CN220289941U - Large-view-field high-flux high-resolution confocal imaging system based on microlens array - Google Patents
Large-view-field high-flux high-resolution confocal imaging system based on microlens array Download PDFInfo
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- 238000010226 confocal imaging Methods 0.000 title claims abstract description 13
- 238000003384 imaging method Methods 0.000 claims abstract description 52
- 238000005286 illumination Methods 0.000 claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 230000003287 optical effect Effects 0.000 claims abstract description 12
- 230000005540 biological transmission Effects 0.000 claims abstract description 4
- 238000006073 displacement reaction Methods 0.000 claims description 12
- 230000021615 conjugation Effects 0.000 abstract description 5
- 238000000799 fluorescence microscopy Methods 0.000 abstract description 2
- 230000003044 adaptive effect Effects 0.000 description 5
- 230000005284 excitation Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 4
- 230000008521 reorganization Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010191 image analysis Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 206010034972 Photosensitivity reaction Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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Abstract
The utility model discloses a large-view-field high-flux high-resolution confocal imaging system based on a micro-lens array, and belongs to the technical field of fluorescence microscopic imaging. The utility model provides a large-view-field high-flux high-resolution confocal imaging system based on a micro-lens array, which comprises an illumination module, a scanning module and a detection module which are sequentially arranged along an optical axis. The scanning module comprises a dichroic mirror positioned in the relay lens group, the illumination module is positioned on the incident light path of the dichroic mirror, the microlens array is positioned on the transmission light path of the dichroic mirror, and the array detector is positioned on the reflection light path of the dichroic mirror. The parallel light is used as illumination light, the micro lens array is used as an imaging objective lens, and multi-focus parallel scanning can be realized, so that large-field imaging is realized. For a large sample, the whole imaging area can be directly covered by the micro lens array, no non-imaging time caused by the movement of a conventional objective lens platform is needed, and the imaging speed is greatly improved. The defocused illumination realizes conjugation of the detection surface and the sample imaging surface, and then the detection surface and the sample imaging surface are combined.
Description
Technical Field
The utility model belongs to the field of fluorescence microscopic imaging, and particularly relates to a large-view-field high-flux high-resolution confocal imaging system based on a micro-lens array.
Background
Confocal laser scanning microscopy (Confocal Laser Scanning Microscope, CLSM) is a novel instrument with high photosensitivity and high resolution that has been developed for over a decade. The laser is used as a light source, and the confocal imaging scanning system, the electron optical system and the microcomputer image analysis system are used for forming the confocal imaging scanning system. The light beam is focused and then falls on tiny points of different depths of a sample (tissue thick slice or cell), and the micro points are moved and scanned, and through electric signal color imaging, the image formed by the reflected light of any point in the sample can be accurately received and generated to be transmitted to a color display, and then is connected with a microcomputer image analysis system for analysis and processing.
Compared with a conventional wide-field microscope, the confocal microscope has better chromatographic capacity and resolution, and is widely applied to the fields of biomedicine, integrated circuit detection and the like. The commercial point scanning confocal microscope adopts point-by-point scanning imaging, the imaging speed is low, the rotary table confocal microscope adopts a Nipkow rotary table to realize multi-focus parallel imaging, and the imaging speed can be improved. However, for imaging a large area sample, because the field of view of the traditional objective lens is limited, the whole large field of view imaging is generally finished by adopting a block splicing imaging mode of a moving translation stage, and the moving and focusing processes of the platform belong to non-imaging time, so that the imaging speed of the system is greatly limited.
Disclosure of Invention
Aiming at the defects or improvement demands of the prior art, the utility model provides a large-view-field high-flux high-resolution confocal imaging system based on a micro-lens array, and aims to solve the technical problem that the imaging speed of the confocal microscopic imaging system is low under the large view field.
In order to achieve the above object, according to one aspect of the present utility model, there is provided a large-field high-flux high-resolution confocal imaging system based on a microlens array, including an illumination module, a scanning module, and a detection module sequentially disposed along an optical axis; the lighting module sequentially comprises a laser and a collimation beam expander group; the scanning module sequentially comprises a relay lens group, a micro lens inclination adjuster, a micro lens array and a sample table, wherein the micro lens array is fixed with the micro lens inclination adjuster, and the distance between an imaging surface of a sample on the sample table and the micro lens array is larger than the distance between a focal plane of a micro lens in the micro lens array and the micro lens array; the detection module comprises an array detector; the scanning module further comprises a dichroic mirror positioned in the relay lens group, the illumination module is positioned on an incident light path of the dichroic mirror, the microlens array is positioned on a transmission light path of the dichroic mirror, and the array detector is positioned on a reflection light path of the dichroic mirror.
