CN114813706B - Blood cell hyperspectral optical tweezers capture energy resonance transfer analyzer - Google Patents
Blood cell hyperspectral optical tweezers capture energy resonance transfer analyzer Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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Abstract
The invention discloses a blood cell hyperspectral optical tweezers trapping energy resonance transfer analyzer, which comprises: the device comprises a carrying device, an RET subsystem, an LCTF subsystem, an optical tweezers subsystem, a spectrum detection subsystem and a main controller, wherein the RET subsystem and the LCTF subsystem are matched with each other, and the pathological molecules and cells where the pathological molecules are located are judged according to image pixel intensity values through LCTF receptor emission wavelength image analysis.
Description
Technical Field
The invention relates to the field of single cell medical analyzers, in particular to a blood cell hyperspectral optical tweezer trapping energy resonance transfer analyzer based on tunable liquid crystal filter (LCTF) laser optical tweezer trapping and tunable Resonance Energy Transfer (RET).
Background
Hemoglobin in erythrocytes of type II diabetic patients combines with free glucose in blood to form glycated hemoglobin, which can be used as a gold standard for analyzing long-term blood glucose detection of type II diabetic patients; hemoglobin of various anemia patients has the characteristics of structural abnormality, erythrocyte morphological change and the like; leukemia comprises a plurality of subclasses, the conventional rapid detection method is to count the white blood cells, the subclasses cannot be identified, and the leukemia subclasses need to be diagnosed through the pathological indexes of molecular tags in the white blood cells. The conventional pathological detection method cannot carry out rapid, accurate, trace and nondestructive detection on the single-cell molecular label.
The acquisition means of the pathological indexes of the blood diseases needs to be combined with refinement and rapidness, and the trace amount and the nondestructive property are combined. The implementation of analytical techniques that can rely on the single cell level. The single cell analysis techniques mainly include micro-spectrochemical analysis (such as molecular vibration spectroscopy methods including raman spectroscopy, resonance raman, surface enhanced raman, coherent raman, infrared micro-spectroscopy, etc.), fluorescence-mediated micro-analysis (such as those requiring fluorescent staining, fluorescence microscopy, fluorescence lifetime imaging, fluorescence-related spectroscopy, etc.), mass spectrometry, nuclear magnetic resonance, etc. Single cell analysis often requires a combination of pre-processing and cell capture techniques to find abnormal cells and perform accurate detection.
The sample preparation process of some existing technical methods is complex, and long analysis time is needed; in addition, in the cell test sample of an actual patient, there are a very large number of cells including both normal cells and abnormal cells, and the abnormal cells have morphological differences, do not have morphological differences, but have changed characteristics of the molecules. In the micro-spectrum and fluorescence-mediated analysis, how to combine with the rapid screening of the normal abnormal cells with high efficiency and the accurate single-cell capture is a very difficult problem.
The laser optical tweezers are combined with a hyperspectral technology, so that the advantages of non-contact, no need of marking, no damage and the like of the optical tweezers are achieved, the atlas unification characteristic of hyperspectral imaging is achieved, accurate spatial and spectral information about a patient, a tissue sample or different disease conditions in more spectral ranges can be provided, external quality characteristics such as the size, the shape and the defects of the sample can be reflected, the difference of the internal physical structure and chemical components of the sample can be reflected, and abnormal cells can be quickly distinguished and captured.
The resonance energy transfer can accurately mark and detect specific pathological molecules, the tunable Raman technology can detect other abnormal molecular characteristics, and the complementation of the integrated laser spectrum can improve the detection accuracy of the specific molecules and realize the full coverage of other pathological molecules.
Disclosure of Invention
The invention provides a blood cell hyperspectral optical tweezers trapping energy resonance transfer analyzer which uses a tunable liquid crystal filter LCTF laser optical tweezers to trap pathological blood cells and combines tunable Resonance Energy Transfer (RET) laser molecular spectrum comprehensive analysis to realize rapid discrimination and trapping of pathological blood cells and rapid, accurate and comprehensive detection of single-cell pathological molecular labels.
