CN116819750A - Real-time three-dimensional microscopic imaging device and method based on super-structured lens array - Google Patents
Real-time three-dimensional microscopic imaging device and method based on super-structured lens array Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/361—Optical details, e.g. image relay to the camera or image sensor
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B30/00—Optical systems or apparatus for producing three-dimensional [3D] effects, e.g. stereoscopic images
- G02B30/20—Optical systems or apparatus for producing three-dimensional [3D] effects, e.g. stereoscopic images by providing first and second parallax images to an observer's left and right eyes
- G02B30/26—Optical systems or apparatus for producing three-dimensional [3D] effects, e.g. stereoscopic images by providing first and second parallax images to an observer's left and right eyes of the autostereoscopic type
- G02B30/27—Optical systems or apparatus for producing three-dimensional [3D] effects, e.g. stereoscopic images by providing first and second parallax images to an observer's left and right eyes of the autostereoscopic type involving lenticular arrays
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N2021/0106—General arrangement of respective parts
- G01N2021/0112—Apparatus in one mechanical, optical or electronic block
Abstract
The invention discloses a real-time three-dimensional microscopic imaging device and method based on a super-structured lens array. The device comprises a plane reflecting mirror, a microfluidic channel and a super-structured lens array from bottom to top; the plane reflector is provided with a metal film and a transparent medium, the metal film is used for realizing the reflection of the light wave, and the transparent medium is used for protecting the metal film and supporting an observed object so that a light field below the observed object is reflected and led out; the super-structure lens array is used for respectively imaging information of a plurality of different visual angles of an observed object on the same focal plane, and has independent transreflective modulation capability on a group of orthogonally polarized light waves; and respectively imaging the information of a plurality of different visual angles through a microscope, and then carrying out three-dimensional image fusion to obtain three-dimensional scene reconstruction. The invention provides a realization method for real-time three-dimensional microscopic imaging of living bodies in biophotonics, and opens up a new idea for the application of super-surfaces and microfluidic biochips.
Description
Technical Field
The invention belongs to the fields of micro-nano optics, three-dimensional microscopic imaging, image fusion algorithm and biological photonics, and particularly relates to a real-time three-dimensional microscopic imaging technology based on a super-structure lens array and an image fusion algorithm, which has great value in the application research fields of observing three-dimensional microbial microscopic imaging and the like.
Background
The microscope is a common optical device for observing the shape and activity of microorganisms, and the development of fields such as microbiology and the like is promoted by the improvement research based on the microscopic technology. The microscope commonly used can magnify the object by about 1000 times, and clearly distinguish submicron particles and microorganism structural details. However, the conventional microscope can only image a plane, and it is difficult to obtain a three-dimensional image of a microscopic object. For many three-dimensional tomography techniques currently in popular use, although three-dimensional information of objects is available and penetration is increased, it requires long scanning and reconstruction, usually only static cells or tissues can be observed, and live three-dimensional imaging of moving microorganisms is not possible. For the currently reported planar excitation in-situ three-dimensional microscope with the confocal alignment of the sweep, the planar excitation in-situ three-dimensional microscope is still a scheme based on three-dimensional chromatography and light sheet microscopy, and has the problems of scanning and reconstruction, and three-dimensional information of an object cannot be obtained in single shooting. In microbiological studies, especially in observing the relative movement, phagocytosis and population activities of small numbers of three-dimensional microorganisms, three-dimensional information is an extremely important information. But there is still a lack of efficient real-time three-dimensional microscopy on living subjects.
Disclosure of Invention
The invention aims at solving the blank of the current three-dimensional real-time microscopic technology, and provides a real-time three-dimensional microscopic imaging device and method based on a super-structured lens array.
