CN108982452A - Multifocal spot scan three-D imaging method and system based on double helix point spread function - Google Patents

Abstract

The invention discloses based on double helix point spread function multifocal spot scan three-D imaging method and system, laser beam exposed in digital micromirror elements with predetermined angle；Digital micromirror elements are projected on sample surface after reflecting laser beam；The light illumination mode for switching digital micromirror elements at equal intervals excites the dot matrix for generating periodic arrangement on sample surface and moves as light illumination mode switches；To sample surface be excited generation fluorescence carry out phase-modulation, the fluorescence signal of Gaussian Profile is converted to the fluorescence signal of duplex form, by detector acquire duplex form fluorescence signal, obtain several width image datas；Double helix point all on every width picture is positioned according to acquired image data and is intercepted and obtains several subprovince domains, wavefront reconstruction processing is carried out to all subprovince domains, obtains sample Three-dimensional Gravity composition.Sample is excited simultaneously by multiple focus points, reduces sample acquisition time, greatly improves the temporal resolution of 3-D image scanning microscopic system.

Description

Multi-focus scanning three-dimensional imaging method and system based on double helix point spread function

technical field

The invention relates to the field of optical microscopy technology, in particular to a multi-focus scanning three-dimensional imaging method and system based on a double helix point spread function.

Background technique

Laser scanning confocal microscopy is an effective technical means to study biological microstructure, and it is widely used in the field of biomedicine. In the confocal microscope system, through a pair of conjugated precision pinholes and a single focal point razor scanning method, the system can suppress the stray light from the non-focus plane, filter out the information outside the focal plane, and obtain high image contrast. Although confocal microscopy can achieve super-resolution imaging, its resolution is affected by the size of the pinhole. The smaller the pinhole, the higher the resolution. However, the signal light that can be collected is weaker, which directly leads to the improvement of resolution Reduce the signal-to-noise ratio of the sample image. In recent years, in order to improve the resolution of the confocal microscope without reducing the signal-to-noise ratio, an image scanning microscope has been proposed, which replaces the photomultiplier tube in the traditional confocal microscope with a CCD. The signal is processed for data, and the double the resolution. When performing three-dimensional imaging on thick samples, the system will conduct tomographic scanning along the Z-axis direction, and splice the information of each layer through algorithms to obtain complete three-dimensional information of the sample.

Although the image scanning microscope has many advantages, it can improve the resolution and obtain a higher signal-to-noise ratio at the same time. However, due to the weak signal ability received by the CCD detector itself, the reading time is long, which leads to the failure of the image scanning microscope system. The imaging speed is slow, and its scanning It takes 60s for a large sample area, and it takes a lot of time to image the three-dimensional structure of the sample.

Thereby prior art still needs to improve and improve.

Contents of the invention

In view of the shortcomings of the above-mentioned prior art, the object of the present invention is to provide a multi-focus scanning three-dimensional imaging method and system based on double helix point spread function, which can simultaneously excite the sample through multiple focus points, improve the imaging range, reduce Sample acquisition time, and through phase modulation of the fluorescence emitted by the sample, the collected point spread function is converted into a double helix form, and then a single two-dimensional scan is achieved to obtain the three-dimensional information of the sample, which greatly improves the image scanning microscope. The time resolution of the system.

In order to achieve the above object, the present invention has taken the following technical solutions:

A kind of multi-focus scanning three-dimensional imaging method based on double helix point spread function, it comprises the steps:

The expanded and collimated laser beam is irradiated onto the digital micromirror element at a preset angle;

The digital micromirror element reflects the laser beam and projects it onto the sample surface;

Switching the illumination mode of the digital micromirror element at equal intervals, exciting the periodically arranged dot matrix on the sample surface and moving as the illumination mode is switched, until all the sample surface is excited to generate fluorescence;

Phase modulation is performed on the fluorescence generated by the excitation of the sample surface, and the fluorescence signal of Gaussian distribution is converted into a fluorescence signal in the form of a double helix. The fluorescence signal in the form of a double helix is collected by the detector every time the illumination mode is switched, and several frames image data;

According to the collected image data, all the double helix points on each picture are located and intercepted to obtain image data of several sub-regions, and the wavefront reconstruction processing is performed on all sub-regions to obtain the three-dimensional reconstruction map of the sample.

In the multi-focus scanning three-dimensional imaging method based on the double helix point spread function, the step of projecting the laser beam onto the sample surface after the digital micromirror element reflects specifically includes:

The laser beam enters the 4f system after being reflected by the digital micromirror element, and is projected onto the sample surface after filtering out the reflected light of redundant diffraction orders through the aperture set on the Fourier surface.

