CN114354552A - Synchronous annular light beam three-dimensional structure and molecular imaging system and method - Google Patents
Synchronous annular light beam three-dimensional structure and molecular imaging system and method Download PDFInfo
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Abstract
The invention discloses a synchronous annular light beam three-dimensional structure and molecular imaging system and a synchronous annular light beam three-dimensional structure and molecular imaging method, which belong to the technical field of three-dimensional imaging of biological tissue structures and molecules, wherein the system comprises a dual-band light source module, a coherent spectrum acquisition module, a Michelson interference module, an oblique right-angle light path framework and a fluorescent signal acquisition module, and the method comprises the steps of resolving blue light and red light from laser; coupling and collimating the blue light and the red light after chromatography to obtain space light acting on the surface of the sample; the light path obliquely enters the sample at an angle of 45 degrees and obliquely exits at an angle of 45 degrees after the spatial light is processed; obtaining a light path carrying biomolecule structure information and collecting the light path by a camera; reconstructing to obtain a three-dimensional image; the imaging system has simple structure, adopts optical/OCT dual-mode imaging, and arranges two orthogonally arranged objective lenses to form an angle of 45 degrees with the surface of the sample, and the A-line of the structure and the molecular imaging can be strictly synchronous.
Description
Technical Field
The invention belongs to the technical field of three-dimensional imaging of biological tissue structures and molecules, and particularly relates to a synchronous annular light beam three-dimensional structure and molecular imaging system and method.
Background
The biological optical imaging has low imaging resolution and does not have the function of imaging structural information. Therefore, multi-mode optical imaging combining optical imaging with other imaging modes is proposed. The CT just makes up the deficiency of optical imaging as a typical structural imaging mode, optical/OCT dual-mode imaging can simultaneously image a three-dimensional structure and molecules, and in the imaging process, structural information obtained by OCT imaging can provide prior information for the optical imaging so as to optimize the optical imaging result.
Scientists have proposed various optical path system configurations, including fluorescence microscopes using two orthogonally arranged objectives, tilted single plane illumination microscopes, etc., but still suffer from low resolution, poor signal-to-noise ratio, small imaging range, etc.
A high-resolution three-dimensional structure of an annular light beam synchronized by oblique angle A-line and a molecular imaging system are characterized in that an oblique angle light path framework and annular light beam shaping are arranged in the imaging system, and the angle and the light field of the light beam can be shaped, so that high-resolution synchronous imaging is realized.
Disclosure of Invention
In order to solve the above-mentioned drawbacks, the present invention provides a synchronous ring beam three-dimensional structure and molecular imaging system and method.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a synchronized ring beam three-dimensional structure and molecular imaging system, the system comprising:
a dual band light source module configured to separate out a blue band light source and a red band light source required for imaging;
a coherent spectrum acquisition module configured to acquire a blue light spectrum and a red light spectrum precipitated from the dual band light source module layer;
the Michelson interference module is configured to couple and collimate the blue light and the red light after chromatography to obtain spatial light acting on the surface of the sample;
an oblique right-angle optical path architecture configured to process the spatial light and then obliquely input the sample at an angle of 45 DEG and obliquely output an optical path at an angle of 45 DEG;
a fluorescence signal acquisition module configured to obtain light carrying biomolecular structural information and to be acquired by a camera.
The system is further improved in that: the dual band light source module contains a polarizing beam splitter configured to disperse laser light through a triple prism and filter a light shielding plate and to perform a polarizing beam splitting to obtain blue light and red light.
The system is further improved in that: the michelson interference module contains a polarization controller configured to detect and process the polarization state of the focused light.
The system is further improved in that: the fluorescence signal acquisition module comprises a filter, and the filter is configured to filter the shaped light path to obtain fluorescence carrying biomolecular structural information.