According to the invention, seven visual angle information of an observed object is collected through the polarization multiplexing super-constructed lens array, different visual angle information is imaged through a traditional microscope, and finally, an image fusion algorithm is used for carrying out three-dimensional reconstruction on the object, so that three-dimensional real-time microscopy on the object is realized. In the placement of the observed object, the invention adopts a microfluidic technology to inject the solution containing the observed object into an observation channel and an observation cavity (microfluidic channel). The invention provides a new research tool for real-time three-dimensional imaging of three-dimensional microbial activities.
The technical scheme adopted for solving the technical problems is as follows:
the real-time three-dimensional microscopic imaging device based on the super-structured lens array comprises a plane reflector, a microfluidic channel and the super-structured lens array from bottom to top;
the plane reflector is provided with a metal film and a transparent medium, the metal film is used for realizing the reflection of the light wave, and the transparent medium is used for protecting the metal film and supporting an observed object so that a light field below the observed object is reflected and led out;
the microfluidic channel is used for guiding an observed object, and the position of the observed object is controlled through liquid flow;
the super-structure lens array is used for respectively imaging information of a plurality of different visual angles of an observed object on the same focal plane, and has independent transreflective modulation capability on a group of orthogonally polarized light waves; and respectively imaging the information of a plurality of different visual angles through a microscope, and then carrying out three-dimensional image fusion to obtain three-dimensional scene reconstruction.
The super-structure lens array re-images the light field information of a plurality of upward and downward visual angles of an observed object on the same focal plane, and each lens has a required depth of field requirement for a shooting visual angle.
The super-structure lens consists of an anisotropic sub-wavelength structure and has independent transmission and reflection coefficient modulation capability for orthogonal polarization states, wherein the super-structure lens has high reflectivity of more than 80 percent, full-wave phase modulation and polarization conversion capability for a first incident light beam, and has high transmittance modulation characteristic of more than 80 percent for a second incident orthogonal polarization state light beam.
The real-time three-dimensional microscopic imaging device is provided with the focal length of the super-structure lens for reflecting the polarization state of light, which is smaller than the distance between the super-surface and the reflecting mirror, and the focal length determines the magnification of the system.
The six super-structure lenses on the periphery collect light of 3 visual angle information emitted upwards by the object, the super-structure lenses on the other 3 opposite sides collect light of 3 visual angle information emitted downwards, and the middle lens collects light of visual angle right above.
The super-structure lens has certain light beam deflection capability, and deflects obliquely incident light waves to the position right above the super-structure lens for output, and deflection angles are respectively designed according to different lens arrangement positions.
The micro-fluidic channel is made of transparent materials, the thickness of the micro-fluidic channel is the focal length of the super-structure lens, the middle and lower parts of the micro-fluidic channel are grooved, and the grooved size is matched with the depth of field and the field size of the super-structure lens; the slot is a microfluidic channel in the device, which is connected with an external microfluidic device for controlling the flow of liquid, so that the solution containing the observed object flows in, and the position of the observed object can be controlled by the pressure of the fluid.
In the real-time three-dimensional microscopic imaging device, the super-structure lens array, the microfluidic channel and the plane mirror are bonded together, and the microfluidic channel is positioned in the field of view of the lens array design through the overlay technology.
A method of real-time three-dimensional microscopic imaging based on a super-structured lens array, comprising the steps of:
the method comprises the steps that (1) a design device is placed below an objective lens of a microscope, so that an imaging surface of the objective lens is overlapped with an imaging surface of a micro lens array, illumination light waves are coupled into the design device through the micro lens array, a micro-fluidic channel is illuminated, the microscope can obtain images of 7 micro lenses collected by the objective lens at the same time, the 7 images are imaged in 7 areas of a microscopic image surface, and the 7 areas are respectively right above the 7 micro lenses;
injecting a solution containing an observed object into a microfluidic device, enabling the observed object to be positioned in a designed view field through flow control, and controlling the position of the observed object through liquid flow;
step (3) filtering stray light with non-designed polarization by using a polaroid, wherein the position of the polaroid is positioned at the diaphragm of the microscope;
shooting an observed object, wherein the shot picture has images of 7 visual angles of the object at 7 positions respectively, dividing 7 areas of the picture, finding a plurality of groups of characteristic points according to imaging parameters and the observation visual angles of each microlens, and recovering a three-dimensional model of the observed object by using a three-dimensional reconstruction algorithm;
and (5) for the real-time three-dimensional microscopic application scene, under the condition of computer computing power permission, the image segmentation and three-dimensional reconstruction algorithm can be inherited into shooting software to carry out three-dimensional real-time reconstruction, and when the computer computing power is insufficient, video recording processing can be carried out firstly, and then three-dimensional reconstruction recovery is carried out on each frame of image to obtain a dynamic image of the three-dimensional model.
