CN111123495B - Three-dimensional total internal reflection microscopic imaging device and method based on annular array light source illumination - Google Patents
Three-dimensional total internal reflection microscopic imaging device and method based on annular array light source illumination Download PDFInfo
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- G02B21/365—Control or image processing arrangements for digital or video microscopes
- G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
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- G06T3/00—Geometric image transformation in the plane of the image
- G06T3/40—Scaling the whole image or part thereof
- G06T3/4053—Super resolution, i.e. output image resolution higher than sensor resolution
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- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/10—Image acquisition modality
- G06T2207/10056—Microscopic image
- G06T2207/10061—Microscopic image from scanning electron microscope
Abstract
The invention discloses a three-dimensional total internal reflection microscopic imaging device and a method based on illumination of an annular array light source, wherein the device comprises: the device comprises an annular LED array, a conical lens, an objective lens, a tube lens and an image acquisition module; a sample is placed on the conical lens; the annular LED array comprises M single-layer annular LED arrays with different radiuses; the side surface of the conical lens consists of M circular truncated cone side surfaces with different inclination angles; the single LED lamp in each layer of annular LED array sequentially emits illumination beams, and the illumination beams enter the conical lens, so that an evanescent field is generated at the interface between the conical lens and the sample; scattered light generated after the sample is illuminated by the evanescent field sequentially passes through the objective lens and the tube lens and is received by the image acquisition module. The three-dimensional total internal reflection microscopic imaging device and method based on annular array light source illumination provided by the invention can realize super-resolution imaging without fluorescence labeling.
Description
Technical Field
The invention relates to the technical field of microscopic imaging, in particular to a three-dimensional total internal reflection microscopic imaging device and method based on illumination of an annular array light source.
Background
In a conventional super-resolution microscope, a fluorescence label is usually needed, the super-resolution effect is achieved by utilizing the nonlinear effect of fluorescence, a marked sample cannot be imaged for a long time due to the photobleaching characteristic of the marked sample, and the fluorescent label can influence the theoretical research of biological characteristic motion influence, so that the unmarked super-resolution imaging is more ideal for observing the interference-free rapid long-time biological motion, and the super-resolution method without the effective fluorescence label is temporarily adopted at present
Therefore, how to provide a method for realizing super-resolution imaging without fluorescent labels is a problem to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a three-dimensional total internal reflection microscopic imaging device and method based on illumination of an annular array light source, which can realize super-resolution imaging without fluorescence labeling.
In order to achieve the purpose, the invention adopts the following technical scheme:
a three-dimensional total internal reflection microscopic imaging device based on annular array light source illumination comprises: the polarization detection module is used for detecting the polarization of the polarized light;
the polarized illumination module comprises the following components in sequence according to the light propagation direction: an annular LED array and a conical lens; wherein a sample is placed on the conical lens;
the annular LED array consists of M single-layer annular LED arrays with different radiuses; the side surface of the conical lens consists of M circular truncated cone side surfaces with different inclination angles;
the polarization detection module sequentially comprises the following components in the light propagation direction: the device comprises an objective lens, a tube lens and an image acquisition module;
the single LED lamp in each layer of annular LED array sequentially emits illumination light beams, the illumination light beams enter the conical lens, and an evanescent field is generated at the interface of the conical lens and the sample; scattered light generated after the sample is illuminated by the evanescent field sequentially passes through the objective lens and the tube lens and is received by the image acquisition module.
Preferably, each layer of annular LED array is composed of N LED lamps which are uniformly distributed, and the positions of the LED lamps on the adjacent layers are correspondingly arranged.
Preferably, the inclination angles of the M circular truncated cone side surfaces forming the conical lens are different, and the inclination angle of the ith circular truncated cone side surface of the conical lens is consistent with the illumination inclination angle of the ith layer of annular LED array, so that the included angle between the illumination light beam vertically incident on the conical lens and the main optical axis of the optical system is different and is larger than the total internal reflection critical angle thetacWherein, thetacArcsin (n), n is the refractive index of the conical lens, and i is an integer from 1 to M.