A synchronized annular beam three-dimensional structure and molecular imaging method, comprising:
blue light and red light are separated from the laser, wherein the blue light is used as a fluorescent light source to measure molecular information of the biological tissue, and the red light is used as a light source of OCT to measure structural information of the biological tissue;
coupling and collimating the blue light and the red light after chromatography to obtain space light acting on the surface of the sample;
the light path obliquely enters the sample at an angle of 45 degrees and obliquely exits at an angle of 45 degrees after the spatial light is processed;
obtaining a light path carrying biomolecule structure information and collecting the light path by a camera;
and reconstructing to obtain a three-dimensional image.
The method is further improved in that: the blue light and the red light to be separated from the laser comprise:
and (3) filtering the laser through triple prism dispersion and a light shielding plate, and carrying out polarization beam splitting to obtain blue light and red light.
The method is further improved in that: the step of coupling and collimating the blue light and the red light after chromatography to obtain the space light acting on the surface of the sample comprises the following steps:
focusing the blue light and the red light;
detecting and processing the polarization state of the focused light;
and coupling and collimating the detected and processed light to obtain space light acting on the surface of the sample.
The method is further improved in that: the optical path for obliquely inputting the sample at an angle of 45 degrees and obliquely outputting the sample at an angle of 45 degrees after the spatial light is processed comprises:
adjusting the space light to obtain Bessel light;
obliquely focusing bezier light onto the sample at a 45 ° angle;
and the reflected light on the sample is obliquely emitted at an angle of 45 degrees, wherein the emitted light path carries the structure information of the biomolecules.
The method is further improved in that: the obtaining the optical path carrying the biomolecule structure information and the acquiring by the camera comprises:
shaping an optical path carrying biomolecular structural information;
filtering the shaped light path to obtain fluorescence carrying biomolecular structural information;
synchronously transmitting fluorescence carrying biomolecular structural information;
the camera collects the fluorescence after synchronous transmission.
Due to the adoption of the technical scheme, the invention has the technical progress that:
the imaging system has simple structure, adopts optical/OCT dual-mode imaging, and arranges two orthogonally arranged objective lenses to form an angle of 45 degrees with the surface of the sample, and the A-line of the structure and the molecular imaging can be strictly synchronous. The transmission distance of the optical signal in the biological tissue is shortened, and the scattering of light is reduced, so that the signal-to-noise ratio is improved. Meanwhile, the CCD camera is superposed with the light path, so that the collection efficiency of the optical signal is improved. And the resolution can be further improved by the annular beam.
Drawings
FIG. 1 is a schematic diagram of a laser microscope system of the present invention;
FIG. 2 is a graph of the Point Spread Function (PSF) of the present invention simulating Gaussian and Bessel beam incidence;
FIG. 3 is a PSF plot of simulated Bessel beam incidence, fluorescence collection of Gaussian beams, and combinations thereof in accordance with the present invention;
FIG. 4 is a PSF plot of simulated Bessel beam incidence, fluorescence collection of Bessel beams, and combinations thereof in accordance with the present invention;
FIG. 5 is a flow chart of the present invention;
SCS, super-continuous light source, F, optical filter, BT, beam trap, PBS, polarization beam splitter, P, prism, B, light shielding plate, M, reflector, DM, D-shaped mirror, OL, objective lens, PC, polarization controller, OFC, optical fiber coupler, L, lens, A, annular aperture, DG, grating, LSC, linear scanning Camera, GM, polarimeter scanning mirror, OAPM, axial parabolic mirror, S, sample, Camera, Camera
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings:
the invention provides a synchronous annular light beam three-dimensional structure and molecular imaging system, as shown in figure 1, the system comprises: two wave band light source module, coherent spectrum collection module, michelson interfere module, oblique right angle light path framework and fluorescence signal acquisition module, wherein:
the dual-waveband light source module is used for separating a blue light waveband light source and a red light waveband light source required by imaging in a layered mode; the coherent spectrum acquisition module is used for acquiring a blue light spectrum and a red light spectrum separated out from the dual-band light source module layer; the Michelson interference module couples and collimates the blue light and the red light after chromatography to obtain space light acting on the surface of the sample; the oblique right-angle light path framework is used for processing the space light, then obliquely inputting the sample at an angle of 45 degrees and obliquely outputting the light at an angle of 45 degrees; the fluorescence signal acquisition module acquires light carrying biomolecular structural information and is acquired by the camera.