The beneficial effects of the invention are as follows:
according to the invention, light field information of different visual angles of microorganisms can be respectively projected to different parts outside the device, all visual field information can be obtained through single shooting of a microscope, and the three-dimensional outline of an original object can be completely restored through a multi-eye image fusion algorithm, so that real-time reconstruction of a three-dimensional scene of the microorganisms is possible. Compared with the traditional three-dimensional chromatographic microscopy, the three-dimensional imaging method does not need scanning, can complete three-dimensional imaging through single shooting, and enables three-dimensional view reconstruction of living microorganism activities to be possible. The invention is a complete planar imaging device, can be directly integrated with the existing microscope, and realizes the integration of a three-dimensional microscopic imaging device.
Drawings
Fig. 1 is a schematic structural diagram of a real-time three-dimensional microscopic imaging device.
Fig. 2 is a schematic diagram of a lens array arrangement.
Fig. 3 is an optical path diagram of a view angle above an observation object.
Fig. 4 is a view of the optical path from the view angle below the observation object.
FIG. 5 (a) is a schematic diagram of a design microstructure;
fig. 5 (b) is a top view of a design microstructure, wherein the design freedom of the microstructure is indicated.
FIG. 6 is a schematic diagram of the conversion of the polarization state of a design light wave in a design device.
Fig. 7 is a schematic diagram of a design of microfluidic channels.
Fig. 8 is an overall structural diagram of a design device.
Detailed Description
The invention is further described below with reference to the drawings and examples.
A real-time three-dimensional microscopic imaging device based on a super-structured lens array comprises a plane reflecting mirror, a microfluidic channel and a super-structured lens array from bottom to top, as shown in a schematic diagram of fig. 1 (the reflecting mirror is a continuous film in fig. 1, and the hexagonal division is used for indicating the positions of different areas of the reflecting mirror corresponding to microlenses above in the schematic diagram).
The plane reflector is provided with a metal film and a transparent medium, wherein the metal film is used for realizing the reflection of light waves, and the transparent medium is used for protecting the metal film and supporting an observed object so that a light field below the observed object is reflected and led out.
The microfluidic channel is used for guiding an observed object, and the position of the observed object is controlled through liquid flow.
The super-structure lens array is used for respectively imaging information of a plurality of different visual angles of an observed object on the same focal plane, and has independent transreflective modulation capability on a group of orthogonally polarized light waves; and respectively imaging the information of a plurality of different visual angles through a microscope, and then carrying out three-dimensional image fusion to obtain three-dimensional scene reconstruction.
In the real-time three-dimensional microscopic imaging device, the super-structured lens array re-images light field information of a plurality of upward and downward visual angles of an observed object on the same focal plane, and each lens has a required depth of field requirement for a shooting visual angle. The super-structure lens array is a regular hexagonal lattice, six lenses are arranged around the central lens as shown in fig. 2, each lens collects light field information of different visual angles of an object, and the corresponding light field information is imaged above the super-structure lens array perpendicular to the plane of the super-structure lens, as shown in fig. 3.