In the multi-focus scanning three-dimensional imaging method based on the double-helix point spread function, the phase modulation is performed on the fluorescence generated by the excitation of the sample surface, and the fluorescence signal of the Gaussian distribution is converted into a fluorescence signal in the form of a double helix, which is passed through the detector Collecting the fluorescent signal in the form of the double helix each time the illumination mode is switched, the steps of obtaining several pieces of image data include:

The fluorescence generated by the excitation of the sample surface is transmitted to the phase modulation unit;

The phase of the double-helix point spread function is loaded on the phase modulation unit, and the phase modulation is performed on the Gaussian distributed fluorescence signal, which is converted into a double-helix fluorescence signal;

The detector collects the fluorescence signal in the form of the double helix every time the illumination mode is switched to obtain several pieces of image data.

In the multi-focus scanning three-dimensional imaging method based on the double-helix point spread function, all the double-helix points on each picture are positioned and intercepted according to the collected image data, and several sub-region image data are obtained. The wavefront reconstruction process is carried out in the area, and the steps of obtaining the three-dimensional reconstruction map of the sample include:

According to the size of the collected image data, generate a zero matrix with a preset multiple size ;

Read the nth image in the collected image data ,right Position and intercept the double helix point in the image to obtain image data of several sub-regions , there is only one double helix point in each subregion image data;

Parallel processing of all sub-regional image data and obtaining sub-regional image data through double Gaussian fitting Coordinates of the two intensity peaks of the middle double helix point , , attach the numerical pinhole of the Gaussian distribution and calculate the rotation angle of the double helix ;

Deconvolve the double helix points after adding digital pinholes to convert them into Gaussian points, and locate the Gaussian points after deconvolution, and calculate their Coordinate position (x, y) on , copy the intensity distribution of the Gaussian point to (a*x, a*y) position, where a is the preset multiple;

Continue to read the n+1th picture in the image data and perform sub-region interception and data processing until all image data processing is completed, and will The image size is reduced to 1/a to obtain the three-dimensional information image of the sample, and according to the rotation angle of the double helix According to the corresponding relationship with the defocus distance of the sample, the depth of the sample at each scanning position is calculated, and the depth map of the sample is obtained by reconstruction.

In the multi-focus scanning three-dimensional imaging method based on the double helix point spread function, the step of attaching the digital pinholes with Gaussian distribution specifically includes:

According to the formula A numerical pinhole of a double Gaussian distribution is generated and multiplied by the intensity peak coordinates of the double helix point, where c is a constant.

In the multi-focus scanning three-dimensional imaging method based on the double helix point spread function, the preset multiple is twice.

In the multi-focus scanning three-dimensional imaging method based on double helical point spread function, the phase modulation unit is a phase plate or a spatial light modulator.

A multi-focus scanning three-dimensional imaging system based on a double helix point spread function, which includes sequentially arranged along the transmission direction of the optical path:

A laser light source for providing a continuous excitation beam;

The beam expansion collimation reflection module is used to expand and collimate the excitation beam, and reflect the expanded and collimated laser beam to make it exit at a preset angle;

The digital micromirror element is used to reflect the laser beam and project it onto the sample surface according to the imported lighting mode switched at equal intervals, to excite the periodically arranged dot matrix on the sample surface and move with the switching of the lighting mode;

The phase modulation acquisition module is used to phase modulate the fluorescence generated by the excitation of the sample surface, convert the fluorescence signal of Gaussian distribution into the fluorescence signal of the double helix form, and collect the fluorescence of the double helix form each time the illumination mode is switched signal to obtain several pieces of image data;

The control terminal is used to import the preset lighting mode into the digital micromirror element, and locate and intercept all the double helix points on each picture according to the collected image data, so as to obtain several sub-regional image data. All sub-regions are subjected to wavefront reconstruction processing to obtain a three-dimensional reconstruction map of the sample.

In the multi-focus scanning three-dimensional imaging system based on the double helix point spread function, the phase modulation acquisition module includes sequentially arranged along the transmission direction of the optical path:

The phase modulation unit is used to phase modulate the fluorescence generated by the excitation of the sample surface, and convert the fluorescence signal of the Gaussian distribution into the fluorescence signal of the double helix form;

The detector is used to collect the fluorescent signal in the form of the double helix each time the illumination mode is switched, and obtain several pieces of image data.

In the multi-focus scanning three-dimensional imaging system based on double helical point spread function, the phase modulation unit is a phase plate or a spatial light modulator.

