Differential imaging microscope based on virtual point spread function engineering

Abstract

The application provides a differential imaging microscope based on a virtual point spread function, which comprises a laser, a shaping lens, a polarizer, a 1 / 2 glass, a 1 / 4 glass, a phase plate, a light splitting module, an X-Y scanning mechanism, a scanning lens, a tube lens, an objective lens, a nanometer displacement table, an objective lens, a tube lens, a scanning lens, a detection lens and a surface array detector; the X-Y scanning mechanism and the nanometer displacement table can drive the sample so that an illumination point can traverse an imaging area in a light spot; for each scanning point, the surface detector receives complete point spread function and sample convolution data; for one scanning imaging process, the surface detector can record scanning imaging data; and a computer processes the scanning imaging data to obtain differential imaging based on a virtual point spread function; the differential imaging can obtain a system effective point spread function with a smaller half-height width; and finally obtained images are a convolution of a sample and the system effective point spread function; the point spread function determines the imaging resolution of the system; and the imaging image resolution is higher.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microscopic detection instrument design and manufacturing, and particularly relates to a differential imaging microscope based on virtual point spread function engineering. BACKGROUND

[0002] In recent decades, a variety of far-field super-resolution optical microscopes beyond the diffraction limit have emerged, such as PALM, STORM, SIM, STED, etc., which have improved the resolution of optical microscopes to 100 nm or higher. However, these super-resolution techniques are either high in cost, or extremely complex in optical system, or super-long in imaging and data processing time, or subject to dependence on special fluorescent dyes and special sample preparation, which have restricted the popularization of super-resolution techniques. Therefore, developing more general and lower-cost super-resolution techniques is still a research hotspot at present.

[0003] A point spread function is an enlarged image point of a point source after passing through an optical system due to diffraction. In an optical microscopic imaging system, a point spread function is used to describe the analytical ability of the optical system to a point source. Imaging differential based on point spread function engineering is an effective method for improving the resolution of a confocal microscopic system, and the optical system is relatively simple and the data processing speed is also fast. In the point spread function differential process, a solid point spread function is subtracted from a ring point spread function, the ring point spread function eliminates the periphery of the solid point spread function, reduces the full width at half maximum of the effective point spread function, and thus improves the imaging resolution. However, the generation of the ring point spread function by physical modulation is limited by the modulation optical element, and the distribution of the generated ring point spread function has great limitations. The image obtained by the differential process will produce negative values. Generally, the negative part is set to zero, but this processing method will cause distortion of the image. The reason for this problem is that there is a difference between the ring point spread function generated by modulation and the periphery of the solid point spread function. SUMMARY

[0004] The purpose of the present application is to provide a differential imaging microscope based on virtual point spread function engineering with higher resolution.

[0005] To achieve the above purpose, the present application adopts the following technical scheme:

[0006] The application provides a differential imaging microscope based on a virtual point spread function engineering, comprising a laser, and a shaping lens, a polarizer, a 1 / 2 glass, a 1 / 4 glass, a phase plate, a light splitting module, an X-Y scanning mechanism, a scanning lens, a barrel lens, an objective lens and a nanometer displacement table arranged in sequence along a laser beam emitted by the laser, wherein a sample is arranged on the nanometer displacement table, the sample can generate fluorescence after being excited by laser, and the objective lens, the barrel lens, the scanning lens, the X-Y scanning mechanism, the light splitting module, a detection lens and a surface array detector are arranged in sequence along a fluorescence emission direction, and the surface array detector is further electrically connected with a computer.

[0007] The nanometer displacement table and the X-Y scanning mechanism can drive the sample to make an illumination spot traverse an imaging area, for each scanning point, the surface detector receives complete point spread function data, for one scanning imaging process, the surface detector can record scanning imaging data, and the computer processes the scanning imaging data to obtain differential imaging based on a virtual point spread function.

