CN117369106B - Multi-point confocal image scanning microscope and imaging method - Google Patents
Multi-point confocal image scanning microscope and imaging method Download PDFInfo
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- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
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- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
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- G02B21/00—Microscopes
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Abstract
The invention discloses a multipoint confocal image scanning microscope and an imaging method, and relates to the technical fields of optical elements, systems, instruments and imaging. The invention sets imaging light source component, light source collimation component, multi-focus generation component, multi-focus moving component, multi-focus projection component and camera detection component along the light path to realize complete image record. The invention provides a unique multi-focus illumination mode combined with an optical phase-locked detection technology, which can effectively remove defocused stray signals in thick sample imaging and carry out depth imaging; and the multi-focus generating component is switched, so that the switching among wide-field, confocal and super-resolution modes is easily realized. In addition, the super-resolution reconstruction can be performed by adopting a pixel redistribution algorithm, and the multi-image deconvolution reconstruction algorithm can reduce frame reconstruction by utilizing redundant information in an original data set, so that the imaging speed is improved. Compared with the existing method, the method can accelerate imaging speed, improve imaging depth, lighten phototoxicity and widen diversity of imaging modes.
Description
Technical Field
The invention relates to the field of optical elements, systems or instruments and imaging, in particular to a multi-point confocal image scanning microscope and an imaging method.
Background
At present, the microscope imaging technology has wide application in the fields of biomedicine, material science and the like. However, the resolution of conventional microscopes is limited by the optical diffraction limit and cannot meet the high-resolution observation requirements for fine structures. To solve this problem, super-resolution imaging techniques have been proposed and widely studied.
Confocal microscopy is the first technology of biologists in fluorescence imaging because of its excellent optical sectioning capability and versatility. However, limited resolution and relatively slow imaging speeds of confocal optics have prevented its wider application in life sciences. By replacing the single point detector of the confocal microscope with a detector array, the image scanning microscope achieves a double resolution enhancement by combining pixel redistribution and deconvolution. In order to solve the problem that the imaging speed of the image scanning microscope recorded by the single-point excitation and the camera is relatively low, various expansion technologies are developed based on the ideas of multi-point excitation and optical or digital reconstruction.
Rescanning confocal microscopes, optical photon redistribution microscopes, instant structure light illumination microscopes, optical photon redistribution turret confocal microscopes, and the like have extremely complex optical paths depending on the optical reconstruction technique. In addition, pixel redistribution requires descan, rescanning, or passing through a microlens array of fluorescent signals, which can result in a significant decrease in fluorescent signals and a significant increase in phototoxicity. Furthermore, while a complete optical reconstruction may be substituted for a digital reconstruction, the underlying information that is embedded in the image scanning microscope (Image Scanning Microscopy, ISM) has no opportunity to be exploited. The digital reconstruction techniques of multi-focus structured light illumination microscopes, turntable confocal image scanning microscopes and the like ingeniously utilize existing devices or modules, such as digital micromirror devices (Digital Micromirror Device, DMDs), turntables and the like, through which multi-point data acquisition is seamlessly realized. However, the ratio of the scanning step size to the pinhole size is also limited by the digital micromirror device or the rotating disk, and thus too many raw frames are required during reconstruction, limiting the increase in imaging speed. Furthermore, the redundant information collected in these methods remains largely unutilized.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a multi-point confocal image scanning microscope and an imaging method.
In order to achieve the above object, the present invention provides the following.
A multi-point confocal image scanning microscope comprising: the system comprises a signal control assembly, an imaging light source assembly, a light source collimation assembly, a multi-focus generation assembly, a multi-focus movement assembly, a multi-focus projection assembly and a camera detection assembly, wherein the imaging light source assembly, the light source collimation assembly, the multi-focus generation assembly, the multi-focus movement assembly, the multi-focus projection assembly and the camera detection assembly are arranged along a light path.
The imaging light source component is used for outputting a light source; the light source collimation assembly is used for carrying out collimation output on the light source output by the imaging light source assembly to obtain a collimation light spot; the multi-focus generation assembly is used for generating a single collimation light spot into a plurality of excitation focus illumination lights; the multi-focus moving component is used for transversely, longitudinally or obliquely translating the excitation focus illumination light; the multi-focus projection assembly is used for projecting the excitation focus illumination light to the sample surface, and simultaneously realizing multi-focus parallel excitation and scanning of the sample surface in cooperation with the multi-focus moving assembly; the camera detection component is used for detecting and outputting fluorescent signals generated by multi-focus parallel excitation of the sample surface.
The signal control component is respectively connected with the multi-focus moving component and the camera detection component; the signal control component is used for generating a control signal; the control signal is used for controlling the multi-focus moving assembly and the camera detecting assembly to synchronously work.
Optionally, the imaging light source assembly includes a multichannel switched light emitting diode; the light source emitted by the light emitting diode is output through a liquid optical waveguide, a single mode optical fiber or a multimode optical fiber with set size.
Optionally, the light source collimation component is an aspheric lens, a 90-degree off-axis parabolic reflector, an adjustable focal length aspheric lens, an achromatic doublet or an air-spaced doublet.
Optionally, etching small holes arranged in a set size on chromed glass by using a photoetching technology to form the multi-focus generating component by adopting a pinhole array; alternatively, the multi-focus generation assembly is formed using a microlens array, digital micromirror device, grating, and/or diffractive optical element (Diffractive Optical Elements, DOE).
Optionally, the multi-focus moving component is a two-dimensional galvanometer.