Specifically, the optical/OCT dual-mode imaging is adopted, two orthogonally placed objective lenses form an angle of 45 degrees with the surface of a sample, the A-lines of the structure and the molecular imaging can be strictly synchronized, the transmission distance of optical signals in biological tissues is shortened, and the light scattering is reduced, so that the signal-to-noise ratio is improved.
Further, the dual-band light source module is provided with a polarization beam splitter, and is used for dispersing laser through a Mitsubishi mirror and filtering through a light shielding plate, and performing polarization beam splitting to obtain blue light and red light.
Further, the michelson interference module comprises a polarization controller for detecting and processing the polarization state of the focused light.
Furthermore, the fluorescence signal acquisition module comprises a filter for filtering the shaped light path to obtain fluorescence carrying biomolecular structural information.
The method based on the synchronous annular light beam three-dimensional structure and the molecular imaging system comprises the following steps:
blue light and red light are separated from the laser, wherein the blue light is used as a fluorescent light source to measure molecular information of the biological tissue, and the red light is used as a light source of OCT to measure structural information of the biological tissue;
coupling and collimating the blue light and the red light after chromatography to obtain space light acting on the surface of the sample;
the light path obliquely enters the sample at an angle of 45 degrees and obliquely exits at an angle of 45 degrees after the spatial light is processed;
obtaining a light path carrying biomolecule structure information and collecting the light path by a camera;
and reconstructing to obtain a three-dimensional image.
Further, blue light and red light that are layered out of the laser are specified: and (3) filtering the laser through a Mitsubishi mirror and a light shielding plate, and performing polarization beam splitting to obtain blue light and red light.
Further, coupling and collimating the blue light and the red light after chromatography to obtain space light acting on the surface of the sample, wherein the space light comprises: focusing the blue light and the red light; detecting and processing the polarization state of the focused light; coupling and collimating the detected and processed light to obtain space light acting on the surface of the sample; the method comprises the following specific steps: the spatial light acting on the sample is obtained by: after being split by a polarization beam splitter, laser emitted by a super-continuous light source is separated from each monochromatic light in the laser by triangular prisms P1 and P2, red light and blue light are obtained under the action of a light shielding plate B, then the red light and the blue light are sent to a D-shaped reflector DM by a reflector M1 to be separated, then the red light and the blue light are focused by an OL1 and sent to a polarization controller to change the polarization characteristics of the red light and the blue light, and space light capable of acting on a sample is obtained after the space light is coupled by an optical fiber coupler OFC and collimated by a collimator L4.
Further, the optical path for obliquely incident sample at an angle of 45 ° and obliquely emitted at an angle of 45 ° after the spatial light treatment includes: adjusting the space light to obtain Bessel light; obliquely focusing bezier light onto the sample at a 45 ° angle; the reflected light on the sample obliquely exits at an angle of 45 degrees, wherein the exiting light path carries the structure information of the biomolecules;
further, obtaining an optical path carrying information on the structure of the biomolecule and captured by a camera comprises: shaping an optical path carrying biomolecular structural information; filtering the shaped light path to obtain fluorescence carrying biomolecular structural information; synchronously transmitting fluorescence carrying biomolecular structural information; the camera collects the fluorescence after synchronous transmission, which is as follows: before being collected, the Bessel light beam carrying biomolecular structure information is separated into fluorescence and excitation light by a filter, the separated fluorescence with information passes through a lens L7 and is focused on a straight line for synchronous transmission, so that the biomolecular and structure information can be synchronously received, and then the biomolecular and structure information is guided to an objective OL4 in front of a camera by a lens L8, a polarization scanning mirror GM2, lenses L9 and L10 and a reflector M5, and finally the camera receives an optical signal focused by the objective OL 4. Since the camera coincides with the optical path of the signal transmission, light carrying the information of the structure of the biomolecules can be completely received.
