CN114354552A - Synchronous annular light beam three-dimensional structure and molecular imaging system and method - Google Patents

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

A synchronized ring beam three-dimensional structure and molecular imaging system and method

technical field

The invention belongs to the technical field of three-dimensional imaging of biological tissue structures and molecules, and in particular relates to a synchronous annular beam three-dimensional structure and molecular imaging system and method.

Background technique

Because the imaging resolution of biological optical imaging is not high, it does not have the function of imaging structural information. Therefore, multimodal optical imaging that combines optical imaging with other imaging modalities is proposed. As a typical structural imaging mode, CT just makes up for the deficiency of optical imaging. Optical/OCT dual-mode imaging can simultaneously image three-dimensional structures and molecules, and during the imaging process, the structural information obtained by OCT imaging can provide advanced optical imaging. The test information is used to optimize the optical imaging results.

Scientists have proposed a variety of optical path system configuration methods, including fluorescence microscopes using two orthogonally arranged objective lenses, tilted single-plane illumination microscopes, etc. However, there are still problems such as low resolution, poor signal-to-noise ratio, and small imaging range.

The oblique right-angle A-line synchronized ring beam high-resolution 3D structure and molecular imaging system is to set the oblique right-angle optical path structure and annular beam shaping in the imaging system, which can shape the beam angle and light field to achieve high-resolution synchronous imaging .

SUMMARY OF THE INVENTION

In order to solve the above-mentioned defects, the present invention proposes a synchronous annular beam three-dimensional structure and molecular imaging system and method.

For solving the above-mentioned technical problems, the technical scheme adopted in the present invention is:

A synchronized ring beam three-dimensional structure and molecular imaging system, the system includes:

The dual-band light source module is configured to be a blue-band light source and a red-band light source required for tomographic imaging;

a coherent spectrum acquisition module configured to acquire blue light spectrum and red light spectrum separated from the dual-band light source module layer;

a Michelson interference module, which is configured to couple and collimate the tomographic blue and red light to obtain spatial light acting on the surface of the sample;

an oblique right-angle optical path structure, which is configured to obliquely enter the sample at an angle of 45° and exit the optical path at an angle of 45° after processing the spatial light;

A fluorescence signal acquisition module configured to acquire light carrying biomolecular structural information and acquired by the camera.

A further improvement of the system is that: the dual-band light source module includes a polarization beam splitter, which is configured to filter the laser light through the prismatic dispersion and the light shield, and perform polarization beam splitting to obtain blue light and red light. .

A further improvement of the present system is that the Michelson interference module contains a polarization controller configured to detect and process the polarization state of the focused light.

A further improvement of the system is that: the fluorescence signal acquisition module includes a filter, and the filter is configured to filter the shaped optical path to obtain fluorescence carrying biomolecular structure information.

A synchronous annular beam three-dimensional structure and molecular imaging method, characterized by comprising:

Blue light and red light will be separated from the laser, wherein the blue light is used as the fluorescent light source to measure the molecular information of biological tissues, and the red light is used as the light source of OCT to measure the structural information of biological tissues;

Coupling and collimating blue light and red light after chromatography to obtain space light acting on the surface of the sample;

After processing the space light, the incident sample is inclined at an angle of 45° and the light path is inclined at an angle of 45°;

The light path carrying the biomolecular structure information is obtained and captured by the camera;

A three-dimensional image is obtained by reconstruction.

A further improvement of the method is that: the blue light and red light that will be layered from the laser include:

The laser is filtered through prismatic dispersion and light-shielding plate and polarized and split to obtain blue and red light.

A further improvement of the method is that: coupling and collimating the chromatographic blue light and red light to obtain the spatial light acting on the surface of the sample includes:

Focus blue and red light;

Detect and process the polarization state of the focused light;

The detected and processed light is coupled and collimated to obtain spatial light acting on the surface of the sample.

A further improvement of the method is that: after the spatial light processing is performed, the light path of the incident sample inclined at an angle of 45° and the light path inclined at an angle of 45° includes:

Adjust the space light to get Bessel light;

Focus the Bessel light on the sample obliquely at a 45° angle;

The reflected light on the sample is inclined at a 45° angle to the outgoing light path, wherein the outgoing light path carries the biomolecular structure information.

A further improvement of the method is that: the obtaining of the light path carrying the biomolecular structure information and being collected by the camera includes:

Shaping the light path carrying biomolecular structure information;

Filter the shaped light path to obtain fluorescence carrying biomolecular structure information;

Synchronous transmission of fluorescence carrying biomolecular structural information;

The camera acquires the fluorescence after synchronous transmission.

