a second conjugate objective lens 8, and a focusing device 9. Among them, the RET subsystem 2 includes an ultraviolet continuous laser 21, a photonic crystal fiber 22, a RET frequency selector 23, and a pump beam expander 24; the LCTF subsystem 3 includes a first conjugate objective lens 31, an LCTF tube lens 32, an LCTF lens 33, a frame CCD 34, and an LCTF filter 35; the optical tweezers system 4 includes an optical tweezers laser 41, an energy regulator 42, and an optical tweezers beam expander 43; the spectral detection subsystem 5 includes a RET conjugate cut-off filter 51, a coupling mirror 52, and an optical fiber spectrometer 53; the focusing device 9 includes a linear guide rail 91, a first moving motor 92, a second moving motor 93, and a motor controller 94.

[0044] In this embodiment, the analyzer is used for cell pathological molecule detection. Specifically, it includes:

[0045] S100: Pretreatment and sample preparation of the sample to be measured;

[0046] Specifically, the S100 includes:

[0047] S101: According to the most important pathogenic molecule to be measured in the cell (in this embodiment, glycated hemoglobin in the red blood cells of type II diabetes patients), determine its RET donor antigen molecule (in this embodiment, the β-chain of glycated hemoglobin HbA1C in type II diabetes patients), and select the donor fluorescent protein antibody (in this embodiment, its labeled fluorophore is Methoxycoumarin, excitation wavelength 360 nm, emission wavelength 410 nm);

[0048] S102: According to its RET donor antigen molecule, determine the RET receptor antigen molecule (in this embodiment, the glycated dipeptide at the N-terminus of the β-chain of glycated hemoglobin HbA1C in type II diabetes patients), and select the receptor fluorescent protein antibody (in this embodiment, its labeled fluorophore is Pacific Orange, excitation wavelength 400 - 410 nm, emission wavelength 551 nm);

[0049] S103: Add an appropriate amount of donor fluorescent protein and receptor fluorescent protein to the red blood cell suspension to be tested, add a diluent, place it on a stirrer and stir. After filtration and removal of the excess fluorescent protein, smear it as a single-layer cell and prepare a sample to be tested, and fix it on the loading device 1.

[0050] S200: Cause the donor protein molecule and the receptor protein molecule in the sample to be tested to undergo resonance energy transfer, obtain the real image signal generated by the fluorescence emission of the receptor protein, thereby obtain the position of the cell with the highest concentration of pathogenic molecules in the sample to be tested, and obtain the cell through an optical tweezer;

[0051] Specifically, the S200 includes:

[0052] S201: The main controller 6 issues an instruction to turn on the ultraviolet continuous laser 21 and adjust the output wavelength of the RET frequency selector 23 to be equal to the excitation wavelength of the donor fluorescent protein (360 nm in this embodiment). The single-frequency ultraviolet continuous laser emitted by the ultraviolet continuous laser 21 along the RET optical axis is broadened in the ultraviolet band by the photonic crystal fiber 22, output by the RET frequency selector 23, and then reflected by the perforated ultraviolet total reflector 7, and then passes through the central hole of the perforated optical tweezer reflector along the main optical axis, and then passes through the second conjugate objective lens 8 and irradiates the sample to be tested. Under the irradiation of the ultraviolet laser, the most important pathogenic molecule in the pathological cells of the sample to be tested causes its donor fluorescent protein to emit fluorescence (410 nm in this embodiment). Also, because the distance between the donor protein molecule and the receptor protein molecule is very small (within 10 nm), the RET condition is satisfied, resonance energy transfer occurs, the donor emits fluorescence to excite the receptor fluorescent protein molecule, and the receptor emits fluorescence (551 nm in this embodiment), and this fluorescence only exists in the pathological cells.