[0008] In some embodiments, by adjusting positions of the 1 / 2 glass, the 1 / 4 glass and the phase plate, modulation of polarization, phase and intensity of a light spot of the illumination point spread function received by the surface array detector is realized, and the surface array detector obtains a group of image scanning imaging data k=1, 2,..., N, wherein N represents different modulation modes, N Xdim *N Ydim represents a number of scanning points in the X direction, N Xdim scanning points in the Y direction, N Ydim represents a number of scanning points in the X direction, N Ximage *N Yimage represents a resolution of the camera.

[0009] In some embodiments, the surface array detector is provided with different virtual pinholes, and by modulation of the virtual pinholes, a group of images j=1, 2,..., M, M images, wherein, one point

[0010] is a weighting coefficient corresponding to the jth virtual pinhole.

[0011] In some embodiments, the computer obtains images corresponding to a virtual solid point spread function required for differential imaging by linear combination obtains images corresponding to a virtual ring point spread function required for differential imaging by linear combination obtains a final image with improved resolution by difference 实心-I 环形 .

[0012] In some embodiments, the area detector is a wide-field detector, which includes an EMCCD or an sCMOS.

[0013] The application has the advantages of the above technical solutions:

[0014] The differential imaging microscope based on virtual point spread function engineering provided by the application comprises a laser, a shaping lens, a polarizer, a 1 / 2 glass, a 1 / 4 glass, a phase plate, a light splitting module, an X-Y scanning mechanism, a scanning lens, a barrel lens, an objective lens, a nanometer displacement table, an objective lens, a barrel lens, a scanning lens, a detection lens and an area array detector, the area array detector is further electrically connected with a computer, the nanometer displacement table can drive the sample so that an illumination spot can traverse an imaging area, for each scanning point, the area detector receives complete point spread function data, for one scanning imaging process, the area detector can record scanning imaging data, the computer processes the scanning imaging data to obtain differential imaging based on a virtual point spread function, the differential imaging can obtain a system effective point spread function with a smaller full width at half maximum, and finally obtained image is a convolution of a sample and the system effective point spread function, the point spread function determines the imaging resolution of the system, and the imaging image resolution is higher.

[0015] The differential imaging microscope based on virtual point spread function engineering provided by the application provides a general and low-cost super-resolution imaging technology, is favorable for popularization of super-resolution technology, promotes development of basic researches such as tumors, provides a scientific basis for early treatment of tumors, and promotes development of medical level and human health civilization. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The differential imaging microscope based on virtual point spread function engineering provided by the application provides a general and low-cost super-resolution imaging technology, is favorable for popularization of super-resolution technology, promotes development of basic researches such as tumors, provides a scientific basis for early treatment of tumors, and promotes development of medical level and human health civilization.

[0017] Figure 2 Some illumination point spread functions obtained by polarization and phase modulation for the embodiment.

[0018] Figure 3 A data structure diagram obtained by area array point scanning imaging provided by the embodiment.

[0019] Figure 4 Several possible virtual pinhole schematic diagrams provided by the embodiment. DETAILED DESCRIPTION

[0020] Please refer to Figure 1The high-resolution parallel microscopic imaging instrument based on the microlens array provided by the embodiment of the present application comprises a laser 110, a shaping lens 120, a polarizer 130, a 1 / 2 glass 140, a 1 / 4 glass 150, a phase plate 160, a light splitting module 170, an X-Y scanning mechanism 180, a scanning lens 190, a cylindrical lens 210, an objective lens 220 and a nanometer displacement table 230 arranged in sequence along the laser beam emitted by the laser 110, wherein the nanometer displacement table 230 is provided with a sample, the sample can generate fluorescence after being excited by laser, the objective lens 220, the cylindrical lens 210, the scanning lens 190, the X-Y scanning mechanism 180, the light splitting module 170, a detection lens 240 and a surface array detector 250 are arranged in sequence along the fluorescence emission direction, and the surface array detector 250 is further electrically connected with a computer 260.

