CN110967817A - Image scanning microscopic imaging method and device based on double micro-lens array - Google Patents
Image scanning microscopic imaging method and device based on double micro-lens array Download PDFInfo
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- CN110967817A CN110967817A CN201911198164.9A CN201911198164A CN110967817A CN 110967817 A CN110967817 A CN 110967817A CN 201911198164 A CN201911198164 A CN 201911198164A CN 110967817 A CN110967817 A CN 110967817A
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
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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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
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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
Abstract
The invention relates to an image scanning microscopic imaging method and device based on a double-microlens array, belonging to the technical field of optical microscopic measurement; the array multi-focus generated by the micro-lens array illuminates a sample, the imaging speed is improved, the influence of defocused fluorescence is eliminated through the pinhole array, the second micro-lens array zooms each focused light beam 1/2, full optical realization of subsequent digital information processing of the traditional image scanning microscopy is realized, the resolution is improved by two times, and the problem of low imaging speed of the traditional image scanning microscopy system is solved. The laser beam reaches the first micro lens array after being expanded to generate array type illumination beams, and after the array type illumination beams are emitted to the surface of a sample, the objective lens collects multi-focus fluorescence from the sample and emits the multi-focus fluorescence to the pinhole array to eliminate the influence of the defocused fluorescence. The second micro lens array partially shrinks each focusing point, the original focusing direction of the light beam is kept, and the fluorescence focus after the zooming processing is imaged on the sCMOS camera under the deflection action of the scanning galvanometer.
Description
Technical Field
An image scanning microscopic imaging method and device based on a double-microlens array belongs to the field of optical microscopic measurement, and mainly relates to a method and device for carrying out non-contact rapid imaging on a fine structure of a biological sample based on a microstructure optical element.
Background
Modern fluorescence microscopes combine imaging speed, molecular specificity and contrast to visualize the cell imaging process, but diffraction limits the resolution of wide-field microscopic fluorescence microscopes, while super-resolution imaging techniques break through the traditional diffraction limit, but decrease in speed, imaging time, field of view, and the like. For example, single molecule imaging counting and stimulated emission depletion microscopes allow the spatial resolution of cells to be less than 100nm, but the imaging speed is limited to 0.01-1 Hz.
Compared with a single-molecule imaging counting microscope and a stimulated emission loss microscope, the structured light illumination microscope loads object high-frequency information into a detection passband of an optical system in a spatial frequency mixing mode, can reach twice of the original resolution limit, and provides faster image acquisition speed and capability of removing stray light compared with the structured light illumination microscope. However, the structured light illumination imaging microscopy technology requires acquisition of a plurality of original images, and digital combination of the acquired images through an algorithm to generate a single two-dimensional high-resolution image, and this method requiring acquisition and combination of a plurality of original images on each plane fundamentally limits the imaging speed of the structured light illumination microscope relative to the conventional microscope.
The other structured light imaging method taking diffraction limited light spots as an illumination light source is called an image scanning microscopic imaging technology, each pixel point of an area array detector is utilized to record a fluorescence image excited by the light spots, and a super-resolution image is reconstructed through operations such as pixel movement and zooming.
Therefore, the problems to be solved by the skilled person are: how to apply the advantages of high resolution, simple equipment and strong robustness to the defects of experimental equipment of image scanning microscopic imaging to carry out quick super-resolution imaging on a sample, and simultaneously reduce or abandon the subsequent complicated image reconstruction process to realize quicker image reconstruction.
Disclosure of Invention
In order to solve the problems of low imaging speed and complex subsequent reconstruction algorithm of the image scanning microimaging technology, the invention designs an image scanning microimaging method and device based on a double-microlens array, wherein an excitation light beam generates an array-type excitation light beam through a first microlens array, the simultaneous multifocal scanning of a sample is realized, the scanning speed is improved, an original image is zoomed to 1/2 of the original image by using a second scanning lens, the optical image processing is realized, the subsequent digital image processing operation is omitted, and the reconstruction time is shortened. The whole process does not need to acquire and store a plurality of camera exposure images, and the data acquisition efficiency is greatly improved while the double resolution ratio is kept.