[0053] S202: The main controller 6 issues an instruction to turn on the LCTF wavelength controller. The LCTF wavelength controller adjusts the central wavelength of the LCTF filter 35 to the emission wavelength of the acceptor fluorescent protein (551 nm in this embodiment). At this time, the fluorescence emitted by the sample to be measured, after passing through the first conjugate objective lens 31, can form an enlarged real image imaged at infinity. After passing through the LCTF tube lens 32, the enlarged real image at infinity is pulled closer to the front side of the LCTF tube lens 32 to form a primary real image. The primary real image is then imaged by the LCTF lens 33 to form a secondary real image signal in front of the area array CCD 34, and the secondary real image signal is sent to the main controller 6. The main controller 6 calculates the high-frequency energy of the two-dimensional Fourier transform image of the secondary real image in real time. At the same time, the main controller 6 issues an instruction to start the motor controller 94. The motor controller 94 sends control instructions to the first moving motor 92 and the second moving motor 93 to drive the first conjugate objective lens 31 and the second conjugate objective lens 8 to slide in a mirror-symmetrical manner. When the high-frequency energy of the image obtained by Fourier transform analysis is the largest, simultaneous focusing of the first conjugate objective lens 31 and the second conjugate objective lens 8 is achieved. After focusing is completed, the main controller 6 continues to analyze the secondary real image to find the pixel point with the largest gray value. The motor controller 94 is used to send control instructions to the sample stage device 1 (the sample stage device 1 is a two-dimensional electric sample stage), and the sample stage device 1 drives the sample to be measured to perform two-dimensional fine movement (move left and right in the illustrated direction) until the pixel point with the largest gray value coincides with the principal optical axis. Among them, the pixel point with the largest gray value means that the emission fluorescence intensity of the acceptor fluorescent protein molecule is the largest, and the position of this point is the position of the cell with the highest concentration of pathogenic molecules.

[0054] S203: The main controller 6 issues an instruction to start the energy regulator 42. The energy regulator 42 turns on to adjust the output energy of the optical tweezer laser 41. The laser emitted by the optical tweezer laser 41 is focused on the cell with the highest concentration of pathogenic molecules in the sample to be measured, and the cell with the highest concentration of pathogenic molecules is captured using the optical tweezer effect.

[0055] S300: Obtain the spectral signal converted after filtering and coupling of the scattering generated by this cell, and perform synchronous analysis of ultraviolet resonance Raman and fluorescence on the spectral signal to determine the content of the main pathogenic molecule in the cell with the highest concentration of pathogenic molecules.

[0056] Specifically, step 300 includes:

[0057] S301: The motor controller 94 sends control instructions to the two-dimensional electric sample stage to drive the sample to be measured to perform two-dimensional movement. Due to the optical tweezer effect, the cell with the highest concentration of pathogenic molecules that is captured always remains in place and does not move with the two-dimensional electric sample stage. In this way, this cell is gradually pulled out from the sample to be measured. After being pulled out, the two-dimensional electric sample stage stops moving;

[0058] S302: The cut-off wavelength of the RET conjugate cut-off filter 51 controlled and adjusted by the main controller 6 is always equal to the output wavelength of the RET frequency selector 23, and this wavelength is the excitation wavelength of the donor fluorescent protein (360 nm in this embodiment); the backward scattering (scattering towards the direction of the spectral detection subsystem 5) generated by the cell with the highest pathogenic molecule concentration is filtered by the RET conjugate cut-off filter 51 to remove the Rayleigh scattering of the corresponding wavelength of the RET frequency selector 23, and is coupled into the fiber optic spectrometer 53 through the coupling mirror 52 and converted into a spectral signal. This spectral signal is transmitted to the main controller 6, and the main controller 6 analyzes the spectral signal corresponding to the emission wavelength of the acceptor fluorescent protein (551 nm in this embodiment), and determines the content of the most important pathogenic molecule (glycated hemoglobin in the red blood cells of type II diabetic patients in this embodiment) in the cell with the highest pathogenic molecule concentration based on its intensity.

[0059] S303: The main controller 6 synchronously adjusts the cut-off wavelengths of the RET frequency selector 23 and the RET conjugate cut-off filter 51 to perform frequency sweeping in the spectral range of the photonic crystal fiber 22 (213 - 400 nm in this embodiment) with a certain step size (1 nm in this embodiment); at each wavelength point, the main controller 6 synchronously analyzes the ultraviolet resonance Raman and fluorescence of the spectral signal of the fiber optic spectrometer 53 at different wavelengths, so as to realize the analysis of the types and contents of other pathogenic molecules in the pathological cells except for the most important pathogenic molecule.