The invention adopts the following technical scheme:
a blood cell hyperspectral optical tweezers capture energy resonance transfer analyzer comprises: the object carrying device is used for placing a sample to be detected; the RET subsystem is used for transmitting laser with preset wavelength to a sample to be detected so that resonance energy transfer occurs between donor protein molecules and acceptor protein molecules and the acceptor protein emits fluorescence; the LCTF subsystem forms a real image signal according to the fluorescence emitted by the receptor protein; the optical tweezers system emits laser to focus on the cells with the maximum concentration of pathogenic molecules of the sample to be detected and captures the cells; a spectrum detection subsystem for acquiring the spectrum signal of the cell; the main controller is electrically connected with the carrying device, the RET subsystem, the LCTF subsystem, the optical tweezers subsystem and the spectrum detection subsystem, and is used for controlling the carrying device to be matched with the LCTF subsystem to judge the cell position with the maximum concentration of pathogenic molecules; and the content of the pathogenic molecules is judged through the spectrum signal.
Preferably, the RET subsystem comprises: the ultraviolet continuous laser is used for emitting single-frequency ultraviolet continuous laser; the photonic crystal fiber is used for broadening the wavelength of the ultraviolet continuous laser; and the RET frequency selector is used for outputting the stretched certain monochromatic light.
Preferably, the system further comprises a perforated ultraviolet total reflection mirror positioned between the RET subsystem and the carrying device.
Preferably, the LCTF subsystem comprises: the first conjugate objective is used for imaging a sample to be detected at an infinite distance; the LCTF tube lens draws the amplified real image at infinity to one side of the LCTF tube lens and forms a primary real image; the LCTF lens is used for forming a first-level real image into a second-level real image on one side of the LCTF lens; and the area array CCD is electrically connected with the main controller and is used for imaging the secondary real image and transmitting the secondary real image to the main controller.
Preferably, the device further comprises a second conjugate objective lens, and the first conjugate objective lens and the second conjugate objective lens are symmetrically positioned on two sides of the carrying device.
Preferably, the focusing device is used for controlling the first conjugate objective lens and the second conjugate objective lens to move symmetrically.
Preferably, the focusing device includes: a linear guide rail; the first moving motor is positioned at one end of the linear guide rail and connected to the first conjugate objective lens; the second moving motor is positioned at the other end of the linear guide rail and connected to the second conjugate objective lens; and the motor controller is electrically connected with the first moving motor and the second moving motor and is used for controlling the first conjugate objective lens and the second conjugate objective lens to symmetrically move relative to the sample to be detected.
Preferably, an LCTF filter electrically connected to a wavelength controller is disposed between the first conjugate objective lens and the LCTF tube lens, and the wavelength controller is electrically connected to a main controller.
Preferably, the optical tweezers system comprises: the optical tweezers laser is used for emitting laser; the energy regulator is positioned on one side of the optical tweezers laser and is used for regulating the energy of the laser emitted by the optical tweezers laser; and the optical tweezers beam expander is positioned on the other side of the optical tweezers laser and is used for expanding the laser emitted by the optical tweezers laser.
Preferably, the spectral detection subsystem comprises: the RET conjugate cut-off sheet is used for filtering light scattering of the corresponding wavelength of the RET frequency selector in the light scattering generated by the sample to be detected; a coupling mirror for coupling the residual light passing through the RET conjugate cut-off sheet; and the fiber spectrometer converts the coupled residual light into a spectrum signal and transmits the spectrum signal to the main controller.
Compared with the prior art, the invention has the following advantages: the invention provides a blood cell hyperspectral optical tweezers capture energy resonance transfer analyzer, which adopts a RET subsystem and an LCTF subsystem to be matched with each other, judges pathological molecules and cells where the pathological molecules are located according to image pixel intensity (namely image gray scale) values through LCTF receptor emission wavelength image analysis, adopts adjustable energy optical tweezers to capture the pathological cells, judges the content of pathogenic molecules through sweep frequency output in the RET subsystem and synchronous analysis of ultraviolet resonance Raman and fluorescence in spectral signals, and realizes quick discrimination and capture of pathological blood cells and quick, accurate and comprehensive detection of pathological molecule labels of single cells through the technical route.
Drawings
FIG. 1 is a schematic view of the present invention.
In the figure, an object carrying device 1, a RET subsystem 2, an ultraviolet continuous laser 21, a photonic crystal fiber 22, a RET frequency selector 23, a pump beam expander 24, an LCTF subsystem 3, a first conjugate objective lens 31, an LCTF tube lens 32, an LCTF lens 33, an area array CCD34, an LCTF filter 35, an optical tweezer subsystem 4, an optical tweezer laser 41, an energy regulator 42, an optical tweezer beam expander 43, a spectrum detection subsystem 5, a RET conjugate cut-off sheet 51, a coupling mirror 52, a fiber spectrometer 53, a main controller 6, a perforated ultraviolet total reflection mirror 7, a second conjugate objective lens 8, a focusing device 9, a linear guide rail 91, a first moving motor 92, a second moving motor 93 and a motor controller 94.