The six super-structure lenses on the periphery collect 3 visual angle information of light emitted upwards by the object, and the other 3 super-structure lenses on the opposite sides collect 3 visual angle information of light emitted downwards, as shown in fig. 3 and 4.
The super-structured lens is composed of an anisotropic sub-wavelength structure, has abundant structural design freedom, for example, the structures shown in fig. 5 (a) and 5 (b) respectively have independent modulation capability of a transmission and reflection coefficient for orthogonal polarization states, and has high reflectivity (more than 80%), full-wave (0-2 pi) phase modulation and polarization conversion capability for a first incident light beam, and high transmissivity (more than 80%) modulation characteristic for a second incident orthogonal polarization state light beam. The evolution of the polarization state of a light wave in an optical system is shown in fig. 6.
The real-time three-dimensional microscopic imaging device is provided with the focal length of the super-structure lens for reflecting the polarization state of light, which is smaller than the distance between the super-surface and the reflecting mirror, and the focal length determines the magnification of the system.
In the real-time three-dimensional microscopic imaging device, the super-structured lens has certain light beam deflection capability, and deflects obliquely incident light waves to the position right above the super-structured lens for output, and deflection angles are respectively designed according to different lens arrangement positions. As shown in fig. 3 and 4, a light beam obliquely incident on each microlens outputs an image directly above each microlens.
The plane reflector consists of a metal film and a coating protection material with a certain thickness, so that an observed object is always above a designed distance above the reflector, and light field information below the object is obtained through reflection of the plane reflector.
In the real-time three-dimensional microscopic imaging device, the microfluidic channel is made of transparent materials, the thickness of the microfluidic channel is the focal length of the super-structure lens, the middle and lower parts of the microfluidic channel are slotted, and the size of the slotted channel is matched with the depth of field and the size of the field of the super-structure lens as shown in fig. 7; the slot is a microfluidic channel in the device, which is connected with an external microfluidic device (such as a high-precision microfluidic injection pump or a high-precision injector) for controlling the flow of liquid, so that the solution containing the observed object flows in, and the position of the observed object can be controlled by the pressure of the fluid.
The real-time three-dimensional microscopic imaging device is used for designing the super-structured lens array, the microfluidic channel and the plane reflecting mirror to be bonded together (such as ultraviolet glue curing process), and the microfluidic channel is positioned in the field of view of the lens array design through the alignment technology. A schematic diagram of a system consisting of a planar mirror, a super-structured lens array and microfluidic channels is shown in fig. 8.
The three-dimensional image fusion algorithm carries out fusion reconstruction on shot light field information of six visual angles and light field information of a central visual field to realize reconstruction of a three-dimensional scene of an object, wherein a multi-order SLAM (Simultaneous Location and Mapping, instant positioning reconstruction) algorithm and the like are used.
The embodiments in the foregoing description may be further combined or replaced, and the embodiments are merely illustrative of the preferred embodiments of the present invention and are not intended to limit the spirit and scope of the present invention, and various changes and modifications made by those skilled in the art to which the present invention pertains without departing from the spirit of the present invention. The scope of the invention is given by the appended claims and any equivalents thereof.
Claims (9)
1. The real-time three-dimensional microscopic imaging device based on the super-structured lens array is characterized by comprising a plane reflecting mirror, a microfluidic channel and the super-structured lens array from bottom to top;
the plane reflector is provided with a metal film and a transparent medium, the metal film is used for realizing the reflection of the light wave, and the transparent medium is used for protecting the metal film and supporting an observed object so that a light field below the observed object is reflected and led out;
the microfluidic channel is used for guiding an observed object, and the position of the observed object is controlled through liquid flow;
the super-structure lens array is used for respectively imaging information of a plurality of different visual angles of an observed object on the same focal plane, and has independent transreflective modulation capability on a group of orthogonally polarized light waves; and respectively imaging the information of a plurality of different visual angles through a microscope, and then carrying out three-dimensional image fusion to obtain three-dimensional scene reconstruction.
