CN107941775B - Multispectral microscopic imaging system - Google Patents
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- CN107941775B CN107941775B CN201711460224.0A CN201711460224A CN107941775B CN 107941775 B CN107941775 B CN 107941775B CN 201711460224 A CN201711460224 A CN 201711460224A CN 107941775 B CN107941775 B CN 107941775B
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- 238000003384 imaging method Methods 0.000 title claims abstract description 76
- 230000003595 spectral effect Effects 0.000 claims abstract description 33
- 239000006185 dispersion Substances 0.000 claims abstract description 30
- 230000003287 optical effect Effects 0.000 claims abstract description 22
- 238000005286 illumination Methods 0.000 claims abstract description 7
- 238000001914 filtration Methods 0.000 claims abstract description 4
- 230000007480 spreading Effects 0.000 claims abstract description 3
- 238000000386 microscopy Methods 0.000 claims description 13
- 230000004044 response Effects 0.000 claims description 10
- 102000034287 fluorescent proteins Human genes 0.000 claims description 7
- 108091006047 fluorescent proteins Proteins 0.000 claims description 7
- 238000013519 translation Methods 0.000 claims description 6
- 239000000523 sample Substances 0.000 abstract description 28
- 239000012472 biological sample Substances 0.000 abstract description 7
- 239000000463 material Substances 0.000 description 9
- 238000000701 chemical imaging Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 230000008569 process Effects 0.000 description 6
- 239000003550 marker Substances 0.000 description 5
- 230000005284 excitation Effects 0.000 description 4
- 238000004624 confocal microscopy Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000004061 bleaching Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 206010036618 Premenstrual syndrome Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
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- 238000012549 training Methods 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a multispectral microscopic imaging system, which comprises: the microscope is used for amplifying and imaging the microscopic sample to an image surface leading-out port; the modulation mask is positioned on an output image surface of the microscope to restrict the range of a post-stage imaging field of view and is added with random mask modulation; the optical filter is positioned behind the field diaphragm to perform narrow-band filtering on illumination information from the microscopic sample and restrict the imaging spectral range of the system; the dispersion unit is used for spatially spreading the spectral information of each point on the sample; and the image sensor is used for recording the sample image modulated by the previous stage so as to reconstruct various mark images on one or two sheets through dispersion and mask modulation. The system can reconstruct various marked images by only adopting one or two images, improves the imaging speed, improves the image resolution, reduces the damage of laser to photosensitive samples and biological samples, and has simple structure and low cost.
Description
Technical Field
The invention relates to the technical field of computational photography, in particular to a multispectral microscopic imaging system.
Background
Remote sensing imaging, life science and material science research put higher demands on multispectral imaging. The observation target or scene can emit signals with different spectral curves because different regions contain different materials or fluorescent labeled proteins. Multispectral imaging is to calculate and reconstruct various material or fluorescent protein images by using the spectral response differences, and the main difficulty is that spectral response curves of different materials or labeled proteins may overlap, so that the splitting of the material or fluorescent labeled images cannot be realized simply by adding optical filters of different spectral bands.
At present, in the macroscopic field, multispectral imaging mainly includes adding optical filters of different spectral bands to an imaging optical path for multiple imaging or simultaneously imaging the spectral bands by using dichroic mirrors of the different spectral bands, and then calculating and reconstructing different materials or labeled images by using the spectral imaging stacks. In the field of microscopic imaging, there are multispectral imaging based on wide-field microscopy, multispectral microscopic imaging based on confocal microscopy and light sheet illumination. Based on multispectral imaging of a wide-field microscope, a lighting end is excited by monochrome or multicolor, different spectral filters are added on an imaging light path for multiple imaging or multiple dichroic mirrors with different spectral bands are added for simultaneous imaging, spectral imaging stacks are used for calculating and reconstructing different materials or marked images, and the influence of overlapping of spectral response curves is reduced. Based on the multispectral microscopic imaging of confocal microscopy, a fluorescence sample is scanned point by point, a dispersive device is added at the acquisition end, and the spectral information of the point is recorded by a plurality of PMTs (photomultiplier tubes). Based on the multispectral microscopy of confocal and optical sheet illumination, a line scanning optical sheet is adopted for illumination, a dispersion device is added in an imaging optical path, and then a camera is used for recording fluorescence spectra excited on the whole line. The whole field of view spectral information is obtained through confocal microscopy point-by-point scanning or line-by-line scanning, and then various marker structure images are calculated and reconstructed.
However, the related art still has the disadvantages, such as that the multispectral imaging based on the confocal microscope requires strong excitation light, which easily causes fluorescence bleaching, and the strong light irradiation may damage the sample cell or tissue structure; for another example, the imaging process requires scanning or multiple times of shooting, the time efficiency is low, the method is suitable for photosensitive samples and biological samples, the imaging speed is low, and the dynamic process cannot be recorded, so that the problem is to be solved.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art.