Detailed Description
In order to facilitate understanding of the technical solutions of the present invention, the following detailed description is made with reference to the accompanying drawings and specific embodiments.
Example 1
As shown in fig. 1, a blood cell hyperspectral optical tweezer trapping energy resonance transfer analyzer comprises:
a carrying device 1 for placing a sample to be tested and moving in the horizontal direction; the carrying device 1 is a two-dimensional electric carrying plate which is of a hollow design and facilitates front-back detection along a main optical axis;
the RET subsystem 2 emits laser with a preset wavelength to a sample to be detected, so that resonance energy transfer is generated between donor protein molecules and acceptor protein molecules, and the acceptor protein emits fluorescence;
specifically, the RET subsystem 2 includes an ultraviolet continuous laser 21, a photonic crystal fiber 22, an RET frequency selector 23, and a pump beam expander 24; the single-frequency ultraviolet continuous laser (in this embodiment, the wavelength is 213 nm) emitted by the ultraviolet continuous laser 21 along the RET optical axis is broadened in an ultraviolet section (in this embodiment, 213nm to 400 nm) by the photonic crystal fiber 22, a certain monochromatic light (in this embodiment, a certain monochromatic light within the range of 213nm to 400 nm) is output by the RET frequency selector 23 according to the frequency selection of the RET frequency selector 23, the output wavelength is equal to the excitation wavelength of the donor fluorescent protein in the sample cell to be detected), the light output by the RET frequency selector 23 is reflected by the ultraviolet total reflection mirror with holes 7, passes through the central hole of the light-absorbing reflector along the main optical axis, and then passes through the second conjugate objective 8 to be focused on the sample to be detected, the most main pathogenic molecules in the pathological cells of the sample to be detected are irradiated by the ultraviolet laser, the donor fluorescent protein thereof is excited to emit fluorescence, and the distance between the donor protein molecules and the acceptor protein molecules is small, so that the RET condition is satisfied, resonance energy transfer occurs, the donor fluorescent protein molecules excite the acceptor fluorescent protein molecules, and the acceptor fluorescent protein molecules are excited to emit fluorescence.
The ultraviolet total reflection mirror 7 with the hole can reflect the light in the ultraviolet wide spectrum wavelength range after the photonic crystal fiber 22 is widened, and transmits the light in the wavelength range larger than the wavelength range.
The light-shooting reflecting mirror with holes and the ultraviolet full reflecting mirror 7 with holes are mutually parallel and are arranged in an inclined way at 45 degrees.
The LCTF subsystem 3 forms a real image signal based on the fluorescence emitted by the receptor protein.
The LCTF subsystem 3 comprises a first conjugate objective lens 31, an LCTF tube lens 32, an LCTF lens 33, an area array CCD34 and an LCTF filter 35;
the first conjugate objective 31 is a microscope objective (this embodiment is an infinite imaging, 20-magnification, 200-800nm wavelength ultraviolet enhancement, long working distance microscope objective).
After passing through the first conjugate objective 31, the sample to be measured at the focus of the first conjugate objective 31 can form an amplified real image imaged at infinity, the amplified real image at infinity is zoomed in to the front side of the LCTF tube lens 32 after passing through the LCTF tube lens 32 to form a primary real image, the primary real image is imaged to the front side of the area array CCD34 through the LCTF lens 33 to form a secondary real image signal, and the area array CCD34 sends the secondary real image signal to the main controller 6.
The LCTF filter 35 is located between the LCTF tube lens 32 and the first conjugate objective lens 31, and selects an imaging wavelength, wherein the final imaging wavelength is the central wavelength of the LCTF filter 35, and the central wavelength is equal to the fluorescence wavelength emitted by the receptor fluorescent protein in the sample cell to be detected.
In this embodiment, the center wavelength of the LCTF filter 35 is controlled by the LCTF wavelength controller.
The first conjugate objective lens 31 and the second conjugate objective lens 8 are symmetrically positioned at two sides of the carrying device 1, and the first conjugate objective lens 31 and the second conjugate objective lens 8 are controlled to symmetrically move by the focusing device 9.