Preferably, the image acquisition module includes: a camera.
A three-dimensional total internal reflection microscopic imaging method based on illumination of an annular array light source is suitable for the three-dimensional total internal reflection microscopic imaging device based on illumination of the annular array light source, and comprises the following steps:
step a: starting from the outermost layer in the annular LED array, sequentially lighting a single LED lamp one by one to enable the lighting beam to move on the circumferential radius of the corresponding radius, lighting an LED lamp with different lighting angles each time, shooting an image once by the image acquisition module, sequentially lighting all N LED lamps in the annular LED array to realize 360-degree circumferential scanning lighting on the sample to obtain N scattered light intensity distribution images of the sample to be tested with the same evanescent field lighting depth and different lighting azimuth angles, repeating the process until the 360-degree circumferential scanning lighting of the annular LED array on the innermost layer on the sample is completed, and obtaining M multiplied by N scattered light intensity distribution images { I } of the sample to be tested with different evanescent field lighting depths and different lighting azimuth anglesi,j(x, y) }; wherein, i is 1,2,3, …, M, j is 1,2,3, …, N, x is 1,2,3, …, Px；y＝1,2,3,…,PyY, where x, y are the row and column numbers of the image pixels, PxIs the total number of pixels per line of the image, PyIs the total number of pixels per column of the image;
step b: calculating M-order autocorrelation quantity of each pixel point at the same position of N light intensity distribution images obtained by lighting the ith layer of annular LED array by using the following formula to obtain M super-resolution images C with improved resolutionmObtaining three-dimensional data sets
i＝1,2,3,…M，x＝1,2,3,…,Px，y＝1,2,3,…,Py,}；
Wherein x, y represent pixel position, Ii,j(x, y) represents an image acquired by the image acquisition module under the illumination of the jth LED lamp in the ith layer of annular LED array, N is the number of images acquired by one 360-degree circumferential scanning, m represents a calculation order, and m is a positive integer not greater than 4;
step c: for M super-resolution images
Respectively carrying out iterative deconvolution, wherein i is 1,2,3, … M, M represents the calculation order, and then taking
Eliminating the nonlinear effect by the power to obtain M two-dimensional super-resolution images with improved resolution;
step d: and calculating and separating three-dimensional chromatographic information of the image under different depth evanescent field illumination based on the M two-dimensional super-resolution images and the Split-Bregman method, thereby realizing three-dimensional super-resolution imaging.
Preferably, step a further comprises: and performing deconvolution denoising pretreatment on the obtained MXN scattering light intensity distribution images of the sample to be detected.
Preferably, in step c, the obtained super-resolution image is subjected to iterative deconvolution by using the following formula:
wherein h is a system point spread function, y is an image after deconvolution, and during the first iteration,
i is an integer from 1 to M, FFT and iFFT are fast Fourier transform and fast inverse Fourier transform, respectively, and j is the number of iterations.
Preferably, j has a maximum value of 100.
Compared with the prior art, the invention discloses a three-dimensional total internal reflection microscopic imaging device and method based on annular array light source illumination, and the device and method have the following technical advantages:
1. there is no other intervention on the observation sample than the lighting, for example: dyeing can more truly observe the sample dynamic;
2. the bleaching characteristic similar to fluorescence imaging is avoided, and long-time imaging can be realized;
3. the super-resolution can be realized without marking, and the resolution can be improved by m times according to different orders m.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a three-dimensional total internal reflection micro-imaging device based on illumination of a circular array light source provided by the invention;
FIG. 2 is a schematic diagram of an annular LED array provided by the present invention;
FIG. 3 is an enlarged view of the side of a conical lens provided by the present invention;
FIG. 4 is a flow chart of a three-dimensional total internal reflection micro-imaging method based on illumination of a circular array light source provided by the invention.