Therefore, the invention aims to provide a multispectral microscopic imaging system which can improve the imaging speed, improve the image resolution and reduce the damage of laser to photosensitive samples and biological samples, and has simple structure and low cost.
In order to achieve the above object, an embodiment of the present invention provides a multispectral microscopic imaging system, including: the microscope is used for amplifying and imaging the microscopic sample to an image surface leading-out port; the modulation mask is positioned on an output image surface of the microscope to restrict the range of a post-stage imaging field of view and is added with random mask modulation; the optical filter is positioned behind the field diaphragm to perform narrow-band filtering on the illumination information from the microscopic sample and restrict the imaging spectral range of the system; the dispersion unit is used for spatially spreading the spectral information of each point on the sample; and the image sensor is used for recording the sample image modulated by the previous stage so as to reconstruct various mark images on one or two sheets through dispersion and mask modulation.
The multispectral microscopic imaging system provided by the embodiment of the invention can realize single or multiple exposure data acquisition under a single camera, namely, a monochrome marking image can be recovered, and various marking images can be reconstructed by only adopting one or two images, so that the imaging speed is improved, the image resolution is improved, the damage of laser to a photosensitive sample and a biological sample is reduced, and the multispectral microscopic imaging system is simple in structure and low in cost.
In addition, the multispectral microscopic imaging system according to the above embodiment of the present invention may have the following additional technical features:
further, in an embodiment of the present invention, the method further includes: and the reconstruction module is used for obtaining an image through the dispersion and mask modulation and reconstructing the tissue structure marked by each fluorescent protein of the sample through the marked fluorescent protein spectral response curve.
Further, in one embodiment of the present invention, the microscope may be a wide field fluorescence microscope.
Further, in one embodiment of the present invention, the modulation mask is located on the image plane and the dispersion unit is located on the fourier plane.
Further, in an embodiment of the present invention, the fourier plane at the dispersive unit and the image plane at the image sensor are implemented by a 4f system composed of lenses or lenses.
Further, in one embodiment of the present invention, the 4f system is used to add dispersion modulation and imaging magnification adjustment.
Further, in an embodiment of the present invention, the optical filter is located in the imaging optical path after the 4f system first stage lens, or directly added inside the microscope or at other positions in the optical path, so as to restrict the imaging spectral range.
Further, in one embodiment of the present invention, the random mask adjusts the mask lateral position by means of translation.
Further, in one embodiment of the present invention, the dispersion unit is an amoxis dispersion prism.
Further, in one embodiment of the present invention, the image sensor is a SCMOS monochrome sensor.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a schematic structural diagram of a multi-spectral microscopy imaging system according to an embodiment of the invention;
fig. 2 is a schematic structural diagram of a multispectral microscopy imaging system according to an embodiment of the invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
A multispectral microscopic imaging system proposed according to an embodiment of the present invention is described below with reference to the drawings.
Fig. 1 is a schematic structural diagram of a multispectral microscopic imaging system according to an embodiment of the present invention.
As shown in fig. 1, the multispectral microscopy imaging system 10 includes: a microscope 100, a modulation mask 200, a filter 300, a dispersion unit 400, and an image sensor 500.
The microscope 100 is used for magnifying and imaging a microscopic sample to an image plane exit. The modulation mask 200 is positioned at the output image plane of the microscope 100 to constrain the post-stage imaging field of view and incorporate random mask modulation. The filter 300 is positioned behind the field stop to perform narrow-band filtering on the illumination information from the microscopic sample and constrain the system imaging spectral range. The dispersion unit 400 is used to spatially spread the spectral information at each point on the sample. The image sensor 500 is used to record the pre-modulated sample image to reconstruct the various mark images on one or both sheets by dispersion and mask modulation. The system 10 of the embodiment of the invention can reconstruct various marked images by only adopting one or two images, improves the imaging speed, improves the image resolution, reduces the damage of laser to photosensitive samples and biological samples, and has simple structure and low cost.
In an embodiment of the present invention, the system 10 of the embodiment of the present invention further includes: and a reconstruction module. The reconstruction module obtains an image through dispersion and mask modulation, and reconstructs the tissue structure marked by each fluorescent protein of the sample through the spectral response curve of the marked fluorescent protein.
It can be understood that, in the embodiment of the present invention, different marker images can be calculated and reconstructed by using a plurality of images obtained by single shooting or mask translation, and combining the spectral response curves of the fluorescent markers. The calculation and reconstruction process of the reconstruction module can be realized on hardware systems such as a common PC or a workstation.
That is, the multispectral microscopic imaging system 10 of the present embodiment of the invention mainly includes two parts, image acquisition and computational reconstruction.