Optionally, the multi-focal projection assembly includes a first lens, a second lens, a dichroic mirror, a tube lens, and a microscope objective disposed along the optical path.
The excitation focus illumination light passes through the first lens, is transversely, longitudinally or obliquely translated by the multi-focus moving assembly, and then enters the second lens.
The tube lens, the microscope objective and the sample surface form a microscope host; the microscope host is used for realizing the nominal magnification of the microscope objective and clamping the sample.
The dichroic mirror is used for separating the excitation focus illumination light emitted by the second lens and fluorescent signals of the sample surface; the excitation focus illumination light emitted by the second lens is reflected into the tube lens; fluorescent signals of the sample surface are transmitted into the camera detection assembly.
Optionally, the camera detection assembly includes a third lens and a camera disposed along the optical path.
Fluorescent signals of a sample surface are incident into the camera through the third lens; the camera is connected with the signal control component; the camera is used for detecting and outputting fluorescent signals generated by multi-focus parallel excitation of the sample surface.
Further, the invention also provides an imaging method using the multi-point confocal image scanning microscope, which comprises the following steps: and acquiring a detection result by using the provided multi-point confocal image scanning microscope, and taking the detection result as an original image.
Based on the original image, adopting a pixel redistribution algorithm or a multi-image deconvolution algorithm to realize imaging reconstruction.
Optionally, based on the original image, the process of implementing imaging reconstruction by adopting a pixel redistribution algorithm includes:
and positioning the pixel-level center coordinates based on a method for taking the pixels with the maximum neighborhood gray values to obtain a candidate center set.
A plurality of sub-images are truncated from the de-defocused image based on the candidate center set, forming a sub-image stack.
And carrying out sub-pixel center positioning on the sub-image to obtain sub-pixel level coordinates of the sub-image center.
And generating a two-dimensional Gaussian mask with the same size as the sub-image by taking the sub-pixel level coordinates as the center.
The two-dimensional gaussian mask is multiplied with the sub-image as a digital pinhole.
And integrating the sub-images obtained by multiplication into canvas with the same size as the detection result according to the sub-pixel level coordinates or the pixel level center coordinates to obtain a confocal image.
Optionally, based on the original image, the process of implementing imaging reconstruction by adopting a multi-graph deconvolution algorithm comprises: constructing an imaging model; the imaging model is as follows:the method comprises the steps of carrying out a first treatment on the surface of the In (1) the->Representing an array of pinholes->Representing airspace coordinates >Indicate acquisition +.>Relative position of illumination mode of picture +.>And->Representing the real object and the detected image, respectively, < >>And->Representing the excitation point spread function and the detection point spread function, respectively,/->Representing convolution operations +.>Representing the hadamard product operation.
And estimating a pinhole array based on the imaging model.
And completing iterative solution of the imaging model based on the constructed imaging model and the estimation result of the pinhole array to obtain a reconstructed image.
According to the specific embodiment provided by the invention, the invention discloses the following technical effects: according to the invention, through the imaging light source assembly, the light source collimation assembly, the multi-focus generation assembly, the multi-focus moving assembly, the multi-focus projection assembly and the camera detection assembly which are arranged along the light path, the generated multi-focus illumination is combined with the optical phase-locked detection technology, so that the unique multi-focus illumination mode of the defocusing signal in the depth imaging can be removed, and the phototoxicity is lower than that of a conventional structured light illumination super-resolution microscope. Also, by providing the multi-focus generation assembly, free switching between wide-field, confocal, and super-resolution modes can be easily performed.
The invention adopts a pixel redistribution algorithm or a multi-image deconvolution algorithm to reconstruct super-resolution imaging, and the redundant information is utilized by the multi-image deconvolution algorithm to reduce the number of reconstruction frames required to reconstruct super-resolution, thereby improving the imaging speed. Compared with the existing method, the three-dimensional resolution can be improved, the imaging speed can be increased, the imaging depth can be increased, phototoxicity can be reduced, and the diversity of imaging modes can be enhanced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic diagram of a multi-point confocal image scanning microscope employing a pinhole array according to the present invention.
Fig. 2 is a schematic structural diagram of a multi-point confocal image scanning microscope employing a microlens array according to the present invention.
Fig. 3 is a matrix diagram of multi-point confocal image scanning microscope adaptive to various types of Light source effects of single-mode laser, multi-mode laser and high-power Light Emitting Diode (LED).
Fig. 4 is a schematic diagram of the detection principle and effect of optical phase lock in the algorithm process provided by the invention. Fig. 4 (a) is an original data diagram, fig. 4 (b) is a schematic diagram of defocus of element data, fig. 4 (c) is a schematic diagram of fourier transform of original data, fig. 4 (d) is a comparison diagram of the result of defocus background processing of original data, fig. 4 (e) is a schematic diagram of widening field of an original data stack, and fig. 4 (f) is a schematic diagram of defocus widening field of an original data stack.
FIG. 5 is a schematic representation of the results and resolution of a wide field, confocal and multi-point confocal image scanning microscope under a fixed actin filament sample of the present invention. Where fig. 5 (a) is a wide field result plot under a fixed actin filament sample, fig. 5 (b) is a confocal result plot under a fixed actin filament sample, fig. 5 (c) is a multi-point confocal image scanning microscopy result plot under a fixed actin filament sample, and fig. 5 (d) is a resolution schematic under a fixed actin filament sample.