[0060] In this embodiment, laser optical tweezers capture combined with LCTF and RET is adopted. By analyzing the LCTF image at the emission wavelength of the RET receptor, the cell with the highest content of the most important pathogenic molecule is judged, and the tunable energy optical tweezers are used for capture and extraction. Through the frequency-swept output in the ultraviolet RET subsystem, the synchronous detection of ultraviolet frequency-swept resonance Raman and fluorescence is completed. Frequency sweeping is performed in the spectral range with a certain step size. At each wavelength point, the spectral signal is synchronously analyzed for ultraviolet resonance Raman and fluorescence at different wavelengths, so as to realize the analysis of the types and contents of other pathogenic molecules in the pathological cells except for the most important pathogenic molecule.

[0061] In summary, the present invention proposes a method for detecting cell pathological molecules, which adopts the mutual cooperation of the RET subsystem and the LCTF subsystem. By analyzing the LCTF receptor emission wavelength image, based on the image pixel intensity, that is, the image gray value, the pathological molecule and the cell where the pathological molecule is located are judged. The tunable liquid crystal filter LCTF laser optical tweezers are used for capturing pathological cells. Through the frequency-swept output in the RET subsystem, the spectral signal is synchronously analyzed for ultraviolet resonance Raman and fluorescence, so as to realize the rapid identification and capture of pathological blood cells, and the rapid, accurate and comprehensive detection of the pathological molecule tags of single cells.

[0062] The above are only the preferred embodiments of the present invention. The protection scope of the present invention shall be subject to the scope defined by the claims. Several improvements and refinements made by those skilled in the art without departing from the spirit and scope of the present invention shall also be regarded as within the protection scope of the present invention.

Claims

1. A method for detecting cell pathological molecules based on LCTF optical tweezers and RET, characterized in that, S100: Pretreatment and sample preparation of the sample to be tested; S200: Cause resonance energy transfer between the donor protein molecules and the acceptor protein molecules in the sample to be tested, obtain the real image signal generated by the fluorescence emitted by the acceptor protein, thereby obtain the position of the cell with the highest concentration of pathogenic molecules in the sample to be tested, and capture the cell through optical tweezers; The S200 includes: S201: The main controller issues an instruction to turn on the ultraviolet continuous laser and adjust the output wavelength of the RET frequency selector to be equal to the excitation wavelength of the donor fluorescent protein. The single-frequency ultraviolet continuous laser emitted by the ultraviolet continuous laser along the RET optical axis is broadened in the ultraviolet band by the photonic crystal fiber, output by the RET frequency selector, and then reflected by the perforated ultraviolet total reflector, and then passes through the central hole of the perforated optical tweezers mirror along the main optical axis, and then passes through the second conjugate objective lens and irradiates the sample to be tested. Under the irradiation of the ultraviolet laser, the donor fluorescent protein in the pathological cells of the sample to be tested is excited to emit fluorescence, and resonance energy transfer occurs. The fluorescence emitted by the donor excites the acceptor fluorescent protein molecules, and the acceptor is excited to emit fluorescence; S202: The main controller issues an instruction to turn on the LCTF wavelength controller. The LCTF wavelength controller adjusts the central wavelength of the LCTF filter to the emission wavelength of the acceptor fluorescent protein; at this time, the fluorescence emitted by the sample to be tested forms an enlarged real image imaged at infinity after passing through the first conjugate objective lens. After passing through the LCTF tube lens, the enlarged real image at infinity is pulled closer to the front side of the LCTF tube lens to form a primary real image. The primary real image is then imaged by the LCTF lens to the front side of the area array CCD to form a secondary real image signal, and the secondary real image signal is sent to the main controller; the main controller calculates the high-frequency energy of the two-dimensional Fourier transform of the secondary real image in real time; at the same time, the main controller issues an instruction to start the motor controller. The motor controller sends control instructions to the first moving motor and the second moving motor to drive the first conjugate objective lens and the second conjugate objective lens to slide symmetrically in the mirror direction. When the high-frequency energy of the image obtained by Fourier transform analysis is the largest, simultaneous focusing of the first conjugate objective lens and the second conjugate objective lens is achieved; after focusing is completed, the main controller continues to analyze the secondary real image, finds the pixel point with the largest gray value, and the motor controller is used to send control instructions to the sample stage device. The sample stage device drives the sample to be tested to move two-dimensionally and finely until the pixel point with the largest gray value coincides with the main optical axis; S203: The main controller issues an instruction to start the energy regulator. The energy regulator turns on to adjust the output energy of the optical tweezers laser; the laser emitted by the optical tweezers laser is focused on the cell with the highest concentration of pathogenic molecules in the sample to be tested, and the cell with the highest concentration of pathogenic molecules is captured by using the optical tweezers effect; S300: Obtain the spectral signal converted by filtering and coupling the scattering generated by the cell, and perform synchronous analysis of ultraviolet resonance Raman and fluorescence on the spectral signal to judge the content of the main pathogenic molecule in the cell with the highest concentration of pathogenic molecules.

