CN112255776B - Point light source scanning illumination method and detection device - Google Patents
Point light source scanning illumination method and detection device Download PDFInfo
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Abstract
The invention relates to a point light source scanning illumination method and a detection device, the method realizes multi-angle illumination by using an XYZ precision displacement table to control the same point light source to scan, can arrange different illumination modes of known space coordinates, and applies the space coordinates determined in advance to a Fourier laminated reconstruction algorithm so as to improve the reconstruction resolution. The detection device consists of a CCD camera, a tube lens, a microscope objective, a point light source, an XYZ precision displacement platform and a computer. The invention is based on the Fourier laminated microscopic imaging technology, can overcome the problem of reconstruction quality deterioration caused by the inconsistent light intensity of point light source illumination samples and the position error of LED light source space coordinates, is easy to realize the contrast optimization of various illumination modes, has more flexible illumination modes and improves the applicability of the system.
Description
Technical Field
The invention relates to the technical field of optical microscopic imaging, in particular to a point light source scanning illumination method and a detection device.
Background
The Fourier stacked imaging (FPM) technology is a computational imaging method applied to the field of optical microscopy, effectively solves the problem that high resolution and wide field of view are simultaneously realized in the traditional microscopy imaging system, has the advantages of low system cost, high imaging resolution, large imaging field range and the like, and has wide application prospect in various microscopy imaging fields such as medical imaging, bioscience and the like.
The structure of the existing FPM system generally adopts an LED array light source to carry out multi-angle illumination on a sample to be detected, but the LED array light source has some defects in illumination. The light emitting intensity of each LED light source in the LED array light source is different, and the larger the angle of the LED from the sample is, the lower the contrast of the acquired low-resolution image is. Before the high-resolution image is restored and reconstructed, the relative coordinate position of each LED light source relative to the sample needs to be calibrated with high precision to determine the translation coordinate of the frequency spectrum, so that the influence of the error of the LED position on the high-resolution reconstruction result is reduced.
Therefore, the LED array light source has the following problems: (1) due to technical reasons such as LED production and the like, the luminous intensity of each LED light source is different; (2) errors in the observed position of the LED relative to the sample can affect the reconstructed result; (3) the fixed LED light source distribution is difficult to be suitable for different samples and microscope objectives, and the flexibility is lacked.
Disclosure of Invention
In order to solve the technical problems in the background technology, the invention provides a point light source scanning illumination method and a detection device which have reasonable conception, can overcome the problem of reconstruction quality deterioration caused by inconsistent light intensity of point light source illumination samples and position errors of LED light source space coordinates based on a Fourier laminated microimaging technology, are easy to realize contrast optimization of various illumination modes, have more flexible illumination modes and improve the applicability of the system.
The technical scheme provided by the invention is as follows:
the point light source scanning illumination method is characterized in that before system measurement, the same point light source is controlled to move by using an XYZ precision displacement table so as to arrange different illumination modes with known space coordinates; then, determining the space position coordinate of the point light source by ensuring the optimization of diffraction spectrum overlapping when the point light source illuminates the sample; and then according to different preset illumination modes with known space coordinates, the XYZ precision displacement table is controlled by the computer to sequentially translate the point light source for scanning illumination, meanwhile, the CCD camera is controlled to sequentially shoot images formed when the samples are illuminated at different space positions, and then the computer obtains a microscopic image with high resolution and a large view field by utilizing a Fourier laminated reconstruction algorithm.
The point light source scanning illumination method comprises the following steps: the point light source adopts an LED light source or a laser point light source.
The point light source scanning illumination method comprises the following steps: the translation of the XYZ precise displacement table, the illumination of the point light source and the image acquisition of the CCD camera are all completed by computer programming control.
The point light source scanning illumination method comprises the following specific implementation steps: (1) determining the translation range of a point light source according to the optimization result of diffraction spectrum overlapping when the point light source illuminates a sample, planning the scanning track of the point light source through a computer algorithm, recording the space coordinate position of the point light source, and collecting low-resolution image data when the point light source illuminates the sample at different space positions; (2) iteratively reconstructing an original low-resolution image by using an original GS; (3) and (3) image reconstruction, namely performing FPM algorithm reconstruction on the collected low-resolution images, and performing super-resolution reconstruction by selecting a classical GS phase recovery algorithm.