Fig. 6 is a schematic representation of the results and resolution of a wide field, confocal, and multi-point confocal image scanning microscope provided by the invention in the YZ plane of a fixed mouse kidney slice sample. Where (a) of fig. 6 is a view of a wide field result under the YZ plane of a fixed mouse kidney slice sample, (b) of fig. 6 is a view of a confocal result under the YZ plane of a fixed mouse kidney slice sample, (c) of fig. 6 is a view of a multi-point confocal image scanning microscope result under the YZ plane of a fixed mouse kidney slice sample, (d) of fig. 6 is a schematic view of a resolution of a fixed mouse kidney slice sample along the Z axis, and (e) of fig. 6 is a schematic view of a resolution of a fixed mouse kidney slice sample along the Y axis.
Fig. 7 is a graph comparing the results of reduced frame reconstruction provided by the present invention with the results of wide field, confocal and classical image scanning microscope reconstruction of normal frame reconstruction. Fig. 7 (a) is a schematic diagram of a wide field of raw data, fig. 7 (b) is a confocal result graph of normal frame number reconstruction, fig. 7 (c) is a classical image scanning microscope reconstruction result graph of normal frame number reconstruction, fig. 7 (d) is a joint RL deconvolution result graph of reduced frame reconstruction, and fig. 7 (e) is a fast iterative shrinkage threshold algorithm-group sparsity deconvolution result graph of reduced frame reconstruction.
Fig. 8 is a flowchart of an imaging method using a multi-point confocal image scanning microscope according to the present invention.
Symbol description:
10-imaging light source assembly, 20-light source collimation assembly, 30-multifocal generation assembly, 40-multifocal movement assembly, 50-multifocal projection assembly, 51-first lens, 52-second lens, 53-dichroic mirror, 54-tube lens, 55-microscope objective, 56-sample plane, 60-camera detection assembly, 61-third lens, 62-camera, 70-signal control assembly.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention aims to provide a multi-point confocal image scanning microscope and an imaging method, which can improve three-dimensional resolution, speed up imaging, increase imaging depth, lighten phototoxicity and enhance diversity of imaging modes.
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention will be rendered by reference to the appended drawings and appended detailed description.
As shown in fig. 1 and 2, the multi-point confocal image scanning microscope provided by the present invention includes: a signal control assembly 70, and an imaging light source assembly 10, a light source collimation assembly 20, a multi-focus generation assembly 30, a multi-focus movement assembly 40, a multi-focus projection assembly 50, and a camera detection assembly 60 disposed along the optical path.
The imaging light source assembly 10 is used to excite a fluorescent sample (i.e., to emit a light source). The light source collimation assembly 20 is used for collimating and outputting the imaging light source assembly 10, so as to realize uniform illumination of large light spots.
The single collimated light spot output by the light source collimation assembly 20 passes through the multi-focal generation assembly 30 to generate a plurality of excitation focal illumination light.
The multi-focus moving component 40 is configured to translate the multi-focus illumination light generated by the multi-focus generating component 30 laterally, longitudinally, or obliquely, so as to achieve uniform scanning of the entire sample.
The multi-focal projection assembly 50 comprises a first lens 51, a second lens 52, a dichroic mirror 53, a tube lens 54 and a microscope objective 55. The multi-focal projection assembly 50 is configured to project the generated multi-focal illumination light onto the sample surface 56 while simultaneously achieving multi-focal parallel excitation and uniform scanning of the sample surface 56 in conjunction with the multi-focal movement assembly 40. The tube lens 54, the microscope objective 55 and the sample surface 56 form a microscope host for achieving a nominal magnification of the microscope objective and clamping of the sample.
The camera detection assembly 60 includes a third lens 61 and a camera 62. The camera detection assembly 60 is used to enable detection and output of fluorescent signals. The signal control assembly 70 is used to generate the required control signals and output signals to control the synchronous operation of all the instruments and devices. Wherein the fluorescent signal generated by excitation of the sample surface 56 passes through the microscope objective 55, tube lens 54 and dichroic mirror 53 in the multi-focal projection assembly 50 before entering the third lens 61 and camera 62.
In practical applications, the imaging light source assembly 10 may employ a multi-channel fast switching LED, and other single-mode lasers and multi-mode lasers are also suitable. The effect of adapting to various types of light sources such as a single-mode laser, a multimode laser, an LED and the like is shown in figure 3.
When using a multi-channel fast switching high power LED, the LED is output through a liquid optical waveguide of a set size (e.g. 3 mm) or through a single mode fiber, multimode fiber. LEDs and multimode lasers have advantages in eliminating speckle artifacts, providing large field of view illumination, and high power. Liquid optical waveguides and multimode optical fibers have advantages in terms of transmission efficiency.
Further, the light source collimation assembly 20 may be an aspheric lens. At this time, the light output from the liquid optical waveguide is collimated by the aspherical lens. In practical applications, the light source collimation assembly 20 may also employ 90 ° off-axis parabolic mirrors, adjustable focal length aspheric mirrors, achromatic doublets, air-spaced doublets, and the like for collimation. The light output by single mode and multimode fibers can also be collimated in the manner described above.
Further, the multiple focal spot generating assembly 30 may be configured in two ways, one employing an array of pinholes (as shown in FIG. 1). Specifically, small holes in a set size arrangement are etched on chromed glass by utilizing a photoetching technology, and light spots emitted by a light source collimation assembly are irradiated onto a pinhole array to form multi-focus illumination. Different samples were adapted by adjusting the pinhole diameter and spacing. The other is formed by a microlens array (as shown in fig. 2), DMD, grating, and DOE to form multi-focal illumination.