Compared with the prior art, in the multi-focus scanning three-dimensional imaging method and system based on the double helix point spread function provided by the present invention, the laser beam after beam expansion and collimation is irradiated on the digital micromirror element at a preset angle; The digital micromirror element reflects the laser beam and projects it onto the sample surface; the illumination mode of the digital micromirror element is switched at equal intervals to excite the periodically arranged dot matrix on the sample surface and move with the illumination mode switch until The entire sample surface is excited to generate fluorescence; phase modulation is performed on the fluorescence generated by the excitation of the sample surface, and the fluorescence signal of Gaussian distribution is converted into a double helix fluorescence signal, and the double helix is collected by the detector every time the illumination mode is switched According to the collected image data, all the double helix points on each picture are located and intercepted, and several sub-region image data are obtained, and the wavefront reconstruction processing is performed on all sub-regions. , to obtain the three-dimensional reconstruction map of the sample. The sample can be excited at the same time through multiple focal points, which improves the imaging range and reduces the sample acquisition time, and through the phase modulation of the fluorescence emitted by the sample, the collected point spread function is converted into a double helix form, thereby achieving a single shot Two-dimensional scanning obtains three-dimensional information of the sample, which greatly improves the time resolution of the image scanning microscope system.

Description of drawings

FIG. 1 is a flow chart of the multi-focus scanning three-dimensional imaging method based on the double helix point spread function provided by the present invention.

Fig. 2a is a schematic diagram of the illumination mode in the multi-focus scanning three-dimensional imaging method based on the double helix point spread function provided by the present invention.

Fig. 2b is a diagram of the laser light intensity distribution generated on the sample in the multi-focus scanning three-dimensional imaging method based on the double helix point spread function provided by the present invention.

Fig. 2c is a phase-modulated intensity distribution diagram of the fluorescence signal generated by the excited sample in the multi-focus scanning three-dimensional imaging method based on the double helix point spread function provided by the present invention on the detector.

Fig. 3 is a comparison diagram of double helix point spread function and standard point spread function imaging at different depths.

FIG. 4 is a flow chart of image data processing in an application embodiment of a multi-focus scanning three-dimensional imaging method based on a double helix point spread function provided by the present invention.

Figure 5 (a) is a reconstruction of a kidney cell sample using the multi-focus scanning three-dimensional imaging method based on the double helix point spread function provided by the present invention.

Figure 5 (b) is the sample depth map corresponding to (a).

FIG. 6 is an optical path diagram of a multi-focus scanning three-dimensional imaging system based on a double helical point spread function provided by the present invention.

Detailed ways

In view of the shortcomings of the slow imaging speed of the three-dimensional image scanning phase system in the prior art, the purpose of the present invention is to provide a multi-focus scanning three-dimensional imaging method and system based on double helix point spread function, which can simultaneously excite the sample through multiple focus points, The imaging range is improved, the sample acquisition time is reduced, and by phase modulation of the fluorescence emitted by the sample, the collected point spread function is converted into a double helix form, thereby achieving a single two-dimensional scan to obtain the three-dimensional information of the sample, greatly Improved temporal resolution of image scanning microscopy systems.

In order to make the object, technical solution and effect of the present invention more clear and definite, the present invention will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

Please refer to Fig. 1, the multi-focus scanning three-dimensional imaging method based on the double helix point spread function provided by the present invention includes the following steps:

S10. The expanded and collimated laser beam is irradiated onto the digital micromirror element at a preset angle;

S20. The digital micromirror element reflects the laser beam and projects it onto the sample surface;

S30, switch the illumination mode of the digital micromirror element at equal intervals, excite the periodically arranged dot matrix on the sample surface and move as the illumination mode is switched, until all the sample surfaces are excited to generate fluorescence;

S40. Perform phase modulation on the fluorescence generated by the excitation of the sample surface, convert the fluorescence signal of Gaussian distribution into the fluorescence signal of the double helix form, collect the fluorescence signal of the double helix form through the detector every time the illumination mode is switched, and obtain Several pieces of image data;

S50. Locate and intercept all double helix points on each picture according to the collected image data, obtain image data of several sub-regions, perform wavefront reconstruction processing on all sub-regions, and obtain a three-dimensional reconstruction image of the sample.