The point light source scanning illumination method, wherein the step (3) specifically comprises the following steps:
firstly, carrying out interpolation processing on an image shot under the irradiation of a point light source on an optical axis of a microscope objective to serve as an initial estimation value of a space domain;
performing Fourier transform on the initial estimation value of the space domain to obtain an initial estimation value of a frequency domain;
selecting a sub-numerical aperture area from the obtained spectrogram to perform inverse Fourier transform to obtain a target complex amplitude image, wherein the target complex amplitude image comprises amplitude information and phase information;
keeping the phase information of the target complex amplitude image unchanged, and replacing the amplitude information of the target complex amplitude image with a low-resolution image shot by a point light source corresponding to the space coordinate position to obtain an updated target complex amplitude image;
performing Fourier transform on the updated target complex amplitude image to obtain an updated spectrogram, mapping according to the spatial coordinate position to obtain a frequency domain spatial coordinate, and replacing the frequency spectrum region of the corresponding sub-value space of the initial spectrogram by using the updated spectrogram;
sixthly, repeating the step three to the step five to complete the frequency spectrum updating of all the sub-numerical apertures;
and seventhly, repeating the step three to converge the iterative process to obtain a high-resolution frequency spectrum image of the image, and then performing inverse Fourier transform to obtain a high-resolution image of a space domain.
A point light source scanning illumination detection device comprises a CCD camera, a tube lens, a microscope objective, a point light source, an XYZ precision displacement table and a computer; the point light source is installed on the XYZ precise displacement table in a matching mode, the computer is respectively and electrically connected with the point light source and the XYZ precise displacement table, the microscope objective is located on the light output side of the point light source in a matching mode, the test sample is placed between the light output side of the point light source and the light input side of the microscope objective in a matching mode, the tube lens is located on the light output side of the microscope objective in a matching mode, and the CCD camera is located on the light output side of the tube lens in a matching mode; and light emitted by the point light source passes through the test sample and then sequentially passes through the microscope objective and the tube lens to be focused on the image plane of the CCD camera.
The point light source scanning illumination detection device, wherein: the point light source adopts an LED light source or a laser point light source.
The point light source scanning illumination detection device, wherein: the translation of the XYZ precise displacement table, the illumination of the point light source and the image acquisition of the CCD camera are all completed under the control of the computer program.
Has the advantages that:
the point light source scanning illumination method has reasonable conception, can overcome the problem of reconstruction quality deterioration caused by the inconsistent light intensity of point light source illumination samples and the position error of the LED light source space coordinate based on the Fourier laminated microimaging technology, has completely consistent light intensity of the point light source illumination samples, is easy to realize the contrast optimization of various illumination modes, can apply high-precision space coordinates to the reconstruction process, has more flexible illumination mode, and improves the applicability of the system.
The point light source scanning illumination detection device is simple and reasonable in structure, the XYZ precise displacement table is used for translating the same point light source to illuminate a test sample in multiple angles, the problem of uneven illumination intensity is fundamentally solved, the intensity of each low-resolution image is more uniform, and the influence of uneven illumination intensity on a reconstruction result is weakened; determining the spatial position coordinates of the point light source by ensuring optimization of the diffraction spectrum overlap when the point light source illuminates the sample; meanwhile, the space coordinate position of the point light source corresponding to each graph is known, and reconstruction quality reduction caused by the deviation of the space coordinate position of the LED light source can be reduced.
Drawings
Fig. 1 is a schematic structural diagram of a point light source scanning illumination detection device according to the present invention.
Detailed Description
The invention will be described in further detail with reference to the drawings and the working principle.
The invention discloses a point light source scanning illumination method, which is based on a Fourier laminated microscopic imaging technology, and is characterized in that before system measurement, an XYZ precision displacement table is used for controlling the same point light source to move so as to arrange different illumination modes with known space coordinates; then, determining the space position coordinate of the point light source by ensuring the optimization of diffraction spectrum overlapping when the point light source illuminates the sample; and then according to the pre-designed illumination modes of different known space coordinates, the XYZ precision displacement table is controlled by the computer to sequentially translate the point light source for scanning illumination, meanwhile, the CCD camera is controlled to sequentially shoot images formed when the samples are illuminated at different space positions, and then the computer obtains a microscopic image with high resolution and a large view field by utilizing a Fourier laminated reconstruction algorithm.