In practical applications, the diameter of the pinhole array is sized to pass through the multifocal projection assembly, requiring filling of the entrance pupil of the microscope objective 55. In the present optical system setup, a 40 μm diameter pinhole is most suitable, and a smaller excitation pinhole may enhance the resolution of imaging to some extent, but severely reduce the signal-to-noise ratio. The focused light spot should also satisfy a diameter of 40 μm or less, for example, a square microlens with a unit size of 125um×125um is used, the focal length is 2.7mm, and the numerical aperture NA is 0.033.
Further, the multi-focal spot moving assembly 40 may employ a two-dimensional galvanometer, which scans to uniformly cover the plane with the resulting multi-focal spot. The two-dimensional galvanometer comprises two galvanometers. The two galvanometer are fixed together through a mechanical structure to realize two-dimensional scanning, and two paths of analog signals generated by the signal control assembly 70 control the two galvanometers to move. The scanning of the whole plane can be realized by using a one-dimensional galvanometer through the inclined pinhole arrangement, and the scanning can also be realized by using an MEMS galvanometer, a resonance mirror or a rotating mirror. The stepping of the galvanometer is severely limited and the step of the scanning beam through the second lens 52 at its focal point is typically equal to or less than half the pinhole diameter. A smaller step would result in too many raw images being acquired, extending the final imaging time and increasing phototoxicity. While a larger step increases the imaging speed but decreases the resolution with artifacts.
Further, the dichroic mirror 53 serves to separate excitation light and emission light, reflect the aforementioned excitation light into the tube lens 54, and transmit the aforementioned fluorescence emission light into the camera detection assembly 60.
Further, the multifocal shift assembly 40 is generally located between the first lens 51 and the second lens 52. This position is rigidly determined and is conjugated to the entrance pupil of the microscope objective 55 via the second lens 52 and the tube lens 54 in the multifocal projection assembly. In this position the beam should theoretically remain stationary, but due to the assembly of the two-dimensional galvanometer, there is no way for the two lenses to guarantee coincidence, so the spot at the entrance pupil of the microscope objective 55 will move. In addition, the two-dimensional galvanometer can be disassembled, a 4F imaging system is formed by two lenses, and the two galvanometers are conjugated.
Further, the tube lens 54 in the camera detection assembly 60 may use a single lens to collect the returned fluorescence. The single lens can realize large-field equal-proportion conjugate imaging. The scientific camera with large target surface can detect the returned fluorescence with high sensitivity. Other relay lens groups can be used to realize conjugate imaging, and a common camera can be used to realize fluorescence detection.
Further, in the present invention, the signal control unit 70 may output three analog signals and two digital signals, and the two analog signals control the multi-focal shift unit 40 for realizing the scanning in the x-direction and the y-direction. The other path of analog signals controls the camera in the camera detection assembly 60, the two paths of digital signals are used as address codes of the data distributor 74HS138, global exposure timing output of the camera is connected with an enabling port of the data distributor 74HS138, and output of the data distributor 74HS138 is connected with external trigger of the imaging light source assembly to realize wavelength switching and switching of a light source. Because of the multi-point parallel excitation scan, the total scan length needs to be exactly equal to the pinhole spacing in the multi-focus generation assembly. In addition, the multipoint parallel excitation causes the scanning angle of the vibrating mirror to be about 40 mu rad, and under a small mechanical scanning angle, the quick starting and stopping of the vibrating mirror is extremely easy to cause uneven scanning steps of each step. The scanning non-uniformity can be corrected and the total voltage can be accurately adjusted through voltage compensation, so that image artifacts caused by the non-uniformity of scanning step sizes are avoided.
Further, the invention also provides an imaging method using the multi-point confocal image scanning microscope, as shown in fig. 8, comprising a step 100 and a step 101.
Wherein, step 100: and acquiring a detection result by using the provided multi-point confocal image scanning microscope, and taking the detection result as an original image. Before the detection result is obtained by adopting the multi-point confocal image scanning microscope, parameters such as exposure time, multi-focus stepping distance and step number, laser power, shooting frame number, whether multicolor and imaging vision field are required to be set, and the pinhole diameters and distances adopted by different sample types are different, so that the set multi-focus stepping distance and step number are also different, and for a thin sample (the thickness is less than 20 um), 40um is generally adopted: 120um diameter and pitch ratio, the number of lateral and longitudinal steps was set to 7. For thicker samples (20 um < thickness <80 um), 40um is typically used: 200um diameter and pitch ratio, the number of lateral and longitudinal steps was set to 11. For very thick samples (thickness >80 um), 40um is typically used: 240um diameter and pitch ratio, the number of lateral and longitudinal steps was set to 14. For live cell imaging of tissue samples, 40um is typically used: 160um diameter and pitch ratio, the number of lateral and longitudinal steps was set to 9. The exposure time and laser power are generally determined based on the sample brightness.
Step 101: based on the original image, adopting a pixel reassignment algorithm or a multi-image deconvolution algorithm to realize imaging reconstruction.
Further, in the above step 101 of the present invention, the process of implementing the imaging reconstruction using the pixel reassignment algorithm based on the original image includes steps 1 to 5.
Wherein, step 1: defocus background processing (optical lock-in detection).