The device comprises an annular LED array 1, a conical lens 2, a sample 3, an objective 4, an objective 5, a tube lens 6 and an image acquisition module.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1 to 3, an embodiment of the present invention discloses a three-dimensional total internal reflection microscopic imaging device based on illumination of an annular array light source, including: the polarization detection module is used for detecting the polarization of the polarized light;
the polarized illumination module includes according to light propagation direction in proper order: an annular LED array 1 and a conical lens 2; wherein, a sample 3 is placed on the conical lens 2;
the annular LED array 1 consists of M single-layer annular LED arrays with different radiuses; the side surface of the conical lens 2 consists of M truncated cone side surfaces with different inclination angles;
the polarization detection module sequentially comprises the following components in the light propagation direction: an objective lens 4, a tube lens 5 and an image acquisition module 6;
the single LED lamp in each layer of single-layer annular LED array sequentially emits illumination beams, the illumination beams enter the conical lens 2, and an evanescent field is generated at the interface of the conical lens 2 and the sample 3; scattered light generated after the sample 3 is illuminated by the evanescent field passes through the objective lens 4 and the tube lens 5 in sequence and is received by the image acquisition module 6, wherein preferably, the image acquisition module 6 comprises: a camera.
It should be noted here that the illumination inclination angle of the annular LED array layer is consistent with the inclination angle of a certain circular truncated cone side surface of the corresponding conical lens 2, and the generatrix of the circular truncated cone is not larger than the size of the LED lamp (the LED lamp is divergent light), so that the light beam can be ensured to vertically enter the conical lens 2.
In order to further optimize the technical scheme, each layer of single-layer annular LED array is composed of N LED lamps which are uniformly distributed, and the positions of the LED lamps on the adjacent layers are correspondingly arranged.
In order to further optimize the technical scheme, the inclination angles of M circular truncated cone side surfaces forming the conical lens 2 are different, and the inclination angle of the ith circular truncated cone side surface of the conical lens 2 is consistent with the illumination inclination angle of the ith layer of annular LED array, so that the included angle between the illumination light beam vertically incident on the conical lens 2 and the main optical axis of the optical system is different and is larger than the total internal reflection critical angle thetacWherein, thetacArcsin (n), n is the refractive index of the conical lens, and i is an integer from 1 to M.
Referring to fig. 4, the embodiment of the present invention further discloses a three-dimensional total internal reflection microscopic imaging method based on illumination of a circular array light source, which is suitable for the three-dimensional total internal reflection microscopic imaging apparatus based on illumination of a circular array light source, and includes the following steps:
step a: starting from the outermost layer in the annular LED array 1, sequentially lighting a single LED lamp one by one to enable the lighting beam to move on the circumferential radius of the corresponding radius, lighting an LED lamp with different lighting angles each time, shooting an image once by the image acquisition module 6, sequentially lighting all N LED lamps in the single-layer annular LED array to realize 360-degree circumferential scanning lighting on the sample to obtain N scattered light intensity distribution images of the sample to be tested with the same evanescent field lighting depth and different lighting azimuth angles, repeating the process until the 360-degree circumferential scanning lighting of the sample by the single-layer annular LED array on the innermost layer is completed, and obtaining M x N scattered light intensity distribution images { I } of the sample to be tested with different evanescent field lighting depths and different lighting azimuth anglesi,j(x, y) }; wherein, i is 1,2,3, …, M, j is 1,2,3, …, N, x is 1,2,3, …, Px；y＝1,2,3,…,PyY, where x, y are the row and column numbers of the image pixels, PxIs the total number of pixels per line of the image, PyIs the total number of pixels per column of the image;
step b: calculating M-order autocorrelation quantity of each pixel point at the same position of N light intensity distribution images obtained by single-layer annular LED array illumination by using the following formula to obtain M super-resolution maps with improved resolutionImage CmObtaining three-dimensional data sets
i＝1,2,3,…M，x＝1,2,3,…,Px，y＝1,2,3,…,Py,}；
Wherein x, y represent pixel position, Ii,j(x, y) represents an image acquired by the image acquisition module (6) under the illumination of the jth LED lamp in the ith layer of annular LED array, N is the number of images acquired by one 360-degree circumferential scanning, m represents a calculation order, and m is a positive integer not greater than 4;
step c: for M super-resolution images
Respectively carrying out iterative deconvolution, wherein i is 1,2,3, … M, M represents the calculation order, and then taking
Eliminating the nonlinear effect by the power to obtain M two-dimensional super-resolution images with improved resolution; in step c, the obtained super-resolution image is subjected to iterative deconvolution by using the following formula:
wherein h is a system point spread function, y is an image after deconvolution, and during the first iteration,
i is an integer from 1 to M, FFT and iFFT are fast fourier transform and fast inverse fourier transform, respectively, j is the number of iterations, and the maximum value of j is typically 100.