Owing to having adopted the above-mentioned technical scheme, the technical progress that the present invention obtains is:

The imaging system of the invention has a simple structure, adopts optical/OCT dual-mode imaging, and sets two orthogonally placed objective lenses at an angle of 45° with the sample surface, and the A-line of structure and molecular imaging can be strictly synchronized. The transmission distance of optical signals in biological tissues is shortened, the scattering of light is reduced, and the signal-to-noise ratio is improved. At the same time, because the CCD camera overlaps with the optical path, the collection efficiency of the optical signal is improved. And the ring beam can further improve the resolution.

Description of drawings

1 is a schematic diagram of a laser microscope system of the present invention;

Fig. 2 is the point spread function (PSF) diagram of simulated Gaussian beam and Bessel beam incidence in the present invention;

Fig. 3 is the PSF figure that simulates Bessel beam incidence, the fluorescence collection of Gaussian beam and their combination in the present invention;

Fig. 4 is the PSF figure that simulates Bessel beam incidence, Bessel beam fluorescence collection and their combination in the present invention;

Fig. 5 is the flow chart of the present invention;

Among them, SCS, supercontinuum light source, F, filter, BT, beam trap, PBS, polarizing beam splitter, P, prism, B, shading plate, M, reflector, DM, D mirror, OL, objective lens, PC, Polarization Controller, OFC, Fiber Coupler, L, Lens, A, Annular Aperture, DG, Grating, LSC, Line Scan Camera, GM, Polarimeter Scanning Mirror, OAPM, Axial Parabolic Mirror, S, Sample, Camera, camera

Detailed ways

Below in conjunction with accompanying drawing, the present invention is described in further detail:

The present invention proposes a synchronous annular beam three-dimensional structure and molecular imaging system, as shown in Figure 1, the system includes: a dual-band light source module, a coherent spectrum acquisition module, a Michelson interference module, an oblique right-angle optical path structure, and a fluorescence signal acquisition module, where:

The dual-band light source module is used to tomography the blue-band light source and the red-band light source required for imaging; the coherent spectrum acquisition module is used to collect the blue and red light spectra from the dual-band light source module; the Michelson interference module will The separated blue light and red light are coupled and collimated to obtain the space light acting on the surface of the sample; the oblique right-angle optical path structure treats the space light with a 45° angle to the sample and a 45° angle to the light path; the fluorescence signal The acquisition module obtains light carrying biomolecular structure information and is collected by the camera.

Specifically, the present invention adopts optical/OCT dual-mode imaging and sets two orthogonally placed objective lenses at a 45° angle to the sample surface, so that the A-line of structural and molecular imaging can be strictly synchronized, shortening the optical signal in biological tissue. The transmission distance is reduced, the light scattering is reduced and the signal-to-noise ratio is improved.

Further, the dual-band light source module has a polarization beam splitter, which is used to filter the laser light through the Mitsubishi mirror dispersion and light shielding plate and perform polarization beam splitting to obtain blue light and red light.

Further, the Michelson interference module contains a polarization controller for detecting and processing the polarization state of the focused light.

Further, the fluorescence signal acquisition module includes a filter, which is used for filtering the shaped optical path to obtain fluorescence carrying biomolecular structure information.

Based on the above-mentioned method for a synchronous annular beam three-dimensional structure and a molecular imaging system, the method includes:

Blue light and red light will be separated from the laser, wherein the blue light is used as the fluorescent light source to measure the molecular information of biological tissues, and the red light is used as the light source of OCT to measure the structural information of biological tissues;

Coupling and collimating blue light and red light after chromatography to obtain space light acting on the surface of the sample;

After processing the space light, the incident sample is inclined at an angle of 45° and the light path is inclined at an angle of 45°;

The light path carrying the biomolecular structure information is obtained and captured by the camera;

A three-dimensional image is obtained by reconstruction.

Further, the blue light and red light will be separated from the laser. Specifically: the laser is filtered through the Mitsubishi mirror dispersion and light shielding plate, and polarized beam splitting is performed to obtain blue light and red light.

Further, coupling and collimating the chromatographic blue light and red light to obtain the spatial light acting on the surface of the sample contains: focusing the blue light and red light; detecting and processing the polarization state of the focused light; Coupling and collimating the processed light to obtain the space light acting on the surface of the sample; the details are as follows: The space light acting on the sample is obtained in this way: the laser light emitted by the supercontinuum light source is split by the polarization beam splitter, and then passes through the The triangular prisms P1 and P2 separate the monochromatic light in the laser, get red light and blue light under the action of the shading plate B, and then send it to the D-shaped mirror DM by the reflector M1 to separate the red light and blue light, and then focus and send it through OL1. The input polarization controller changes the polarization characteristics of red light and blue light, and then is coupled by the optical fiber coupler OFC and collimated by the collimator L4 to obtain the space light that can act on the sample.