The focusing device 9 comprises a linear guide rail 91, a first moving motor 92, a second moving motor 93 and a motor controller 94; the first moving motor 92 is located at one end of the linear guide 91 and connected to the first conjugate objective lens 31; the second moving motor 93 is positioned at the other end of the linear guide rail 91 and is connected to the second conjugate objective lens 8; the motor controller 94 is electrically connected to the first moving motor 92 and the second moving motor 93, and is configured to control the first conjugate objective lens 31 and the second conjugate objective lens 8 to symmetrically move with respect to the sample to be measured, so as to achieve simultaneous focusing of the first conjugate objective lens 31 and the second conjugate objective lens 8, and the determination of the focus point depends on the two-dimensional fourier transform of the two-level real image of the LCTF subsystem 3, and when the high-frequency energy of the image obtained by the fourier transform analysis is maximum, it indicates that the image is in the focus point. In addition, the motor controller 94 is further configured to send a control command to the object carrying device 1, so that the object carrying device drives the sample to be measured to perform two-dimensional fine movement.
The optical tweezers system 4 is used for emitting laser to focus on the cells with the maximum concentration of pathogenic molecules of the sample to be detected and capturing the cells;
the optical tweezers system 4 comprises an optical tweezers laser 41, an energy regulator 42 and an optical tweezers beam expander 43; the optical tweezers laser 41 is used for emitting laser (532 nm in this embodiment); the energy regulator 42 is located at one side of the optical tweezers laser 41, and is used for regulating the energy of the laser emitted by the optical tweezers laser 41 to achieve an optimal capture effect; the optical tweezers beam expander 43 is located on the other side of the optical tweezers laser 41 and is used for expanding the laser beam emitted by the optical tweezers laser 41, the expanded laser beam is reflected by the holed light shooting reflector, and the second conjugate objective lens 8 is focused on pathological cells in the sample to be detected and captures the pathological cells by using the optical tweezers effect.
A spectrum detection subsystem 5 for acquiring the spectrum signal of the pathological cell;
the spectrum detection subsystem 5 comprises a RET conjugate cut-off sheet 51, a coupling mirror 52 and a fiber spectrometer 53; the RET conjugate cut-off sheet 51 is used for filtering light scattering of the corresponding wavelength of the RET frequency selector 23 in the light scattering generated by the sample to be measured; the coupling mirror 52 is used for coupling the residual light passing through the RET conjugate cut-off plate 51; the fiber spectrometer 53 converts the coupled residual light into a spectral signal and transmits the spectral signal to the main controller 6, the back scattering generated by the sample to be tested passes through the second conjugate objective 8 along the main optical axis, passes through the central holes of the light pickup reflector with holes and the ultraviolet total reflection mirror 7 with holes, then passes through the RET conjugate cut-off sheet 51 to filter the wavelength corresponding to the RET frequency selector 23, and then is coupled by the coupling mirror 52 and enters the fiber spectrometer 53 to be converted into a spectral signal, and the spectral signal is transmitted to the main controller 6 for analysis.
The main controller 6 is electrically connected with the loading device 1, the RET subsystem 2, the LCTF subsystem 3, the optical tweezers subsystem 4, the spectrum detection subsystem 5 and the focusing device 9, and is used for controlling the start and stop of the LCTF wavelength controller, the ultraviolet continuous laser 21, the energy regulator 42 and the motor controller 94, regulating the output wavelength of the RET frequency selector 23 and the cut-off wavelength of the RET conjugate cut-off plate 51 equal to the output wavelength, receiving the two-dimensional real image data output by the area array CCD34 and the spectrum data output by the fiber spectrometer 53, and judging the content of pathogenic molecules by analyzing the ultraviolet resonance raman and fluorescence signals of the spectrum.
The above is only a preferred embodiment of the present invention, and the scope of the present invention is defined by the scope defined by the claims, and a plurality of modifications and amendments made by those skilled in the art without departing from the spirit and scope of the present invention should be considered as the scope of the present invention.