Step d: and calculating and separating three-dimensional chromatographic information of the image under different depth evanescent field illumination based on the M two-dimensional super-resolution images and the Split-Bregman method, thereby realizing three-dimensional super-resolution imaging.
In order to further optimize the above technical solution, step a further includes: and performing deconvolution denoising pretreatment on the obtained MXN scattering light intensity distribution images of the sample to be detected.
The technical scheme provided by the invention applies a multilayer annular LED array and matched conical lenses to generate illumination beams with different incident angles and azimuth angles, so as to construct an illumination evanescent field with controllable propagation direction and adjustable illumination depth; exciting single wave vector evanescent fields in different directions by using the asymmetry of the sample structure to generate polar scattering and the autocorrelation cumulant difference of adjacent observation points, thereby realizing super-resolution imaging; and calculating and separating three-dimensional chromatographic information of the image under the illumination of evanescent fields of different depths by using a weak constraint Split-Bregman method, thereby realizing three-dimensional imaging. The technical scheme provided by the invention has the advantages of simple adjustment, low observation cost and capability of realizing total internal reflection super-resolution microscopic imaging without marking a sample.
In addition, it should be noted that, in order to meet the sampling requirement after increasing the resolution by m times after calculation, the magnification of the system should make the pixel size of the original image smaller than the resolution of the original system
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. A three-dimensional total internal reflection microscopic imaging method based on illumination of a circular array light source is characterized by being suitable for a three-dimensional total internal reflection microscopic imaging device based on illumination of a circular array light source, and the method comprises the following steps:
step a: starting from the outermost layer in the annular LED array (1), sequentially lighting a single LED lamp one by one to enable the lighting beam to move on the circumferential radius of the corresponding radius, lighting an LED lamp with different lighting angles each time, shooting an image once by the image acquisition module (6), sequentially lighting all N LED lamps in the annular LED array of the layer to realize 360-degree circumferential scanning lighting on the sample to obtain N scattered light intensity distribution images of the sample to be tested with the same evanescent field lighting depth and different lighting azimuth angles, repeating the process until the 360-degree circumferential scanning lighting of the annular LED array of the innermost layer on the sample is completed to obtain M multiplied by N scattered light intensity distribution images of the sample to be tested with different evanescent field lighting depths and different lighting azimuth angles, namely obtaining a four-dimensional data set { I }i,j(x,y)，i＝1,2,3,…M，j＝1,2,3,…N，x＝1,2,3,…,Px，y＝1,2,3,…,PyY, where x, y are the row and column numbers of the image pixels, PxIs the total number of pixels per line of the image, PyIs the total number of pixels per column of the image;
step b: calculating M-order autocorrelation quantity of each pixel point at the same position of N light intensity distribution images obtained by lighting the ith layer of annular LED array by using the following formula to obtain M super-resolution images with improved resolution
I.e. obtaining a three-dimensional data set
Wherein x, y represent pixel position, Ii,j(x, y) represents an image acquired by the image acquisition module (6) under the illumination of the jth LED lamp in the ith layer of annular LED array, N is the number of images acquired by one 360-degree circumferential scanning, m represents a calculation order, and m is a positive integer not greater than 4;
step c: for M super-resolution images
Respectively carrying out iterative deconvolution, wherein i is 1,2,3, … M, M represents the calculation order, and then taking
Eliminating the nonlinear effect by the power to obtain M two-dimensional super-resolution images with improved resolution;