The invention relates to a point light source scanning illumination method, which comprises the following specific implementation steps:
(1) determining the translation range of a point light source according to the optimization result of diffraction spectrum overlapping when the point light source illuminates a sample, planning the scanning track of the point light source through a computer algorithm, recording the space coordinate position of the point light source, and collecting low-resolution image data when the point light source illuminates the sample at different space positions;
(2) iteratively reconstructing an original low-resolution image by using an original GS;
(3) and (3) image reconstruction, namely performing FPM algorithm reconstruction on the plurality of acquired low-resolution images, and performing super-resolution reconstruction by selecting a classic GS phase recovery algorithm.
The image reconstruction in the step (3) specifically includes the following steps:
firstly, carrying out interpolation processing on an image shot under the irradiation of a point light source on an optical axis of a microscope objective to serve as an initial estimation value of a space domain;
performing Fourier transform on the initial estimation value of the airspace to obtain an initial estimation value of a frequency domain;
selecting a sub-numerical aperture area from the obtained spectrogram to perform inverse Fourier transform to obtain a target complex amplitude image, wherein the target complex amplitude image comprises amplitude information and phase information;
keeping the phase information of the target complex amplitude image unchanged, and replacing the amplitude information of the target complex amplitude image with a low-resolution image shot by a point light source corresponding to the space coordinate position to obtain an updated target complex amplitude image;
performing Fourier transform on the updated target complex amplitude image to obtain an updated spectrogram, mapping according to the spatial coordinate position to obtain a frequency domain spatial coordinate, and replacing the frequency spectrum region of the corresponding sub-value space of the initial spectrogram by using the updated spectrogram;
sixthly, repeating the step three to the step five to complete the frequency spectrum updating of all the sub-numerical apertures;
and seventhly, repeating the step three to converge the iterative process to obtain a high-resolution frequency spectrum image of the image, and then performing inverse Fourier transform to obtain a high-resolution image of a spatial domain.
As shown in fig. 1, the point light source scanning illumination detection device of the present invention is based on the point light source scanning illumination method of the present invention, and comprises a CCD camera 1, a tube lens 2, a microscope objective 3, a point light source 4, an XYZ precision displacement stage 5, and a computer 6.
The point light source 4 is installed on an XYZ precision displacement table 5 in a matching mode, the computer 6 is respectively and electrically connected with the point light source 4 and the XYZ precision displacement table 5, the microscope objective 3 is located on the light output side of the point light source 4 in a matching mode, the test sample 7 is located between the light output side of the point light source 4 and the light input side of the microscope objective 3 in a matching mode, the tube lens 2 is located on the light output side of the microscope objective 3 in a matching mode, and the CCD camera 1 is located on the light output side of the tube lens 2 in a matching mode.
Light emitted by the point light source 4 passes through the test sample 7, then sequentially passes through the microscope objective 3 and the tube lens 2, and is focused on an image plane of the CCD camera 1.
The point light source 4 is an LED light source or a laser point light source.
The translation of the XYZ precision displacement table 5, the illumination of the point light source 4 and the image acquisition of the CCD camera 1 are all completed by the programming control of the computer 6.
Wherein, in the FPM system, the reconsitution quality of high resolution image receives the contrast influence of the low resolution image of collection, and when using different LED to carry out the multi-angle illumination to the sample, the illumination intensity of illumination sample is inconsistent, can produce the inhomogeneous error of intensity, and this has just influenced the quality of reexamination.
The invention has reasonable conception, can overcome the problem of reconstruction quality deterioration caused by inconsistent light intensity of point light source illumination samples and position error of LED light source space coordinates based on the Fourier laminated microscopic imaging technology, easily realizes the contrast optimization of various illumination modes, has more flexible illumination modes and improves the applicability of the system.
The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, substitutions, combinations, equivalent variations and modifications made according to the technical spirit of the present invention should be equivalent replacement ways, and still fall within the protection scope of the technical solution of the present invention.