The image acquisition part is composed of a microscope 100, a modulation mask 200, an optical filter 300, a dispersion unit 400 and an image sensor 500. The microscope 100 can achieve multi-labeled sample fluorescence excitation and magnification imaging. The modulation mask 200 incorporates random binary modulation at the image plane. The dispersion unit 400 spreads the spectral information of each point on the image plane in space on the fourier plane surface of the 4f system, and the 4f system realizes the magnification adjustment and dispersion range adjustment of the imaging system.
The calculation and reconstruction part utilizes a plurality of pictures obtained by single shooting or mask translation, namely, images of a plurality of marked samples can be obtained through single shooting or twice shooting, the imaging speed is high, and different marked images can be calculated and reconstructed by combining spectral response curves of all fluorescent marks. The calculation and reconstruction process of the reconstruction module can be realized on hardware systems such as a common PC or a workstation.
Further, in one embodiment of the present invention, the microscope 100 may be a wide field fluorescence microscope.
It will be appreciated that the microscope 100 of embodiments of the present invention is a wide field fluorescence microscope to achieve first order amplification of the fluorescence sample, with the sample image plane being derived from the exit for post-processing.
Further, in one embodiment of the present invention, the modulation mask 200 is located on the image plane and the dispersion unit is located on the fourier plane.
Further, in one embodiment of the present invention, the fourier plane at the dispersion unit 400 and the image plane at the image sensor 500 are implemented by a 4f system of lenses or lenses.
Further, in one embodiment of the present invention, a 4f system is used to incorporate dispersion modulation and imaging magnification adjustment.
Further, in one embodiment of the present invention, the filter 300 is located in the imaging optical path 4f behind the first stage lens of the system, or directly inside the microscope 100 or elsewhere in the optical path, to restrict the imaging spectral range.
It is understood that the filter 300 of the present embodiment may be located after the 4f system first stage lens after the modulation mask 200, and the filter 300 is used for spectral range restriction of the optical signal from the sample. That is, after passing through the filter 300, the optical signal from the sample can pass only within a certain bandwidth, thereby defining a subsequent dispersion range. It should be noted that, in the actual imaging optical path, the filter 300 is not strictly limited, i.e., is adjustable in position.
The 4f system can be two lenses or lenses with different focal lengths, and the rear focal plane of the front lens coincides with the front focal plane of the rear lens. If the image plane of the imaging optical path is positioned on the front focal plane of the front lens, an image plane which is enlarged or reduced is obtained at the rear focal plane of the rear lens, and the enlargement and reduction ratios of the image plane are determined by the focal length ratios of the rear lens and the front lens.
Further, in one embodiment of the present invention, the random mask may be adjusted in lateral position by translation.
It will be appreciated that the modulation mask 200 is located at the microscope output image plane, and that the modulation mask 200 serves to limit the field of view and incorporates a random mask modulation which can adjust the lateral position of the mask by translation.
Optionally, in one embodiment of the invention, the dispersive unit is an amoxist dispersive prism.
Alternatively, in one embodiment of the present invention, image sensor 500 may be a SCMOS monochrome sensor.
It is understood that the image sensor 500 may be located at the back focal plane of the second lens of the 4f system, and the image sensor 500 is used to record the mask modulated and dispersion modulated imaging results, wherein the image sensor 500 employs a SCMOS monochrome camera.
In summary, in the embodiments of the present invention, a microscope objective lens may be used to add binary random mask modulation to the output sample fluorescence image plane, constrain the spectral range with the optical filter 300, add dispersion modulation to the fourier plane, adjust the magnification and dispersion range with a 4f system, and the image sensor 500 records the modulated image. The system 10 of the embodiment of the invention has low power of the excitation light source, can be applied to imaging of photosensitive samples and biological samples, reduces fluorescent bleaching and damage to the samples, and has high imaging speed and high image reconstruction resolution.
For example, as shown in FIG. 2, in one embodiment of the invention, the system comprises: microscope 100, modulation mask 200, filter 300, dispersion unit 400, and image sensor 500.
Specifically, as shown in connection with fig. 2, the microscopic sample 107 is located on the focal plane of the microscope objective 106 of the microscope 100; the excitation light emitted from the laser light source 104 is reflected by the dichroic mirror 105, and then focused by the objective lens 106 to excite the fluorescence signal of the sample 107. The fluorescence signal passes through the objective lens 106, then passes through the dichroic mirror 105, then passes through the reflecting mirror 103 and the tube lens 102, and then is imaged to the image surface exit 101; placing a random mask at the image surface position for modulation, limiting the field range and adding modulation; further relaying the image to a dispersing prism 400 through a 4f system first lens and filter 300; the dispersed optical signal is imaged on the image sensor 500 of the back focal plane through the second lens of the 4f system.