[0021] It can be understood that the confocal microscope imaging system provided by the present application adopts a point scanning mode for imaging.

[0022]

[0023] The intensity distribution of the confocal image of the sample, wherein S is the density distribution of the marker in the scanning imaging area, H eff (r) is the effective point spread function of the confocal system:

[0024]

[0025] wherein H ill is the system illumination point spread function; is the effective detection point spread function of the system, which is the convolution of the system detection point spread function H det and the pinhole transfer function P. The system point spread function of the confocal system is jointly determined by the illumination point spread function H ill and the effective detection point spread function When the zero pinhole is selected, the confocal system will reach its limit resolution, at this time its effective point spread function is as follows:

[0026]

[0027] The differential imaging microscope based on the virtual point spread function engineering works as follows:

[0028] The nano displacement stage 230 and the X-Y scanning mechanism 180 can drive the sample so that the illumination spot can traverse the imaging area in a spot, and for each scanning point, the area detector 250 receives complete point spread function data, and for a scanning imaging process, the area detector 250 can record scanning imaging data, and the computer 260 processes the scanning imaging data to obtain virtual point spread function-based differential imaging.

[0029] In some embodiments, by adjusting the positions of the 1 / 2 glass, the 1 / 4 glass, and the phase plate, the polarization, phase, and intensity modulation of the spot of the illumination point spread function received by the area array detector is realized, and the area array detector obtains a set of image scanning imaging data k = 1, 2,... N, where N represents different modulation modes, N Xdim *N Ydim representing the number of scanning points in the X direction, N Xdim scanning points in the Y direction, N Ydim scanning points in the Y direction, N Ximage *N Yimage representing the resolution of the camera.

[0030] Please refer to Figure 2 , some illumination point spread functions obtained by polarization and phase modulation in this embodiment. By modulating the illumination point spread function with polarization, phase, and intensity control, a variety of illumination point spread functions are obtained, including solid point spread functions, ring point spread functions, and virtual point spread functions. Among them, the virtual point spread function is obtained by mathematical operation of the solid point spread function.

[0031] Please refer to Figure 3 , the data structure diagram obtained by the area array point scanning imaging provided in this embodiment.

[0032] In some embodiments, the area array detector 250 is provided with different virtual pinholes, and by modulating the virtual pinholes, a j = 1, 2,... M, M images, where, one point in

[0033] is the weighting coefficient corresponding to the jth virtual pinhole. Please refer to Figure 4 , several possible virtual pinhole schematic diagrams provided in this embodiment.

[0034] In some embodiments, the computer obtains the images corresponding to the virtual solid point spread functions required for differential imaging by linear combination by linear combination of the images corresponding to the virtual ring point spread functions required for differential imaging The final resolution enhanced image I = I 实心 -I 环形 .

[0035] It can be understood that, in the differential imaging process, although continuously increasing the subtraction coefficient can continuously reduce the half width of the point spread function, negative values will appear in the point spread function, which is impossible in practice. The conventional processing method is to directly assign the part less than zero to zero, but this processing method brings the problem that the reconstructed image is a non-real image, that is, an artifact.

[0036] The application obtains a group of point spread functions based on virtual pinhole modulation of area array imaging, and then obtains a solid point spread function and a ring point spread function for differential imaging through linear combination.

[0037] Further, the coefficients of linear combination are obtained through pre-optimization solution of the computer 260. In the optimization solution, there are two targets: the final half width of the point spread function and the high similarity of the periphery of the virtual solid point spread function and the virtual ring point spread function generated by combination.

[0038] The imaging process of the imaging method of the application is analyzed from the point spread function, and the final obtained image is the convolution of the sample and the effective point spread function. The point spread function determines the imaging resolution of the system.

[0039] In some embodiments, the area detector is a wide-field detector, which includes an EMCCD or an sCMOS.