2. The real-time three-dimensional microscopic imaging device according to claim 1, wherein the super-structured lens array re-images the light field information of a plurality of upward and downward view angles of the observed object on the same focal plane, and each lens has a required depth of field requirement for a shooting view angle.
3. The real-time three-dimensional microscopic imaging device according to claim 1, wherein the super-structured lens is composed of anisotropic sub-wavelength structures, and has independent transmission and reflection coefficient modulation capability for orthogonal polarization states, and the super-structured lens has high reflectivity, full-wave phase modulation and polarization conversion capability of more than 80% for a first incident beam, and has high transmittance modulation characteristic of more than 80% for a second incident orthogonal polarization state beam.
4. The apparatus of claim 1, wherein the focal length of the super-lens for the polarization of the reflected light is set to be smaller than the distance between the super-surface and the reflecting mirror, and the focal length determines the magnification of the system.
5. The real-time three-dimensional microscopic imaging device according to claim 1, wherein the six super-lenses are arranged at the periphery, wherein 3 super-lenses at opposite sides collect light of 3 visual angles information emitted upwards by the object, the other 3 super-lenses at opposite sides collect light of 3 visual angles information emitted downwards, and the middle lens collects light of visual angles right above.
6. The real-time three-dimensional microscopic imaging device according to claim 1, wherein the super-structured lens has a certain light beam deflection capability, and deflects obliquely incident light waves to the position right above the super-structured lens for output, and the deflection angles are respectively designed according to different lens arrangement positions.
7. The real-time three-dimensional microscopic imaging device according to claim 1, wherein the microfluidic channel is made of transparent material, the thickness of the microfluidic channel is the focal length of the super-structured lens, the middle and lower parts of the microfluidic channel are slotted, and the size of the slotted channel is matched with the depth of field and the size of the field of view of the super-structured lens; the slot is a microfluidic channel in the device, which is connected with an external microfluidic device for controlling the flow of liquid, so that the solution containing the observed object flows in, and the position of the observed object can be controlled by the pressure of the fluid.
8. The real-time three-dimensional microscopic imaging device according to claim 1, wherein the super-structured lens array, the microfluidic channel and the planar mirror are bonded together and the microfluidic channel is located within the field of view of the lens array design by an overlay technique.
9. A method of real-time three-dimensional microscopic imaging based on a super-structured lens array, comprising the steps of:
the method comprises the steps that (1) a design device is placed below an objective lens of a microscope, so that an imaging surface of the objective lens is overlapped with an imaging surface of a micro lens array, illumination light waves are coupled into the design device through the micro lens array, a micro-fluidic channel is illuminated, the microscope can obtain images of 7 micro lenses collected by the objective lens at the same time, the 7 images are imaged in 7 areas of a microscopic image surface, and the 7 areas are respectively right above the 7 micro lenses;
injecting a solution containing an observed object into a microfluidic device, enabling the observed object to be positioned in a designed view field through flow control, and controlling the position of the observed object through liquid flow;
step (3) filtering stray light with non-designed polarization by using a polaroid, wherein the position of the polaroid is positioned at the diaphragm of the microscope;
shooting an observed object, wherein the shot picture has images of 7 visual angles of the object at 7 positions respectively, dividing 7 areas of the picture, finding a plurality of groups of characteristic points according to imaging parameters and the observation visual angles of each microlens, and recovering a three-dimensional model of the observed object by using a three-dimensional reconstruction algorithm;
and (5) for the real-time three-dimensional microscopic application scene, under the condition of computer computing power permission, the image segmentation and three-dimensional reconstruction algorithm can be inherited into shooting software to carry out three-dimensional real-time reconstruction, and when the computer computing power is insufficient, video recording processing can be carried out firstly, and then three-dimensional reconstruction recovery is carried out on each frame of image to obtain a dynamic image of the three-dimensional model.
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