The microscope 100 may be a conventional commercial microscope, an inverted microscope or an upright microscope according to different applications, and is not limited to the inverted microscope shown in fig. 2, and the structure and function thereof are known to those skilled in the art and will not be described in detail herein.
In addition, different modulation imaging treatments are carried out on the multi-marker fluorescence sample collected by the embodiment of the invention, and a single marker image can be reconstructed by using a single marker spectral response curve. Firstly, generating an over-complete sparse representation dictionary by utilizing monochromatic marked undispersed sample imaging training; the imaging process is regarded as a convolution process of a true value image and a mark spectral response; and recovering the monochromatic mark imaging by calculating the sparse representation coefficient of the dispersive image on the product of the dispersive kernel and the dictionary.
It should be noted that, the matching of the numerical aperture of each stage system and the numerical aperture provided by the objective lens itself require the selection of an appropriate dispersion kernel and the size of the over-trained complete dictionary.
According to the multispectral microscopic imaging system provided by the embodiment of the invention, the single-color marked image can be recovered by acquiring data through single or multiple exposures of a single camera, and various marked images can be reconstructed by only adopting one or two images, so that the imaging speed is increased, the image resolution is improved, the damage of laser to a photosensitive sample and a biological sample is reduced, and the multispectral microscopic imaging system is simple in structure and low in cost.
In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are therefore not to be considered limiting of the invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In the present invention, unless otherwise expressly stated or limited, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through an intermediate. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (8)
1. A multi-spectral microscopy imaging system, comprising:
the microscope is used for amplifying and imaging the microscopic sample to an image surface leading-out port;
the modulation mask is positioned on an output image surface of the microscope to restrict the range of a post-stage imaging field of view and add random mask modulation, wherein the random mask adjusts the transverse position of the mask in a translation mode;
a field stop;
the optical filter is positioned behind the field diaphragm to perform narrow-band filtering on illumination information from the microscopic sample and restrict the imaging spectral range of the system;
the dispersion unit is used for spatially spreading the spectral information of each point on the sample; and
the image sensor is used for recording the sample image modulated by the preceding stage so as to reconstruct various mark images on one or two sheets through dispersion and mask modulation;
the reconstruction module is used for obtaining an image through the dispersion and mask modulation and reconstructing the tissue structure marked by each fluorescent protein of the sample through the spectral response curve of the marked fluorescent protein; wherein individual images of the multi-labeled sample are obtained by a single shot.
2. The multispectral microscopy imaging system of claim 1, wherein the microscope is a wide-field fluorescence microscope.
3. The multispectral microscopy imaging system of claim 1, wherein the modulation mask is located at an image plane and the dispersive unit is located at a fourier plane.
4. The multispectral microscopy imaging system of claim 3, wherein the Fourier surface at the dispersive unit and the image surface at the image sensor are implemented by a 4f system of lenses or lenses.
5. The multispectral microscopy imaging system of claim 4, wherein the 4f system is configured to incorporate chromatic dispersion modulation and imaging magnification adjustment.
6. The multispectral microscopy imaging system of claim 5, wherein the filter is positioned in the imaging path after the 4f system first stage lens, or directly within the microscope or elsewhere in the optical path, to restrict the imaging spectral range.
7. The multispectral microscopy imaging system of claim 1, wherein the dispersive unit is an amoexi dispersive prism.
8. The multispectral microscopy imaging system according to any one of claims 1 to 7, wherein the image sensor is a SCMOS monochrome sensor.
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| CN202221407U (en) * | 2011-08-26 | 2012-05-16 | 麦克奥迪实业集团有限公司 | Multispectral microscope |
| CN102749834A (en) * | 2012-07-24 | 2012-10-24 | 河北工程大学 | Optical microscopic imaging system and imaging method |
| CN104932092A (en) * | 2015-06-15 | 2015-09-23 | 上海交通大学 | Automatic focusing microscope based on eccentric beam method and focusing method thereof |
| CN107193118A (en) * | 2017-07-04 | 2017-09-22 | 清华大学 | Muti-spectrum imaging system and microscope |
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| CN202221407U (en) * | 2011-08-26 | 2012-05-16 | 麦克奥迪实业集团有限公司 | Multispectral microscope |
| CN102749834A (en) * | 2012-07-24 | 2012-10-24 | 河北工程大学 | Optical microscopic imaging system and imaging method |
| CN104932092A (en) * | 2015-06-15 | 2015-09-23 | 上海交通大学 | Automatic focusing microscope based on eccentric beam method and focusing method thereof |
| CN107193118A (en) * | 2017-07-04 | 2017-09-22 | 清华大学 | Muti-spectrum imaging system and microscope |
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