[0040] The differential imaging microscope based on virtual point spread function engineering provided by the application includes a laser, a shaping lens, a polarizer, a 1 / 2 glass, a 1 / 4 glass, a phase plate, a light splitting module, an X-Y scanning mechanism, a scanning lens, a barrel lens, an objective lens, a nanometer displacement table, an objective lens, a barrel lens, a scanning lens, a detection lens, and an area array detector. The area array detector is further electrically connected with a computer. The nanometer displacement table can drive the sample so that the illumination point can traverse the imaging area. For each scanning point, the area detector receives complete point spread function data. For a scanning imaging process, the area detector can record scanning imaging data. The computer processes the scanning imaging data to obtain differential imaging based on virtual point spread function. The differential imaging can obtain a system effective point spread function with a smaller half width. The final obtained image is the convolution of the sample and the system effective point spread function. The point spread function determines the imaging resolution of the system, and the imaging image resolution is higher.

[0041] The differential imaging microscope based on the virtual point spread function engineering provided by the application provides a general and low-cost super-resolution imaging technology, is favorable for popularization of the super-resolution technology, promotes development of basic researches such as tumors, provides a scientific basis for early treatment of tumors, and promotes development of medical levels and human health civilization.

[0042] Of course, the differential imaging microscope based on the virtual point spread function engineering of the application can also have various transformations and modifications, and is not limited to the specific structure of the above-mentioned embodiments. In summary, the protection scope of the application should include those transformations or substitutions and modifications that are obvious to those skilled in the art.

Claims

1. A differential imaging microscope based on virtual point spread function engineering, characterized in that, The system includes a laser, and a shaping lens, a polarizer, a half-wave plate, a quarter-wave plate, a phase plate, a beam splitter, an XY scanning mechanism, a scanning lens, a tube lens, an objective lens, and a nanometer displacement stage arranged sequentially along the laser beam emitted from the laser. A sample is placed on the nanometer displacement stage, and the sample generates fluorescence upon laser excitation. The objective lens, tube lens, scanning lens, XY scanning mechanism, beam splitter, detection lens, and area array detector are arranged sequentially along the fluorescence emission direction. The area array detector is also electrically connected to a computer. The nanostage and the XY scanning mechanism can drive the sample so that the illumination point can traverse the imaging area. For each scanning point, the surface detector receives complete point spread function data and sample convolution data. For one scanning imaging process, the surface detector can record the scanning imaging data. The computer processes the scanning imaging data to obtain differential imaging based on the virtual point spread function.

2. The differential imaging microscope based on virtual point spread function engineering according to claim 1, characterized in that, By adjusting the half-wave plate, the quarter-wave plate, and the phase plate, the polarization, phase, and intensity of the light spot of the illumination point spread function received by the area array detector are modulated, and the area array detector obtains a set of image scanning imaging data. , Where N represents different modulation methods, N Xdim *N Ydim N represents the number of points in the scanned image, in the X direction. Xdim There are 1 scan point, N in the Y direction. Ydim N scan points Ximage *N Yimage This represents the camera's resolution.

3. The differential imaging microscope based on virtual point spread function engineering according to claim 2, characterized in that, The array detector is equipped with different virtual pinholes, and by modulating the virtual pinholes, a certain value can be obtained. , M images, where One point , is the weighting coefficient corresponding to the j-th type of virtual pinhole.

4. The differential imaging microscope based on virtual point spread function engineering according to claim 3, characterized in that, The computer obtains the image corresponding to the virtual solid dot spread function required for differential imaging through linear combination. The image corresponding to the virtual ring point spread function required for differential imaging is obtained through linear combination. The final image with improved resolution is obtained through differential processing. .

5. The differential imaging microscope based on virtual point spread function engineering according to claim 1, characterized in that, The area detector is a wide-field detector, which includes EMCCD or sCMOS.

Images