The purpose of the invention is realized as follows:
the image scanning microscopic imaging method based on the double micro-lens array comprises the following steps:
a. the exciting light forms an array type exciting focus through a first micro lens array, the sample is subjected to area array scanning, and multi-focus fluorescence is collected from the sample by using the same objective lens;
b. the multi-focus fluorescence is deflected by the scanning galvanometer and then emitted to a pinhole array positioned at the back focal plane of the scanning lens, so that the separation of the off-focus fluorescence is realized;
c. the second microlens array performs a local zoom process on the light transmitted through the pinhole array, zooms each focal point to 1/2 of the original size, and images onto the sCMOS camera through the scanning galvanometer.
In the step a, the focal length of the first microlens array is twice that of the second microlens array, in the step b, the pinhole interval of the micropore array is equal to the distance between the microlenses in the microlens array, and the diameter of the pinhole is slightly larger than that of the microlens; in the step c, the focal length of the second micro lens array is half of that of the first micro lens array, and the zooming processing of the focal point is realized.
The image scanning microscopic imaging device based on the double micro lens array realizes the full optical realization of the traditional image scanning microscopy by using the double micro lens array and a related light path. Characterized in that said device comprises: laser instrument, half-wave plate, microlens array, compensating plate, beam splitter, scanning lens, scanning galvanometer, tubular lens, pinhole array, sCMOS camera, wherein: the laser is used for exciting fluorescence in a sample, and the laser reaches the first micro-lens array after being expanded by the half-wave plate and the light beam to generate array type illuminating light beams. Then a compensating plate is arranged to reduce astigmatism, and the astigmatism is reflected to the surface of the sample through the beam splitter, the first scanning lens, the second scanning lens, the scanning galvanometer and the tubular lens. The same objective lens is used for collecting multi-focus fluorescence from a sample, and the multi-focus fluorescence sequentially passes through the tubular lens, the first scanning lens, the scanning galvanometer and the second scanning lens and is reflected to the pinhole array through the beam splitter so as to eliminate the influence of the defocused fluorescence. The second micro lens array is used for locally shrinking each focusing point, meanwhile, the original focusing direction is kept, and the fluorescence focus after the zooming processing is imaged on the sCMOS camera through the third scanning mirror under the deflection action of the scanning galvanometer.
In the image scanning microimaging device based on the double-microlens array, the focal lengths of the first scanning lens and the second scanning lens are equal to form a 4f system;
in the image scanning microimaging device based on the double-microlens array, the back focal plane of the tubular lens coincides with the front focal plane of the scanning lens 2;
according to the image scanning microimaging device based on the double-microlens array, the excitation light passes through the first microlens array to form the array type excitation focus to carry out area array scanning on the sample, and the fluorescence signal generated by stimulated emission on the sample plane is also in a multi-focus form.
In the image scanning microscopic imaging device based on the double-microlens array, the multifocal fluorescence is deflected by the scanning galvanometer and the beam splitter and then emitted to the pinhole array, and the pinhole interval of the pinhole array is the same as the microlens interval of the microlens array.
In the image scanning microimaging device based on the double-microlens array, the pinhole array is positioned on the front focal plane of the first scanning lens;
in the image scanning microimaging device based on the double microlens arrays, the focal length of the second microlens array is half of that of the first microlens array, so that the original image is zoomed to 1/2;
the image scanning microscopic imaging device based on the double-microlens array is characterized in that each scanning position is recorded by a pixel point of an sCMOS camera sensitive to the position, and images recorded by array focus scanning are overlapped through an algorithm to generate a final high-resolution image;
the image scanning microscopic imaging device based on the double-microlens array is characterized in that the array type focus generated by the microlens array is adopted to illuminate a sample, the influence of defocused fluorescence is eliminated through the pinhole array, the zooming of each focus point 1/2 is realized through the second microlens array, and the all-optical realization of the subsequent digital information processing of the traditional image scanning microscopy is realized.