The method comprises the following specific processes:
in the system shown in fig. 1, two triangular prisms and a light shield are used to separate the blue and red light from the excitation light. The blue light is used as a fluorescent light source to measure the molecular information of the biological tissue, and the red light is used as an OCT light source to measure the structural information of the biological tissue. At the sample, light beams are obliquely focused in the sample through an objective lens OL2 arranged at an angle of 45 degrees with the surface of the sample, an objective lens OL3 at the other side of the sample is also arranged at an angle of 45 degrees with the surface of the sample, the light beams reflected in the depth direction of the sample pass through a micro objective lens OL3, are shaped and filtered, are focused on a straight line after a lens L7, and finally all fluorescence signals are completely collected by a CCD camera superposed with a light path. The method can obtain the structure and molecular information of A-line synchronization, and improve the signal-to-noise ratio and the resolution of the system.
The point spread function PSF at which the gaussian beam and the bessel beam are incident is simulated in the imaging system shown in fig. 2, and the PSF at which the gaussian beam (a-c) and the bessel beam (d-f) are incident is simulated in fig. 2. The numerical aperture of the gaussian beam is set to 0.1, the center z-y and y-x cross-sections of the PSF are as shown in fig. 2(a) and 2(b), and the imaging depth is about 100 μm in the z-direction; in the lateral direction, the full width at half maximum (FWHM) of the intensity profile through the center of the PSF was 3.36 μm, as shown in FIG. 2 (c). In order to keep the area covered by the bessel beam on the objective lens the same as that of the gaussian beam, the numerical aperture of the outer edge of the annular bessel beam is set to 0.5, the numerical aperture of the inner edge is set to 0.4899, the center z-y and y-x cross sections of the PSF are as shown in fig. 2(d) and 2(e), the imaging depth is slightly reduced to 93.6 μm in the z direction, and the FWHM of the intensity profile passing through the center of the PSF is sharply increased to 452nm in the lateral direction as shown in fig. 2 (f).
The PSFs of bessel beam incidence, gaussian beam fluorescence collection, and combinations thereof are simulated in the imaging system shown in fig. 3, where (a) the PSF of bessel beam incidence, (b) the PSF of gaussian beam fluorescence collection, (c) the incident PSF is combined, (d) the intensity profile passes through the center of the PSF in the y-direction, and (e) the intensity profile passes through the center of the PSF in the z-direction. The bessel beam still covers the same area on the objective lens as the gaussian beam. The numerical aperture of the outer edge of the Bessel beam is set to be 0.8, and the numerical aperture of the fluorescence collection objective lens is set to be 0.5. For the fluorescence collected PSF, the FWHM was 678.2nm in the lateral direction and 4.33 μm in the axial direction. For the combination of incident PSF and fluorescence collection PSF, the resolution was 677.6nm in the z-direction and 285nm in the x/y-direction.
Simulating the PSFs of bessel beam incidence, bessel beam fluorescence collection, and combinations thereof in the imaging system shown in fig. 4, in fig. 4 (a) the PSFs of bessel beam incidence, (b) the PSFs of bessel beam fluorescence collection, (c) the combined incident PSFs, and (d) the intensity profile passes through the centers of the PSFs in the y and z directions. The bessel beam still covers the same area on the objective lens as the gaussian beam. The numerical aperture of the outer edge of the annular Bessel beam is set to be 0.5. For the combined PSF, the z-direction resolution was increased to 471nm, the x/y-direction resolution was 285nm, and the imaging depth was 72 μm.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (9)
1. A synchronized ring beam three-dimensional structure and molecular imaging system, comprising:
a dual band light source module configured to separate out a blue band light source and a red band light source required for imaging;
a coherent spectrum acquisition module configured to acquire a blue light spectrum and a red light spectrum precipitated from the dual band light source module layer;
the Michelson interference module is configured to couple and collimate the blue light and the red light after chromatography to obtain spatial light acting on the surface of the sample;
an oblique right-angle optical path architecture configured to process the spatial light and then obliquely input the sample at an angle of 45 DEG and obliquely output an optical path at an angle of 45 DEG;
a fluorescence signal acquisition module configured to obtain light carrying biomolecular structural information and to be acquired by a camera.