Optionally, the size of the light spot detected by the array detector is PSF detecter ，PSF detecter And D is less than or equal to 0.7D, wherein D is the interval between adjacent microlenses of the microlens array.
Optionally, the distance between the sample imaging surface and the focal plane of the microlens is a defocus distance δ,0.05 f+.delta.ltoreq.0.15 f, where f is the focal length of the microlens.
Optionally, the collimating and beam expanding lens group includes a first lens and a second lens.
Optionally, the relay lens group includes a third lens and a fourth lens, and the microlens array is parallel to a focal plane of the fourth lens.
Optionally, the detection module further comprises an optical filter and a fifth lens, which are sequentially positioned between the dichroic mirror and the array detector.
Optionally, the array detector is one of CCD, CMOS, sCMOS.
Optionally, the sample stage is a three-dimensional nano-displacement stage.
Optionally, the system further comprises a control module and an image reconstruction module, wherein the control module is in signal connection with the sample stage and the array detector, and the image reconstruction module is in signal connection with the control module.
In general, the above technical solutions conceived by the present utility model enable the following to be achieved compared with the prior art
The beneficial effects are that:
the parallel light is used as illumination light, the micro lens array is used as an imaging objective lens, and the multi-focus parallel scanning can realize large-field imaging. For a large sample, the whole imaging area can be directly covered by the micro lens array, no non-imaging time caused by the movement of a conventional objective lens platform is needed, and the imaging speed is greatly improved. Meanwhile, the micro lens is located at a small distance below the focal plane and performs defocusing imaging to form a relay excitation lattice, the relay excitation lattice is projected to the camera plane, conjugation of the camera and the sample plane is achieved, and further, the resolution of the system is improved by combining a high-resolution algorithm, the relay illumination lattice is not required to be generated by an expensive spatial light modulator, optical conjugation of the camera and the sample is achieved, cost and complexity of experimental equipment are reduced, the limitation of the number of illumination lattices of the spatial light modulator is avoided, and a larger imaging area can be achieved.
Drawings
FIG. 1 is a schematic diagram of a large field-of-view high resolution confocal imaging system of the utility model based on a microlens array;
FIG. 2 is a schematic view of an imaging optical path of a microlens array of the present utility model;
FIG. 3 is an image of a spot detected by an array detector of the present utility model;
FIG. 4 is a schematic diagram of a scanning mode of a nano-displacement stage according to the present utility model;
FIG. 5 is a schematic of the pixel location of a single spot of the present utility model;
fig. 6 is a representative adaptive pixel rebinning displacement vector diagram for a single spot of the present utility model.
In the figure, an illumination module 110, a laser 111, a collimator-beam expander lens group 110a, a first lens 112, a second lens 113, a scanning module 120, a relay lens group 120a, a third lens 121, a dichroic mirror 122, a fourth lens 123, a microlens array 124, a sample 125, a displacement stage 126, a tilt adjuster 127, a detection module 130, a filter 131, a fifth lens 132, an array detector 133, a control module 140, an image reconstruction module 150, a glass substrate 161, and a microlens surface 162.
Detailed Description
The present utility model will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present utility model more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the utility model. In addition, the technical features of the embodiments of the present utility model described below may be combined with each other as long as they do not collide with each other.
As shown in fig. 1, the present utility model proposes a large-field high-flux high-resolution confocal imaging system based on a microlens array, which includes an illumination module 110, a scanning module 120, and a detection module 130 sequentially disposed along an optical axis. The illumination module 110 sequentially comprises a laser 111 and a collimation beam expander group 110a; the scanning module 120 sequentially comprises a relay lens group 120a, a microlens array 124, a microlens inclination adjuster 127, and a sample stage 126, wherein the microlens array 124 is fixed with the microlens inclination adjuster 127, and the distance between the sample imaging surface and the microlens array 124 is greater than the distance between the focal plane of the microlens array and the microlens array; the detection module 130 includes an array detector 133; the scanning module 120 further includes a dichroic mirror 122 disposed within the relay lens group 120a, the illumination module 110 is disposed on an incident light path of the dichroic mirror 122, the microlens array 124 is disposed on a transmission light path of the dichroic mirror 122, and the array detector 133 is disposed on a reflection light path of the dichroic mirror 122.