step d: calculating and separating three-dimensional chromatographic information of the image under different depth evanescent field illumination based on the M two-dimensional super-resolution images and a Split-Bregman method, and realizing three-dimensional super-resolution imaging;
a three-dimensional total internal reflection microscopic imaging device based on illumination of a ring-shaped array light source is characterized by comprising: the polarization detection module is used for detecting the polarization of the polarized light;
the polarized illumination module comprises the following components in sequence according to the light propagation direction: an annular LED array (1) and a conical lens (2); wherein a sample (3) is placed on the conical lens (2);
the annular LED array (1) consists of M single-layer annular LED arrays with different radiuses; conical lens (2) comprise the round platform that M inclination is different, and the i side surface inclination of the round platform of conical lens (2) is unanimous with the illumination inclination of the annular LED array on the i layer for illumination beam vertical incidence conical lens (2) back is different with optical system primary optical axis contained angle, just the contained angle is greater than the total internal reflection and facesBoundary angle thetacWherein, thetacArcsin (n), n is the refractive index of the conical lens, and i is an integer from 1 to M;
the polarization detection module sequentially comprises the following components in the light propagation direction: the device comprises an objective lens (4), a tube lens (5) and an image acquisition module (6);
the single LED lamp in each layer of annular LED array sequentially emits illumination light beams, the illumination light beams enter the conical lens (2), and an evanescent field is generated at the interface of the conical lens (2) and the sample (3); scattered light generated after the sample (3) is illuminated by the evanescent field sequentially passes through the objective lens (4) and the tube lens (5) and is received by the image acquisition module (6).
2. The three-dimensional total internal reflection microscopic imaging method based on annular array light source illumination of claim 1, wherein each layer of annular LED array is composed of N LED lamps which are uniformly arranged, and the LED lamps of adjacent layers are correspondingly arranged.
3. The three-dimensional total internal reflection microscopic imaging method based on annular array light source illumination according to any one of claims 1-2, characterized in that the image acquisition module (6) comprises: a camera.
4. The three-dimensional total internal reflection microscopic imaging method based on annular array light source illumination of claim 1, wherein in step a, further comprising: and performing deconvolution denoising pretreatment on the obtained MXN scattering light intensity distribution images of the sample to be detected.
5. The three-dimensional total internal reflection microscopic imaging method based on annular array light source illumination of claim 1, wherein in step c, the obtained super-resolution image is subjected to iterative deconvolution by using the following formula:
wherein h is the systemPoint spread function, y is the deconvoluted image, on the first iteration,
i is an integer from 1 to M, FFT and iFFT are fast fourier transform and fast inverse fourier transform, respectively, and j is the number of iterations.
6. The three-dimensional total internal reflection microscopic imaging method based on annular array light source illumination according to claim 5, wherein the maximum value of j is 100.
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| CN112540455A (en) * | 2020-12-23 | 2021-03-23 | 中国科学院长春应用化学研究所 | Reflective annular illumination Fourier laminated high-resolution microscopic imaging system |
| CN112903682B (en) * | 2021-02-03 | 2022-06-21 | 浙江大学 | In-situ controllable frequency shift illumination microscopic chip and rapid high-resolution imaging system |
| CN113820843B (en) * | 2021-07-22 | 2022-08-26 | 西安电子科技大学 | High-resolution quantitative phase microscope system based on annular LED illumination |
| CN113759535A (en) * | 2021-07-26 | 2021-12-07 | 南京理工大学智能计算成像研究院有限公司 | High-resolution microscopic imaging method based on multi-angle illumination deconvolution |
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