2. The simultaneous ring beam three-dimensional structure and molecular imaging system of claim 1, wherein the dual band light source module comprises a polarizing beam splitter configured to filter and polarizedly split laser light through a dispersion and mask of a triple prism to obtain blue and red light.
3. The simultaneous ring-beam three-dimensional structure and molecular imaging system of claim 1, wherein the michelson interference module comprises a polarization controller configured to detect and process the polarization state of the focused light.
4. The simultaneous ring beam three-dimensional structure and molecular imaging system of claim 1, wherein the fluorescence signal collection module comprises a filter configured to filter the shaped optical path to obtain fluorescence carrying biomolecular structural information.
5. A synchronized annular beam three-dimensional structure and molecular imaging method, comprising:
blue light and red light are separated from the laser, wherein the blue light is used as a fluorescent light source to measure molecular information of the biological tissue, and the red light is used as a light source of OCT to measure structural information of the biological tissue;
coupling and collimating the blue light and the red light after chromatography to obtain space light acting on the surface of the sample;
the light path obliquely enters the sample at an angle of 45 degrees and obliquely exits at an angle of 45 degrees after the spatial light is processed;
obtaining a light path carrying biomolecule structure information and collecting the light path by a camera;
and reconstructing to obtain a three-dimensional image.
6. The simultaneous ring beam three-dimensional structure and molecular imaging method of claim 5, wherein said resolving blue and red light from a laser comprises:
and filtering the laser through the dispersion of the triple prism and the light shielding plate, and carrying out polarization beam splitting to obtain blue light and red light.
7. The simultaneous ring-beam three-dimensional structure and molecular imaging method of claim 5, wherein the coupling and collimating the blue and red chromatographed light to obtain the spatial light acting on the surface of the sample comprises:
focusing the blue light and the red light;
detecting and processing the polarization state of the focused light;
and coupling and collimating the detected and processed light to obtain space light acting on the surface of the sample.
8. The simultaneous ring beam three-dimensional structure and molecular imaging method of claim 5, wherein the optical path after processing the spatial light to obliquely enter the sample at an angle of 45 ° and to obliquely exit at an angle of 45 ° comprises:
adjusting the space light to obtain Bessel light;
obliquely focusing bezier light onto the sample at a 45 ° angle;
and the reflected light on the sample is obliquely emitted at an angle of 45 degrees, wherein the emitted light path carries the structure information of the biomolecules.
9. The simultaneous ring beam three-dimensional structure and molecular imaging method of claim 6, wherein the obtaining the optical path carrying the biomolecular structural information and captured by the camera comprises:
shaping an optical path carrying biomolecular structural information;
filtering the shaped light path to obtain fluorescence carrying biomolecular structural information;
synchronously transmitting fluorescence carrying biomolecular structural information;
the camera collects the fluorescence after synchronous transmission.
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| US20210033837A1 (en) * | 2017-06-16 | 2021-02-04 | University Court Of The University Of St Andrews | Three-photon light sheet imaging |
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| US20210033837A1 (en) * | 2017-06-16 | 2021-02-04 | University Court Of The University Of St Andrews | Three-photon light sheet imaging |
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| CN114847882A (en) * | 2022-05-06 | 2022-08-05 | 山东探微医疗技术有限公司 | High-resolution jump type multiband spectrometer and working method |
| CN114847882B (en) * | 2022-05-06 | 2024-08-13 | 山东探微医疗技术有限公司 | High-resolution jump type multiband spectrometer and working method |
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