Specifically, the laser beam emitted from the laser 111 is collimated and expanded by the collimating and expanding lens set 110a to form a collimated and expanded beam, and then is incident on the microlens array 124 through the relay optical path of the relay lens set 120a to form multi-focus illumination. As shown in fig. 2, the distance between the imaging surface of the sample on the sample stage and the microlens array 124 is greater than the distance between the focal plane of the microlenses in the microlens array 124 and the microlens array 124. Sample 125 is placed on sample stage 126, and fluorescence excited by sample 125 is collected and focused on the intermediate imaging plane by microlens array 124, reflected by dichroic mirror 122, and conjugated to array detector 133 through relay lens group 120a to form an array of light spots (as shown in fig. 3). The array detector 133 converts fluorescent signals of many spot arrays collected by the micro lens into electrical signals. The sample 125 is placed on the displacement stage 126 for XYZ three-dimensional scanning, and the control module 140 is used for controlling the nano displacement stage 126 to move for scanning, and simultaneously controlling the array detector 133 to synchronously collect image data, so as to complete the whole sample sampling.
As shown in fig. 4, the single microlens 172 scans with the movement of the displacement stage 126, and its imaging range is shown by a dashed box 171. The micro lens array is illuminated by parallel light, and the micro lens array is used as an imaging objective lens, so that multi-focus parallel scanning can be realized, and large-field imaging is realized. For a large sample, the whole imaging area can be directly covered by the micro lens array, no non-imaging time caused by the movement of a conventional objective lens platform is needed, and the imaging speed is greatly improved. Meanwhile, the micro lens is located at a small distance below the focal plane and performs defocusing imaging to form a relay excitation lattice, the relay excitation lattice is projected to the camera plane, conjugation of the camera and the sample plane is achieved, and further, the resolution of the system is improved by combining a high-resolution algorithm, the relay illumination lattice is not required to be generated by an expensive spatial light modulator, optical conjugation of the camera and the sample is achieved, cost and complexity of experimental equipment are reduced, the limitation of the number of illumination lattices of the spatial light modulator is avoided, and a larger imaging area can be achieved. Optionally, the collimating and beam expanding lens group 110a includes a first lens 112 and a second lens 113.
Alternatively, the relay lens group 120a includes a third lens 121 and a fourth lens 123, and the microlens array 124 is parallel to a focal plane of the fourth lens 123.
Optionally, the detection module 130 further includes a filter 131 and a fifth lens 132 sequentially positioned between the dichroic mirror 122 and the array detector 133.
Optionally, the array detector 133 is one of CCD, CMOS, sCMOS.
Optionally, the sample stage 126 is a three-dimensional nano-displacement stage.
Specifically, the microlens array 126 includes a glass substrate 161 and a plurality of microlens surfaces 162 arrayed on the glass substrate 161.
Optionally, the size of the light spot detected by the array detector is PSF detecter ，PSF detecter And D is less than or equal to 0.7D, wherein D is the interval between adjacent microlenses of the microlens array. The size of the light spot is 0.7 times of the distance between the micro lenses, so that the sufficient resolution improving effect and smaller crosstalk can be obtained, the size of the light spot when the size of the illumination light spot of the sample surface is close to the size of the focal plane is ensured, and the lattice crosstalk influence is reduced.
The spot size of the sample surface is PSF sample =2.44λ× (wd+h)/R, where λ is the excitation light wavelength, WD is the effective working distance of the microlens imaging optical path, R is the microlens diameter, h is the sagittal height of the microlens, and the relay optical path is amplified to the detector face spot size required PSF detecter ＝PSF sample ×M MLA Not more than 0.7D, wherein M MLA Magnification for imaging the microlenses, D being the spacing between adjacent microlenses.
The back focal length BFL may be obtained by the formula:effective working distance of microlens imaging light pathThe distance between the focal plane of the sample imaging plane and the focal plane of the microlens is the defocus distance δ=wd-f, where f is the focal length of the microlens, n MLA Is the refractive index of the microlens. Thus, the sample defocus distance can be achieved by changing the distance (back focal length) of the micro-lens face and the intermediate imaging face.
Preferably, the defocus distance is in the range of 0.05 f.ltoreq.delta.ltoreq.0.15 f. Where f is the focal length of the microlens.
In some embodiments, as shown in fig. 2, the distance between the microlens surface and the intermediate imaging surface (back focal length BFL) is designed to be 1.7mm, corresponding to a working distance wd=145 μm (microlens aperture diameter 68 μm, adjacent microlens spacing 70 μm, microlens focal length 115 μm, microlens refractive index n MLA =1.46, wavelength 0.5 μm, magnification of microlens imaging is 19 times), and finally the defocus distance of the sample imaging plane and the focal plane of the microlens is δ=wd-f=15 μm. At this time, although the focal point of the illumination lattice is not the smallest, the imaging plane resolution near the focal plane is close to the focal plane resolution because the microlens has a lower numerical aperture and a longer axial depth of field. The resolution of the system at the focal plane of the micro lens is about 1.25 mu m (the full width half maximum value of the point spread function), the resolution of the sample imaging surface after the defocusing treatment is about 1.35 mu m, and the defocusing treatment does not obviously deteriorate the resolution.