Claims (4)
1. A point light source scanning illumination method is characterized in that: the scanning illumination method is characterized in that the same point light source is controlled to move by using an XYZ precision displacement table so as to arrange different illumination modes with known space coordinates; then, determining the space position coordinate of the point light source by ensuring the optimization of diffraction spectrum overlapping when the point light source illuminates the sample; then according to different preset lighting modes with known space coordinates, the XYZ precision displacement table is controlled by the computer to sequentially translate the point light source for scanning lighting, meanwhile, the CCD camera is controlled to sequentially shoot images formed when the samples are lighted at different space positions, and then the computer obtains a microscopic image with high resolution and a large view field by utilizing a Fourier laminated reconstruction algorithm;
the point light source adopts an LED light source or a laser point light source;
the translation of the XYZ precision displacement table, the illumination of the point light source and the image acquisition of the CCD camera are controlled by computer programming;
the scanning illumination method comprises the following specific implementation steps:
(1) determining the translation range of a point light source according to the optimization result of diffraction spectrum overlapping when the point light source illuminates a sample, planning the scanning track of the point light source through a computer algorithm, recording the space coordinate position of the point light source, and collecting low-resolution image data when the point light source illuminates the sample at different space positions;
(2) iteratively reconstructing an original low-resolution image by using an original GS;
(3) image reconstruction, namely performing FPM algorithm reconstruction on a plurality of collected low-resolution images, and performing super-resolution reconstruction by using a classical GS phase recovery algorithm;
the step (3) specifically comprises the following steps:
firstly, carrying out interpolation processing on an image shot under the irradiation of a point light source on an optical axis of a microscope objective to serve as an initial estimation value of a space domain;
performing Fourier transform on the initial estimation value of the airspace to obtain an initial estimation value of a frequency domain;
selecting a sub-numerical aperture area from the obtained spectrogram to perform inverse Fourier transform to obtain a target complex amplitude image, wherein the target complex amplitude image comprises amplitude information and phase information;
keeping the phase information of the target complex amplitude image unchanged, and replacing the amplitude information of the target complex amplitude image with a low-resolution image shot by a point light source corresponding to the space coordinate position to obtain an updated target complex amplitude image;
performing Fourier transform on the updated target complex amplitude image to obtain an updated spectrogram, mapping according to the spatial coordinate position to obtain a frequency domain spatial coordinate, and replacing the frequency spectrum region of the corresponding sub-value space of the initial spectrogram by using the updated spectrogram;
sixthly, repeating the step three to the step five to complete the frequency spectrum updating of all the sub-numerical apertures;
seventhly, repeating the step three to converge the iterative process to obtain a high-resolution frequency spectrum image of the image, and then performing inverse Fourier transform to obtain a high-resolution image of a space domain;
the point light source scanning illumination detection device based on the point light source scanning illumination method comprises a CCD camera, a tube lens, a microscope objective, a point light source, an XYZ precision displacement table and a computer;
the point light source is installed on the XYZ precision displacement table in a matching mode, the computer is respectively and electrically connected with the point light source and the XYZ precision displacement table, the microscope objective lens is located on the light output side of the point light source in a matching mode, a test sample is placed between the light output side of the point light source and the light input side of the microscope objective lens in a matching mode, the tube lens is located on the light output side of the microscope objective lens in a matching mode, and the CCD camera is located on the light output side of the tube lens in a matching mode;
light emitted by the point light source passes through a test sample and then sequentially passes through the microscope objective and the tube lens and is focused on an image plane of the CCD camera;
the point light source adopts an LED light source or a laser point light source;
and the translation of the XYZ precision displacement table, the illumination of the point light source and the image acquisition of the CCD camera are controlled and finished by the computer.
2. A point light source scanning illumination detection apparatus based on the point light source scanning illumination method of claim 1, characterized in that: the detection device consists of a CCD camera, a tube lens, a microscope objective, a point light source, an XYZ precision displacement platform and a computer;
the point light source is installed on the XYZ precision displacement table in a matching mode, the computer is respectively and electrically connected with the point light source and the XYZ precision displacement table, the microscope objective lens is located on the light output side of the point light source in a matching mode, a test sample is placed between the light output side of the point light source and the light input side of the microscope objective lens in a matching mode, the tube lens is located on the light output side of the microscope objective lens in a matching mode, and the CCD camera is located on the light output side of the tube lens in a matching mode;
and light emitted by the point light source passes through the test sample and then sequentially passes through the microscope objective and the tube lens to be focused on the image plane of the CCD camera.
3. The point light source scanning illumination detection apparatus of claim 2, wherein: the point light source adopts an LED light source or a laser point light source.
4. The point light source scanning illumination detection apparatus of claim 3, wherein: and the translation of the XYZ precision displacement table, the illumination of the point light source and the image acquisition of the CCD camera are controlled and finished by the computer.
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