Further, after processing the spatial light, the incident sample at an angle of 45° and the light path at an angle of 45° include: adjusting the space light to obtain Bessel light; obliquely focusing the Bessel light at an angle of 45° On the sample; the reflected light on the sample exits the light path at an angle of 45°, wherein the outgoing light path carries the biomolecular structure information;

Further, obtaining the light path carrying the biomolecular structure information and being captured by the camera includes: shaping the light path carrying the biomolecular structure information; filtering the shaped light path to obtain the fluorescence carrying the biomolecular structure information; The fluorescence of the information is transmitted synchronously; the fluorescence after synchronous transmission is collected by the camera, as follows: Before the Bessel beam carrying the biomolecular structure information is collected, the fluorescence and excitation light are separated by a filter, and the separated fluorescence and excitation light are separated. After passing through the lens L7, the fluorescence is focused on a straight line for synchronous transmission, so that the molecular and structural information of the organism can be received synchronously, and then guided by the lens L8, the polarization scanning mirror GM2, the lenses L9, L10, and the mirror M5 to the objective lens in front of the camera OL4, and finally the camera receives the light signal focused by the objective lens OL4. Since the optical paths of the camera and the signal transmission are coincident, the light carrying the structural information of the biomolecules can be fully received.

The specific process of this method is as follows:

In the system shown in Figure 1, blue and red light are separated from the excitation light using two prisms and a light shield. Among them, blue light is used as the fluorescent light source to measure the molecular information of biological tissues, and red light is used as the light source of OCT to measure the structural information of biological tissues. At the sample, the beam is obliquely focused into the sample by an objective lens OL2 placed at an angle of 45° to the sample surface, and the objective lens OL3 on the other side of the sample is also placed at an angle of 45° to the sample surface, the beam reflected in the depth direction of the sample After passing through the microscope objective lens OL3, after shaping and filtering, it is focused on a straight line after the lens L7, and finally all the fluorescent signals are collected by the CCD camera that coincides with the optical path. This method can obtain A-line synchronized structural and molecular information, improving the signal-to-noise ratio and resolution of the system.

In the imaging system shown in Fig. 2, the point spread function PSF of the incident Gaussian beam and the Bessel beam is simulated, and the PSF of the incident Gaussian beam (a-c) and the Bessel beam (d-f) are simulated in Fig. 2. The numerical aperture of the Gaussian beam is set to 0.1, the central z-y and y-x cross-sections of the PSF are shown in Fig. 2(a) and Fig. 2(b), in the z direction, the imaging depth is about 100 μm; in the lateral direction, through the PSF The full width at half maximum (FWHM) of the intensity profile at the center is 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 annular Bessel beam is set to 0.5 at the outer edge and 0.4899 at the inner edge, and the central z-y and y-x cross-sections of the PSF are shown in Figure 2 ( d) and Fig. 2(e), in the z direction, the imaging depth drops slightly to 93.6 μm, and in the lateral direction, the FWHM of the intensity profile passing through the center of the PSF sharply increases to 452 nm, as shown in Fig. 2(f) .

The PSF of Bessel beam incidence, Gaussian beam fluorescence collection, and their combination is simulated in the imaging system shown in Fig. 3, in Fig. 3 (a) Bessel beam incidence PSF, (b) Gaussian beam fluorescence collection PSF, (c) combined incident PSF, (d) intensity profile through the center of the PSF along the y-direction, (e) intensity profile through the center of the PSF along the z-direction. The Bessel beam still covers the same area on the objective as the Gaussian beam. Set the numerical aperture of the outer edge of the Bessel beam to 0.8 and the numerical aperture of the fluorescence acquisition objective to 0.5. For the fluorescence-collected PSF, the FWHM was 678.2 nm in the lateral direction and 4.33 μm in the axial direction. For the combination of incident PSF and fluorescence collection PSF, the resolution is 677.6 nm in the z direction and 285 nm in the x/y direction.

Simulated PSF of Bessel beam incidence, Bessel beam fluorescence collection, and their combination in the imaging system shown in Figure 4, (a) Bessel beam incidence PSF, (b) Bessel beam incidence in Figure 4 PSF collected by beam fluorescence, (c) combined incident PSF, (d) intensity profile along the y and z directions through the center of the PSF. The Bessel beam still covers the same area on the objective as the Gaussian beam. Set the numerical aperture of the outer edge of the annular Bessel beam to 0.5. For the combined PSF, the z-direction resolution was improved to 471 nm, the x/y-direction resolution was 285 nm, and the imaging depth was 72 μm.

The above-mentioned embodiments merely describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various Such deformations and improvements shall fall within the protection scope determined by the claims 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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