Because the illumination light excites the focal plane fluorescent signal and simultaneously excites the defocusing layer sample, and the emitted fluorescent light does not pass through the small holes again, partial background signals exist on the acquired original image, and in order to improve the positioning accuracy of the illumination bright spots and realize optical slicing, defocusing signal removal is required to be carried out on the acquired original data. The characteristics of these signals are that they appear constantly during the scanning process, whereas defocus information appears with the point scan, so the background defocus signal will exhibit a dc characteristic over the time frame. The signal is subjected to Fourier transformation pixel by pixel in the acquisition direction, and the zero frequency contains the sum of all information, namely the sum of a direct current component and all alternating current components, so that the removal of the background defocusing signal can be realized by inhibiting the direct current component. Wherein the optical phase lock detection principle and result are shown in fig. 4.
Step 2: the pixel level is centrally located.
The purpose of this step is mainly to extract the sub-image, which is read at the pixel level, so that in case of a pre-defined sub-image size, it is necessary to locate the pixel level center of the sub-image (i.e. the illumination spot center). After the original image is processed in the step 1, most of defocusing information is removed, and the interference information outside the residual spot illumination can not interfere with the positioning of the center of the illumination bright spot any more, so that no additional pretreatment is needed. If the original image is directly pixel-level centered without performing step 1, the following preprocessing procedure is required.
Firstly, performing Butterworth high-pass filtering on the frequency spectrum of the image to inhibit low-frequency noise of the image and keep high-frequency fluorescence information of the image. Then, by morphological processing, the outline of the illumination point is smoothed by an on operation, fine protrusions are eliminated, and isolated noise pixels are cleaned up.
Then, through median filtering of the image, isolated pixel points in the original image are removed, and Gaussian filtering is carried out, so that gray value-space distribution curves of the illumination points tend to be Gaussian lines, and the condition of locating center coordinates is met.
And screening out the local maximum value points as candidate center point sets of the center coordinates by comparing the gray value of each pixel with the gray value of the eight adjacent areas of each pixel.
Step 3: sub-images are extracted.
Setting a hard threshold, taking the pixels with gray values lower than the threshold in the candidate center point set obtained in the step 2 as the background, deleting the pixels from the candidate center point set, and taking the coordinates higher than the threshold as the center coordinates of the sub-image. Taking the coordinates as the center, a series of sub-images are cut out from the result image processed in the step 1 by properly selecting the side length (the illumination point is completely contained and no other illumination points are contained) to form a sub-image stack.
Step 4: a digital pinhole is applied.
Further, to accurately locate and add digital pinholes, the sub-pixel level center needs to be determined. Firstly, gaussian filtering is carried out on the sub-graph, so that the gray value-space distribution curve of the illumination point tends to be Gaussian, and the gray value-space distribution curve meets the requirement of sub-pixel level center positioning. The sub-pixel level center positioning can use quadratic function fitting to determine the center, and the maximum value coordinates of the quadratic function fitting in two directions are used for determining the center, so that the sub-pixel level coordinates of the sub-image center are obtained. And generating a two-dimensional Gaussian mask with the same size as the sub-image by taking the sub-pixel level coordinates as a center, and multiplying the two-dimensional Gaussian mask with the sub-image as a digital pinhole to realize further defocusing operation in the digital aspect.
Step 5: pixel redistribution and deconvolution.
The coordinate area of the sub-image in the canvas of the original image size is determined according to the sub-pixel level center and the sub-image size, and since the coordinates of the canvas are pixel level coordinates, the sub-image needs to be resampled to the corresponding canvas pixel level coordinate area. And integrating all the sub-images into the canvas in the same way and storing the corresponding results to obtain the theoretical confocal image.
Based on the above description, compared with the processing mode of integrating the image directly processed by confocal into the corresponding position of the original image according to the center point coordinates, the pixel reassignment algorithm needs to interpolate the canvas of the original image size, so that the number of the canvas coordinate indexes is increased by two times, and the generation of non-integer pixel points relative to the original image is equivalent to the expansion of the width and the height of the canvas by two times. And then searching a sub-pixel coordinate point closest to the sub-pixel level center, wherein the corresponding array index value is the pixel level center coordinate of the sub-image in the canvas after expansion, so as to realize pixel redistribution. In order to integrate sub-images into pixel-level coordinates, resampling of the sub-images is required when integrating the sub-images into the expanded canvas. And finally, the resolution can be further improved by a deconvolution algorithm to realize 2-time image resolution improvement.
Further, multiple images are generated from a single sample, each image containing only a portion of the sample information, but the superposition of all images can form a uniform and complete illumination field, and images meeting these conditions can be reconstructed into a single, higher quality image by "joint" deconvolution. The frame reduction reconstruction algorithm can fully utilize redundant information and utilize fewer original frames to realize super-resolution reconstruction. Based on this, the multi-map deconvolution algorithm can be divided into joint RL (joint Richardson-Lucy, jRL) deconvolution and a fast iterative shrink threshold algorithm-Group Sparsity (FISTA-GS) deconvolution.
Further, when the imaging reconstruction is realized using jRL deconvolution based on the original image, the procedure thereof includes the following steps.
Step 1: and constructing an imaging model.