Claims (7)
1. A blood cell hyperspectral optical tweezers trapping energy resonance transfer analyzer is characterized by comprising:
the object carrying device (1) is used for placing a sample to be tested;
the RET subsystem (2) is used for transmitting laser with preset wavelength to a sample to be detected, so that resonance energy transfer is generated between donor protein molecules and acceptor protein molecules, and the acceptor protein emits fluorescence;
an LCTF subsystem (3) for forming a real image signal based on fluorescence emitted by the receptor protein;
the optical tweezers system (4) emits laser to focus on the cells with the maximum concentration of pathogenic molecules of the sample to be detected and captures the cells;
a spectral detection subsystem (5) for acquiring a spectral signal of the cell;
the main controller (6) is electrically connected with the carrying device (1), the RET subsystem (2), the LCTF subsystem (3), the optical tweezers subsystem (4) and the spectrum detection subsystem (5), controls the carrying device (1) and the LCTF subsystem (3) to be matched for judging the cell position with the maximum concentration of pathogenic molecules, and judges the content of the pathogenic molecules through spectrum signals;
wherein the RET subsystem (2) comprises:
an ultraviolet continuous laser (21) for emitting a single-frequency ultraviolet continuous laser;
a photonic crystal fiber (22) for broadening the wavelength of the ultraviolet continuous laser;
the RET frequency selector (23) is used for outputting a certain single-color light after broadening, and the output wavelength of the single-color light is equal to the excitation wavelength of donor fluorescent protein in the sample cell to be detected;
the LCTF subsystem (3) comprises:
the first conjugate objective lens (31) is used for imaging a sample to be detected at infinite distance;
an LCTF tube lens (32) which zooms in the amplified real image at infinity to one side of the LCTF tube lens (32) and forms a primary real image;
the LCTF lens (33) is used for forming the primary real image into a secondary real image on one side of the LCTF lens (33);
the area array CCD (34) is electrically connected with the main controller (6) and is used for imaging the secondary real image and transmitting the secondary real image to the main controller (6);
the LCTF filter (35) is positioned between the LCTF tube lens (32) and the first conjugate objective lens (31), selects imaging wavelength, and finally, the imaging wavelength is the central wavelength of the LCTF filter (35), and the central wavelength is equal to the fluorescence wavelength emitted by receptor fluorescent protein in a sample cell to be detected;
the spectral detection subsystem (5) comprises:
the RET conjugate cut-off sheet (51) is used for filtering light scattering of the corresponding wavelength of the RET frequency selector (23) in the light scattering generated by the sample to be detected;
a coupling mirror (52) for coupling the remaining light passing through the RET conjugate cut-off plate (51);
and the fiber spectrometer (53) converts the coupled residual light into a spectrum signal and transmits the spectrum signal to the main controller (6).
2. The blood cell hyperspectral optical tweezers trapping energy resonance transfer analyzer according to claim 1, further comprising a perforated ultraviolet holomirror (7) between the RET subsystem (2) and the loading device (1).
3. The hyperspectral optical tweezer trapping energy resonance transfer analyzer for blood cells according to claim 1, further comprising a second conjugate objective lens (8), wherein the first conjugate objective lens (31) and the second conjugate objective lens (8) are symmetrically located at two sides of the object carrying device (1).
4. The blood cell hyperspectral optical tweezer trapping energy resonance transfer analyzer according to claim 3, further comprising a focusing device (9) for controlling the symmetrical movement of the first conjugate objective lens (31) and the second conjugate objective lens (8).
5. A blood cell hyperspectral optical tweezer trapping energy resonance transfer analyzer according to claim 4, wherein the focusing device (9) comprises:
a linear guide rail (91);
a first moving motor (92) which is positioned at one end of the linear guide rail (91) and is connected to the first conjugate objective lens (31);
the second moving motor (93) is positioned at the other end of the linear guide rail (91) and is connected to the second conjugate objective lens (8);
and the motor controller (94) is electrically connected with the first moving motor (92) and the second moving motor (93) and is used for controlling the first conjugate objective lens (31) and the second conjugate objective lens (8) to symmetrically move relative to the sample to be measured.
6. The apparatus according to claim 1, wherein the LCTF filter (35) is electrically connected to a wavelength controller, the wavelength controller controls the central wavelength of the LCTF filter (35), and the wavelength controller is electrically connected to the main controller (6).
7. The blood cell hyperspectral optical tweezer trapping energy resonance transfer analyzer according to claim 1, wherein the optical tweezer system (4) comprises:
an optical tweezers laser (41) for emitting laser light;
the energy regulator (42) is positioned on one side of the optical tweezers laser (41) and is used for regulating the energy of the laser emitted by the optical tweezers laser (41);
and the optical tweezers beam expander (43) is positioned on the other side of the optical tweezers laser (41) and is used for expanding the laser emitted by the optical tweezers laser (41).
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