The invention relates to an image scanning microscopic imaging device based on a double micro-lens array, which comprises a laser, a half-wave plate arranged in front of the laser, a beam expander arranged on a direct light path of the laser, a micro-lens array and a compensation plate, wherein multi-focus illumination is collimated through two scanning lenses, a tubular lens and an objective lens are used for exciting fluorescent light beams on a sample plane, a single-shaft scanning galvanometer for bilateral scanning is arranged between a first scanning lens and a second scanning lens, and the conversion of the multi-focus illumination array on the sample plane is realized through the rotation of the scanning galvanometer. The sample excited fluorescence passes through the tubular lens, the first scanning lens, the second scanning lens and the scanning galvanometer from the objective lens in sequence, and reaches the pinhole array positioned at the front focal plane of the first scanning lens from the beam splitter through reflection so as to block the influence of the defocused fluorescence. The fluorescence beam obtained after the pinhole array filtration is transmitted to the second micro lens array, and the fluorescence is locally shrunk to 1/2 with the original size after passing through the micro lens array, and meanwhile, the propagation direction of each focus point is kept. Then, the focus after optical reconstruction is reflected and imaged on an sCMOS camera through a scanning lens. According to the design, the array type focus generated by the micro lens array illuminates a sample, the imaging speed is improved, the influence of defocused fluorescence is eliminated through the pinhole array, the zooming of each focus point is realized through the second micro lens array, the all-optical reconstruction of the subsequent digital information processing of the traditional image scanning microscope is realized, the resolution is improved by two times, and the problem of low imaging speed of the traditional image scanning microscope system is solved.
Drawings
FIG. 1 is a schematic diagram of a scanning microimaging method and device based on a double microlens array
In the figure: the laser comprises a laser 1, a half-wave plate 2, a first reflector 3, a second reflector 4, a first lens 5, a second lens 6, a third reflector 7, a first microlens array 8, a compensation plate 9, a beam splitter 10, a first scanning lens 11, a second scanning lens 12, a tubular lens 13, an objective lens 14, a sample 15, a pinhole array 16, a fourth reflector 17, a fifth reflector 18, a second microlens array 19, a third scanning lens 20, a scanning galvanometer 21, a fourth scanning lens 22, a light filter 23 and a CMOS camera 24.
Detailed Description
The following detailed description of embodiments of the invention refers to the accompanying drawings
The image scanning microimaging method and device based on the double-microlens array comprises a laser 1, a half-wave plate 2 is arranged in front of the laser 1 to achieve intensity control of laser, a beam expander composed of a first lens 5 and a second lens 6 is arranged on a direct light path of the laser, the expanded light is emitted to a first microlens array 8, the focal length of the first microlens array is 2mm, the diameter of the first microlens array is 25mm, and a compensation plate 9 is arranged behind the first microlens array to eliminate astigmatism of a focused light beam. The multifocal illumination is collimated by a first scanning lens 11 and a second scanning lens 12, the first scanning lens 11 and the second scanning lens 12 constituting a 4f system. The excitation light beam of the sample 15 plane is imaged through a tubular lens 13 and an objective lens 14, wherein the back focal plane of the tubular lens 13 is coincident with the front focal plane of a second scanning lens 12, a scanning galvanometer 21 for single-axis double-side scanning is arranged between the first scanning lens 11 and the second scanning lens 12, and the conversion of the multi-focus illumination array on the sample 15 plane is realized through the rotation of the scanning galvanometer 21. The sample 15 excites fluorescence, which starts from an objective lens 14, passes through a tubular lens 13, a first scanning lens 11, a second scanning lens 12 and a scanning galvanometer 21 in sequence, and reaches a pinhole array positioned at the front focal plane of the scanning lens 1 through reflection from a beam splitter 10, the pinhole interval of the micropore array is equal to the microlens interval in the microlens array, the pinhole interval is 200 mu m, and the diameter of the pinhole is 40 mu m so as to block the influence of defocused fluorescence. The fluorescence beam obtained after filtering by the pinhole array 16 is transmitted to the second microlens array 19, the focal length of which is 1mm, and the fluorescence is locally shrunk to 1/2 of the original size after passing through the microlens array 19, while maintaining the propagation direction of each focus point. Then, the focus after optical reconstruction is reflected by the third scanning lens 20 and the scanning galvanometer 21, and is imaged on an sCMOS camera 24 after passing through the fourth scanning lens 22 and the optical filter 23, so that the all-optical reconstruction of the subsequent digital information processing of the traditional image scanning microscopy is realized.
The method and the device for scanning and microimaging an image based on a double-microlens array proposed by the present invention are described in detail above, and the principle and the implementation mode of the present invention are explained in the present document by applying specific examples, and the description of the above examples is only used to help understanding the method and the core idea of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there are changes in the specific embodiments and the application scope, and these changes should be covered by the protection scope of the appended claims. In view of the above, the present disclosure should not be construed as limiting the invention.