The MC-ISM imaging process is as follows: the imaging light source component generates excitation light, the multi-focus generating component generates a multi-focus excitation mode, the multi-focus projection component projects the generated multi-focus illumination excitation mode onto a sample surface, the multi-focus moving component is matched with the multi-focus projection component to realize uniform scanning of the sample, and finally fluorescent signals enter the camera detection component and are detected by the camera after being amplified by the microscope host component. The multi-focus excitation mode can be expressed by convolution of a pinhole array and an excitation point diffusion function, the multi-focus illumination excitation mode irradiates a sample, namely the multi-focus excitation mode is multiplied by the sample function, and finally the multi-focus illumination excitation mode is received by a camera through a detection light path, namely the multi-focus illumination excitation mode is convolved with the detection point diffusion function, and the specific imaging process is shown in the following formula:
。
In the method, in the process of the invention,representing an array of pinholes->Representing airspace coordinates>Indicate acquisition +.>Relative position of illumination mode of picture +.>And->Representing the real object and the detected image, respectively, < >>And->Representing the excitation point spread function and the detection point spread function, respectively,/->Representing convolution operations +.>Representing Hadamard product operation, < >>The multi-focal illumination mode is represented as a convolution of the pinhole array and the excitation point spread function.
Step 2: pinhole array estimation.
In the imaging model (see formula above),as a result of the detection obtained, the real object +.>As the object to be solved, the excitation light wavelength and the emission light wavelength are known, and the excitation point spread function +.>And a detection point spread function->Therefore, there is also a need to solve the pinhole array +.>. After a sample is excited by a multi-focus illumination mode to emit a fluorescent signal, under the convolution of a detection point spread function, the highest point of detected light intensity deviates from the center of a pinhole, and the pinhole array cannot be directly solved in a space domain, so that the frequency domain is selected to solve the pinhole array. The specific solving steps are as follows.
Step 2.1: the original image acquired by each frame is subjected to direct Fourier transform, and the Fourier spectrum of the original image also has periodically distributed characteristic frequency points because of the existence of a space domain periodic lattice, so that the original image can be directly obtained by selecting local highest points. The highest pixel point in both directions is reserved as the basis vector of the frequency domain.
Step 2.2: after the two basic vectors are linearly combined, the corresponding positions of the two basic vectors are also characteristic frequency points, so that the characteristic frequency point positions of the sub-pixel level are obtained through polynomial fitting, and the linear coordinates and the frequency point positions are subjected to least square fitting to obtain the basic vectors of the sub-pixel level.
Step 2.3: and obtaining an accurate space basic vector by transforming the basic vector of the frequency domain through coordinates.
Step 2.4: and generating a space lattice by using the space basic vector, wherein the relative position between the points of the space lattice is correct, but the absolute position has integral displacement with the position before the actual dot is excited, comparing the absolute position with the bright point of the actual image, summing the offset vector between the space lattice point and the center of the nearest space bright point to obtain an offset, solving the offset of the sub-pixel level by polynomial fitting, and adding the offset to the dot matrix generated according to the space basic vector to obtain the position on the sample surface corresponding to the actual pinhole array.
Step 3: and (5) carrying out iterative solution.
jRL deconvolution is based on poisson response and maximum likelihood estimation of photons by the area array detector, and a reconstructed image is obtained through iterative solution. Using imaging processesTo make a simplified representation of the reverse process +. >Indicating (I)>Indicate->Image, sheet of->The iteration number is represented, and the iteration process is shown in the following formula.
。
。
。
In the superscriptMeaning the estimated value of the corresponding parameter, is used to represent each intermediate result in the iterative process.Is->Estimated value generated at the current iteration +.>And->Element-wise division of the measured values to measure the deviation of the two, +.>And->The n-th iteration and the n+1-th iteration are respectively estimated values of the real object.
Further, when the imaging reconstruction is realized by adopting the FISTA-GS deconvolution based on the original image, the process thereof comprises the following steps.
Step 1: and (5) designing an optimization model.
Besides jRL deconvolution, constraint can be applied to the image reconstruction process, the acquired image of the MC-ISM is a dot matrix image, if constraint is applied to each frame, loss of image information can be possibly caused, so that sparse constraint is applied to superimposed images of the dot matrix image by using group sparsity, and reconstruction quality can be further improved. The imaging model constructed when FISTA-GS deconvolution realizes imaging reconstruction is the same as the imaging model constructed in jRL deconvolution realization presentation reconstruction, but FISTA-GS deconvolution does not directly reconstruct a real objectAs solving parameters, the real object is +. >Go up->Information in the corresponding pinhole area +.>As a solving target, +.>Relative position of illumination mode of picture>Directly use subscript->Indicating that the multi-focus excitation pattern is +.>Imaging result is->The multifocal detection diffusion mode is +.>. Based on this, the imaging model can be simplified as: />。
The design optimization model is as follows:。
wherein,representing the original image +.>Representing an objective function. />Is->Is a simplification of (1) to express->The original image is stretched. />The first term to the right of the equation, which is a fidelity term, is expressed to ensure that the reconstructed object is as close as possible to the measured image by the optical system,/->The second term to the right of the equation is represented, which is a set of sparse regularization terms, a priori constraints imposed on the optimization problem, +.>Is a regularization coefficient controlling the extent of both effects, +.>Is a norm.
Step 2: and solving the gradient.
For a slightly and continuously convex functionAnd a closed but not differentiable convex function +.>The optimization problem can be solved by a near-end gradient descent method, and a near-end gradient operator +.>Can be expressed as follows:
。
wherein L represents Lipschitz constant, which plays a role of step distance,representing vectors containing images in an iterative process, +. >Is indicated at->Another value in the neighborhood, ++>A hamiltonian is represented herein as a gradient operation. Similar to the operation of the soft contraction threshold, in +.>Regional, derivative is available->. At->Area, derivative available. At->Regional, derivative is available->。
In the process of solvingWhen it is, it can be decomposed into +.>. To interpret the convolution, define the component +.>Is the coordinates of (a)Component->Is +.>Then +.>Therefore, there are: />。
。
。
In the method, in the process of the invention,representing the imaging of the object to be sought by the optical system, < >>Indicate->The image is a gradient of a parameter carry-over function f, < >>Indicate->The image is a parameter->Gradient of->Indicate->Gradient of the sheet image->Indicate->Multi-focus excitation mode of a sheet of image, +.>Representing the excitation point spread function, +.>And->Respectively->Andsimplified expression of formula,/->And->Respectively->And->Is a component of (a).