2. The method for detecting cell pathological molecules based on LCTF optical tweezers and RET according to claim 1, characterized in that, The S200 includes: S201: By outputting a single-frequency ultraviolet continuous laser equal to the excitation wavelength of the donor fluorescent protein, after broadening and frequency selection, it irradiates the sample to be tested. Resonance energy transfer occurs between the donor protein molecules and the acceptor protein molecules in the sample to be tested. The fluorescence emitted by the donor excites the acceptor fluorescent protein molecules, and the acceptor emits fluorescence upon excitation.

3. The method for detecting cell pathological molecules based on LCTF optical tweezers and RET according to claim 2, characterized in that, The S200 further includes: S202: The fluorescence emitted by the sample to be tested forms a secondary real image after filtering and secondary imaging. Calculate the high-frequency energy of the image of the two-dimensional Fourier transform of the secondary real image, adjust the imaging parameters. When the high-frequency energy of the image is the maximum, find the pixel point with the maximum gray value on the secondary real image, and move the sample to be tested so that the pixel point with the maximum gray value coincides with the principal optical axis. This pixel point is the position where the cell with the highest concentration of pathogenic molecules is located.

4. The method for detecting cell pathological molecules based on LCTF optical tweezers and RET according to claim 3, characterized in that, The S200 further includes: S203: Emit a laser and focus it on the cell with the highest concentration of pathogenic molecules in the sample to be tested, and use the optical tweezer effect to capture the cell with the highest concentration of pathogenic molecules.

5. The method for detecting cell pathological molecules based on LCTF optical tweezers and RET according to claim 3, characterized in that, In the S202, control the first conjugate objective lens and the second conjugate objective lens located on both sides of the sample to be tested to perform mirror-symmetrical sliding. When the high-frequency energy of the image obtained by Fourier transform analysis is the maximum, achieve the simultaneous focusing of the first conjugate objective lens and the second conjugate objective lens. After the focusing is completed, continue to analyze the secondary real image and find the pixel point with the maximum gray value on the secondary real image.

6. The method for detecting cell pathological molecules based on LCTF optical tweezers and RET according to claim 1, characterized in that, The 300 includes: S301: Drive the sample to be tested to move two-dimensionally. Due to the optical tweezer effect, the cell always remains in place and is extracted from the sample to be tested.

7. The method for detecting cell pathological molecules based on LCTF optical tweezers and RET according to claim 6, characterized in that, The 300 further includes: S302: Convert the Rayleigh scattering formed by filtering and coupling the scattering generated by the cell into a spectral signal, and perform synchronous analysis of ultraviolet resonance Raman and fluorescence on the spectral signal to determine the content of the most main pathogenic molecule in the cell with the highest concentration of pathogenic molecules.

8. The method for detecting cell pathological molecules based on LCTF optical tweezers and RET according to claim 7, characterized in that, The 300 further includes: S303: Perform frequency sweeping at a certain step size within the spectral range. At each wavelength point, perform synchronous analysis of ultraviolet resonance Raman and fluorescence at different wavelengths on the spectral signal to achieve the analysis of the types and contents of other pathogenic molecules in the pathological cells except the most main pathogenic molecule.

9. The method for detecting cell pathological molecules based on LCTF optical tweezers and RET according to claim 1, characterized in that, The S100 includes: S101: According to the most main pathogenic molecule to be measured in the cell, determine its RET donor antigen molecule and select the donor fluorescent protein antibody; S102: According to its RET donor antigen molecule, determine the RET acceptor antigen molecule and select the acceptor fluorescent protein antibody; S103: Add an appropriate amount of donor fluorescent protein and acceptor fluorescent protein to the red blood cell suspension to be tested, add a diluent, place it on a stirrer and stir. After filtering and removing the excess fluorescent protein, smear it as a single-layer cell to prepare a sample to be tested.

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