Claims (9)
1. The image scanning microscopic imaging method and device based on the double micro lens array realizes the full optics realization of the traditional image scanning microscopy by using the double micro lens array and the related light path; characterized in that said device comprises: laser instrument, half-wave plate, microlens array, compensating plate, beam splitter, scanning lens, scanning galvanometer, tubular lens, pinhole array, sCMOS camera, wherein: the laser is used for exciting fluorescence in a sample, and reaches the first micro-lens array after being expanded by the half-wave plate and the light beam to generate array type multi-focus illumination light beams; then a compensating plate is arranged to reduce the influence of astigmatism, and the astigmatism is reflected to the surface of the sample through a beam splitter, a first scanning lens, a scanning galvanometer, a second scanning lens and a tubular lens and finally through an objective lens; collecting multi-focus fluorescence from a sample by using the same objective lens, sequentially passing through a tubular lens, a first scanning lens, a scanning galvanometer and a second scanning lens, and reflecting the multi-focus fluorescence to the pinhole array through a beam splitter so as to eliminate the influence of the defocused fluorescence; the second micro lens array is used for locally shrinking each focusing point, meanwhile, the original focusing direction is kept, and the fluorescence focus after the zooming processing passes through the third scanning lens and then is imaged on the sCMOS camera under the deflection action of the scanning galvanometer.
2. The image scanning microscopic imaging method and device based on the double micro lens array are characterized by comprising the following steps:
(1) the excitation light is subjected to area array scanning on the sample through the first micro-lens array, and fluorescence is collected from the sample by using the same objective lens;
(2) the fluorescence signal is deflected by the scanning galvanometer and then emitted to a pinhole array positioned at the back focal plane of the scanning lens, so that the defocusing fluorescence is blocked;
(3) the second micro lens array carries out local zooming processing on the light transmitted through the pinhole array and images the light on the sCMOS camera through the scanning galvanometer.
3. The method and apparatus for scanning microscopy imaging based on two microlens arrays according to claim 1 wherein the focal length of the first scanning lens and the second scanning lens are equal to form a 4f system.
4. The image scanning microimaging method and device based on the double-microlens array as claimed in claim 1, wherein the back focal plane of the tubular lens coincides with the front focal plane of the second scanning lens.
5. The method and apparatus for image scanning microscopy according to claim 2 wherein in step (1), the excitation light is scanned area-wise through the array-like excitation focuses formed by the first microlens array, and the fluorescence signal of stimulated emission generated at the sample plane is also in the form of multi-focus.
6. The method and apparatus for scanning microscopy imaging based on two microlens arrays as claimed in claim 2, wherein in step (2), the multifocal fluorescent light is deflected by the scanning galvanometer and the beam splitter and then emitted to the pinhole array, and the pinhole interval of the pinhole array is the same as the microlens interval of the microlens array.
7. The method and apparatus for scanning microscopy imaging based on two microlens arrays as claimed in claim 2, wherein in step (3), the focal length of the lens of the second microlens array is half of the focal length of the first microlens array, and the light passing through the pinhole array is locally scaled to 1/2, which is the original size.
8. The method and apparatus for scanning microscopy imaging based on two microlens arrays according to claim 1 wherein the pinhole array is located at the front focal plane of the first scanning lens.
9. The multifocal light beam passing through the first micro-lens array scans a sample, each scanned position is recorded by a pixel point of an sCMOS camera sensitive to the position after the multifocal light beam passes through a complete light path, and images recorded by array focus scanning are overlapped through an algorithm to generate a final high-resolution image.
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| CN111879740A (en) * | 2020-07-14 | 2020-11-03 | 哈尔滨工业大学 | All-optical super-resolution microscopic device based on photon reset technology |
| CN112525871A (en) * | 2020-10-28 | 2021-03-19 | 汕头大学 | Non-scanning confocal microscopic system based on micro-LED |
| CN114113019A (en) * | 2021-11-30 | 2022-03-01 | 哈尔滨工业大学 | Array scanning super-resolution microscopic imaging device, method and equipment based on multiple signal classification algorithm and storage medium |
| CN117369106A (en) * | 2023-12-05 | 2024-01-09 | 北京大学 | Multi-point confocal image scanning microscope and imaging method |
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