Step 3: and (5) carrying out iterative solution.
Step 3.1:given an initial value +.>And bias initial value of background +.>Wherein->Can be directly set as +.>，/>Setting to zero initial value to obtain +.>，/>,/>。
Step 3.2: calculating forward propagation results:and calculates the forward propagation result +.>Is a gradient of (a). Wherein (1)>Indicate->Round iteration- >Result of pictures,/->Represent the firstBackground value of round iteration ∈>Representing the number of iterations.
Step 3.3: based on forward propagation resultsAnd imaging of the original image by the optical system +.>Calculating an objective function +.>A loss value is obtained. />
Step 3.4: iterative updating according to FISTA algorithmAnd->Intermediate variables in the iterative process, respectively.
Step 3.5: and (3) circularly performing the steps 3.2-3.4 until the iteration times are met, wherein the steps are as follows:。
based on the above description, the present invention has the following advantages over the prior art:
1) The high-power LED or the multimode laser in the imaging light source assembly can effectively remove speckle artifacts and provide a large illumination field. The exposure time can be shortened with sufficient power.
2) The multi-focus generation assembly uses a photolithographic array of pinholes, which provides greater flexibility in optimizing and adjusting the ratio of pinhole diameters to pitches, and which is also very inexpensive to produce. The efficiency is relatively high when using microlenses to create multiple focal points. Compared with the DMD which generates multiple focuses and provides scanning, the generation of the multiple focuses is decoupled from stepping, and the pinhole size is not limited by the stepping size under the condition that the stepping size meets the Nyquist sampling law, so that a larger imaging field of view can be realized and the scanning step size is reduced. Under the scanning of a small angle (0.2 degrees), the switching speed of the vibrating mirror can reach 6.7KHz, which is similar to the switching speed of the DMD.
3) The third lens 61 in the camera detection assembly uses a single lens that effectively increases the imaging field of view.
4) The change of the intervals of different pinhole arrays or microlens arrays in the multi-focus generation assembly can balance the imaging speed and the removal of defocusing signals, and is suitable for samples with different requirements.
5) Sparse multi-focus illumination generated by the multi-focus generation assembly is matched with an optical phase-locked detection technology, and a digital pinhole technology is combined, so that defocusing signals in depth imaging can be physically removed. The out-of-focus background is removed more cleanly than if the digital pinhole was used alone. Meanwhile, after the background of the original image is removed by the optical phase-locked detection technology, most of defocusing information is removed, and interference information except spot illumination can not interfere with the positioning of the center of the illumination bright point any more, so that the accurate extraction of the sub-image is facilitated (as shown in fig. 4).
6) By using the image scanning microscope principle, a lateral resolution of 110 nm can be achieved on a fixed actin filament sample, achieving a double increase in diffraction limit (as shown in fig. 5). Compared with wide-field confocal imaging scanning microscopy, the multi-point confocal imaging scanning microscopy obviously removes defocusing signal interference, the resolved structure is clearer, the lateral resolution and the axial resolution reach 131 nm and 336 nm respectively, and double three-dimensional resolution improvement is realized (as shown in fig. 6).
7) The principle of realizing imaging reconstruction based on a pixel redistribution algorithm has obviously reduced phototoxicity compared with other super-resolution imaging methods. Single-point scanning confocal is not sample friendly due to the extremely short pixel dwell time, so single-point power is relatively high. The turntable confocal super-resolution microscope has larger fluorescence signal attenuation and thus larger phototoxicity because the detection light path needs to pass through the micro lens array once again. Compared with a classical structure light illumination super-resolution microscope, the multi-point confocal image scanning microscope is equivalently overlapped into two wide fields, and the structure light illumination super-resolution microscope is equivalently overlapped into three wide fields, so that the multi-point confocal image scanning microscope has the lowest phototoxicity.
8) Multiple picturesThe deconvolution algorithm utilizes redundant information to achieve reduced frame reconstruction, reducing the number of original frames required for reconstruction, and thus improving imaging speed (as shown in fig. 7). jRL deconvolution synthesizes sample information contained in a plurality of images at different viewing angles to reconstruct a super-resolution image, and because the deconvolution step considers the poisson response of the area array detector to photons, jRL deconvolution can remove poisson noise in the image to a certain extent. The optimization model of the FISTA-GS deconvolution comprises a fidelity term and a sparse regularization term, and is formed by regularizing coefficients The influence of the two is weighed, compared with the general sparse constraint, the group sparsity applies the sparse constraint to the superimposed image of the dot matrix image, so that the possible loss of image information is avoided, and the reconstruction quality can be further improved. Compared to the wide field image, jRL deconvolution, the Fischer-GS deconvolution achieves a double resolution improvement.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to assist in understanding the methods of the present invention and the core ideas thereof; also, it is within the scope of the present invention to be modified by those of ordinary skill in the art in light of the present teachings. In view of the foregoing, this description should not be construed as limiting the invention.