In some embodiments, the confocal imaging system further includes a control module and an image reconstruction module, where the control module is in signal connection with the sample stage and the array detector, and the control module 140 is configured to control the nano-displacement stage 126 to move and scan, and simultaneously control the array detector 133 to synchronously collect image data, so as to complete the whole sample sampling. The image reconstruction module is in signal connection with the control module and is used for reconstructing the acquired image data.
Optionally, the image reconstruction module may perform super-resolution reconstruction processing on the detected light spot by using an existing subtraction-image scanning technique for performing adaptive pixel reconstruction.
The super-resolution reconstruction processing of the subtraction-image scanning technology of the self-adaptive pixel reorganization comprises digital pinhole confocal processing, self-adaptive pixel reorganization, subtraction processing and deconvolution processing. The digital pinhole confocal processing is to multiply each light spot of the light spot array by a two-dimensional Gaussian function to inhibit background signals and realize digital confocal effect. The self-adaptive pixel reorganization process is to take a pixel area with the size of one Airy spot (1 AU) as a virtual pinhole (N pixels in the Airy spot area as shown in figures 5 and 6) with each light spot taking the center as the origin, and each pixel as an independent point detectorAcquiring sample scanning information and reconstructing an image of a scanning area, wherein the reconstructed image of the most central pixel is taken as a reference image, and the reconstructed images of other pixels of the Airy spot area are all displaced by about a i ＝r i /2。(r i For the i-th pixel to center pixel distance, a i A displacement vector for the i-th pixel). The specific displacement vector of the image acquired by each pixel can be specifically obtained by performing cross-correlation processing on the image acquired by the central pixel. The images acquired by each pixel are finally superimposed, i.e(r is the position of the image, P (r) ISM P for final adaptive pixel rebinned image i The image acquired for each pixel), this process is called adaptive pixel rebinning. Then, the subtraction confocal processing is carried out to further improve the resolution and the contrast ratio: p (r) Sub-ISM ＝P(r) Confocal -γ×P(r) ISM (r is the position of the image, P (r) Sub-ISM P (r) is an image of pixel reorganization subjected to subtraction processing ISM P (r) for the final adaptive pixel rebinned image Confocal And (3) reconstructing a confocal image for the most central pixel of the light spot, wherein gamma is a subtraction coefficient, and the specific conditions are required to be adjusted so that a negative value of a final subtracted image cannot occur), and finally deconvoluting the subtracted image to further improve the resolution.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the utility model and is not intended to limit the utility model, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the utility model are intended to be included within the scope of the utility model.
Claims (8)
1. The large-view-field high-flux high-resolution confocal imaging system based on the micro-lens array is characterized by comprising an illumination module, a scanning module and a detection module which are sequentially arranged along an optical axis;
the lighting module sequentially comprises a laser and a collimation beam expander group;
the scanning module sequentially comprises a relay lens group, a micro lens inclination adjuster, a micro lens array and a sample table, wherein the micro lens array is fixed with the micro lens inclination adjuster, and the distance between a sample imaging surface and the micro lens array is larger than the distance between a focal plane of a micro lens in the micro lens array and the micro lens array;
the detection module comprises an array detector;
the scanning module further comprises a dichroic mirror positioned in the relay lens group, the illumination module is positioned on an incident light path of the dichroic mirror, the microlens array is positioned on a transmission light path of the dichroic mirror, and the array detector is positioned on a reflection light path of the dichroic mirror.
2. The system of claim 1, wherein the array detector detects a spot size of PSF detecter ，PSF detecter And D is less than or equal to 0.7D, wherein D is the interval between adjacent microlenses of the microlens array.
3. The system of claim 1, wherein the distance between the sample imaging surface and the focal plane of the microlens is a defocus distance δ,0.05 f+.delta.ltoreq.0.15 f, where f is the focal length of the microlens.
4. The system of claim 2, wherein the collimating and beam expanding lens group comprises a first lens and a second lens.
5. The system of claim 4, wherein the relay lens group comprises a third lens and a fourth lens, the microlens array being parallel to a focal plane of the fourth lens.
6. The system of claim 1, wherein the detection module further comprises a filter, a fifth lens, positioned in sequence between the dichroic mirror and the array detector.
7. The system of claim 6, wherein the array detector is one of CCD, CMOS, sCMOS.
8. The system of claim 1, wherein the sample stage is a three-dimensional nano-displacement stage.
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