Claims (8)
1. A multi-point confocal image scanning microscope, comprising: the signal control assembly, the imaging light source assembly, the light source collimation assembly, the multi-focus generation assembly, the multi-focus moving assembly, the multi-focus projection assembly and the camera detection assembly are arranged along the light path; wherein, a pinhole array is adopted, and small holes which are arranged in a set size are etched on chromeplated glass by utilizing a photoetching technology so as to form the multi-focus generating component; or forming the multi-focus generation assembly by adopting a micro lens array, a digital micro mirror device and a grating;
The imaging light source component is used for outputting a light source; the light source collimation assembly is used for carrying out collimation output on the light source output by the imaging light source assembly to obtain a collimation light spot; the multi-focus generation assembly is used for generating a single collimation light spot into a plurality of excitation focus illumination lights; the multi-focus moving component is used for transversely, longitudinally or obliquely translating the excitation focus illumination light; the multi-focus projection assembly is used for projecting the excitation focus illumination light to the sample surface, and simultaneously realizing multi-focus parallel excitation and scanning of the sample surface in cooperation with the multi-focus moving assembly; the camera detection component is used for detecting and outputting fluorescent signals generated by multi-focus parallel excitation of the sample surface;
the signal control component is respectively connected with the multi-focus moving component and the camera detection component; the signal control component is used for generating a control signal; the control signal is used for controlling the multi-focus moving assembly and the camera detecting assembly to synchronously work;
the multi-focus projection assembly includes a first lens, a second lens, a dichroic mirror, a tube lens, and a microscope objective disposed along an optical path;
after passing through the first lens, the excitation focus illumination light enters the second lens after being transversely, longitudinally or obliquely translated by the multi-focus moving assembly;
The tube lens, the microscope objective and the sample surface form a microscope host; the microscope host is used for realizing the nominal magnification of the microscope objective and clamping a sample;
the dichroic mirror is used for separating the excitation focus illumination light emitted by the second lens and fluorescent signals of the sample surface; the excitation focus illumination light emitted by the second lens is reflected into the tube lens; the fluorescent signal of the sample surface is transmitted into the camera detection assembly;
the camera detection assembly comprises a third lens and a camera which are arranged along the light path;
fluorescent signals of a sample surface are incident into the camera through the third lens; the camera is connected with the signal control component; the camera is used for detecting and outputting fluorescent signals generated by multi-focus parallel excitation of the sample surface.
2. The multi-point confocal image scanning microscope of claim 1, wherein the imaging light source assembly comprises a multi-channel switched light emitting diode; the light source emitted by the light emitting diode is output through a liquid optical waveguide, a single mode optical fiber or a multimode optical fiber with set size.
3. The multi-point confocal image scanning microscope of claim 1, wherein the light source collimation assembly is an aspheric lens, a 90 ° off-axis parabolic mirror, an adjustable focal length aspheric lens, an achromatic doublet, or an air-spaced doublet.
4. The multi-point confocal image scanning microscope of claim 1, wherein the multi-focal-point moving assembly is a two-dimensional galvanometer.
5. An imaging method using a multi-point confocal image scanning microscope, comprising:
acquiring a detection result by using the multi-point confocal image scanning microscope according to any one of claims 1-4, and taking the detection result as an original image;
based on the original image, adopting a pixel reassignment algorithm to realize imaging reconstruction; the signal is subjected to Fourier transformation pixel by pixel in the acquisition direction, and the zero frequency contains the sum of all information, namely the sum of a direct current component and all alternating current components, so that the background defocusing signal is removed by inhibiting the direct current component.
6. The imaging method using a multi-point confocal image scanning microscope according to claim 5, wherein the process of implementing imaging reconstruction using a pixel reassignment algorithm based on the original image comprises:
positioning pixel-level center coordinates based on a method for taking pixels with maximum neighborhood gray values to obtain a candidate center set;
intercepting a plurality of sub-images from the defocused image based on the candidate center set to form a sub-image stack;
Sub-pixel center positioning is carried out on the sub-image, and sub-pixel level coordinates of the sub-image center are obtained;
generating a two-dimensional Gaussian mask with the same size as the sub-image by taking the sub-pixel level coordinates as the center;
multiplying the two-dimensional gaussian mask with the sub-image as a digital pinhole;
and integrating the sub-images obtained by multiplication into canvas with the same size as the detection result according to the sub-pixel level coordinates or the pixel level center coordinates to obtain a confocal image.
7. An imaging method using a multi-point confocal image scanning microscope, comprising:
acquiring a detection result by using the multi-point confocal image scanning microscope according to any one of claims 1-4, and taking the detection result as an original image;
based on the original image, adopting a multi-image deconvolution algorithm to realize imaging reconstruction.
8. The imaging method using a multi-point confocal image scanning microscope according to claim 7, wherein the process of implementing imaging reconstruction using a multi-map deconvolution algorithm based on the original image comprises:
constructing an imaging model; the imaging model is as follows:
；
in the method, in the process of the invention,representing an array of pinholes->Representing airspace coordinates >Indicate acquisition +.>Relative position of illumination mode of picture +.>And->Representing the real object and the detected image, respectively, < >>And->Representing the excitation point spread function and the detection point spread function, respectively,/->Representing convolution operations +.>Representing a hadamard product operation;
completing the estimation of a pinhole array based on the imaging model;
and completing iterative solution of the imaging model based on the constructed imaging model and the estimation result of the pinhole array to obtain a reconstructed image.
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