The multi-focus scanning three-dimensional imaging method based on the double-helix point spread function provided by the present invention first expands and collimates the laser beam generated by the laser light source, and then irradiates it on the digital micromirror element at a preset angle. In this embodiment, The preset angle is preferably -24 degrees. Of course, other incident angles are also used in other embodiments. The present invention is not limited to this. Afterwards, the laser beam is reflected by the digital micromirror element and then projected onto the sample surface. , the digital micromirror element integrates a reflective micromirror array and a static random-access memory (Static Random-Access Memory, SRAM), and each pixel corresponds to a rotatable micromirror. By rotating the micromirror position to control the outgoing angle of the reflected light, so that each micro-mirror acts as an optical switch, so that the opening and closing of each micro-mirror can be controlled as required to control the bright and dark positions of the reflected light. Therefore, in the digital micro-mirror element After reflecting the laser beam, switch the lighting mode of the digital micromirror element at equal intervals. The switching schematic diagram of the lighting mode is shown in Figure 2a. By switching the lighting mode of the digital element, the laser beam is reflected by it to excite the sample surface. A periodic array of dots is generated on the surface and moves with the switching of the illumination mode until the entire sample surface is excited to generate fluorescence, as shown in Figure 2a, where the illumination mode is a binary image pre-imported into the memory of the digital micromirror element, each The pixel value corresponds to the on-off state of each micro-mirror. Through different illumination modes, a periodically arranged dot matrix can be generated on the sample surface and move according to the switching of the illumination mode. As shown in Figure 2b, when the entire sample surface is excited to generate fluorescence, the phase modulation of the fluorescence is first performed to convert it into a double-helix fluorescence signal, as shown in Figure 2c, and the detector is switched every time In the illumination mode, the fluorescent signals in the double helix form are collected synchronously to obtain several pieces of image data, and then all the collected image data are processed, and all the double helix points on each picture are positioned and determined according to the collected image data. Intercept to obtain several sub-regional image data, so all sub-regions are reconstructed by wavefront to obtain the three-dimensional reconstruction image of the sample, because the excited point spread function of the sample is converted into a double helix point spread function after phase modulation Form, as shown in Figure 3, its light intensity distribution is two opposite Gaussian points, as the defocus distance changes, the double helix point will rotate, and the rotation angle is proportional to the defocus distance, so the double helix point spread function can be Realize three-dimensional nano-positioning with extremely high positioning accuracy, and obtain a high-resolution three-dimensional reconstruction map of the sample. In this embodiment, the preset angle is preferably -24 degrees. Of course, other incident angles are used in other embodiments, which is not limited in the present invention.

Further, after the step S20, it also includes:

S21. The laser beam enters the 4f system after being reflected by the digital micromirror element, and is projected onto the sample surface after filtering out redundant diffraction-order reflected light through an aperture set on the Fourier surface.

In this embodiment, after the laser beam is reflected by the digital micromirror element, it enters the 4f system and is filtered by the aperture on the Fourier surface to filter out the excess diffraction order light, and then projected onto the sample surface, and the excess light is filtered out by the aperture. Diffraction-order light can effectively improve beam quality and improve system imaging resolution.

Specifically, the step S40 includes:

S401. The fluorescence generated by the excitation of the sample surface is transmitted to the phase modulation unit;

S402. Load the phase of the double helix point spread function into the phase modulation unit, perform phase modulation on the Gaussian distributed fluorescent signal, and convert it into a double helix fluorescent signal;

S403. The detector collects the fluorescent signal in the double helix form each time the illumination mode is switched, and obtains several pieces of image data.

In this embodiment, the fluorescence generated after the sample surface is excited by the laser beam is first transmitted to the phase modulation unit, and the phase modulation unit is loaded with the phase of the double helix point spread function. Specifically, the phase modulation unit is a phase plate or a spatial light modulation unit. When a spatial light modulator is used, the double-helical phase plate is imaged on the liquid crystal panel of the spatial light modulator, wherein the double-helical phase plate is a set of Laguerre-Gauss (Laguerre-Gauss, LG ) mode beams are coherently superimposed, and the excited point spread function of the sample becomes the form of double helix point spread function after phase modulation, so as to phase modulate the fluorescence signal of Gaussian distribution and convert it into double helix form of fluorescence After that, the detector collects the fluorescent signal in the double helix form each time the illumination mode is switched, and obtains several pieces of image data, that is, the digital micromirror element will send a trigger signal to the detector while switching the illumination mode, For example, when switching to a new mode, a colleague sends a rising edge signal with a voltage of 3V, and the detector performs acquisition synchronously after receiving the trigger signal, and obtains a series of image data I ₁ , I ₂ ... _In , and completes image data acquisition to for subsequent 3D reconstruction.

Further, after acquiring a series of image data, data processing is performed on it to obtain a three-dimensional reconstructed image of the sample, specifically the step S50 includes:

S501. Generate a zero matrix with a preset multiple size according to the size of the collected image data ;

S502. Read the nth image in the collected image data ,right Position and intercept the double helix point in the image to obtain image data of several sub-regions , there is only one double helix point in each subregion image data;

S503. Perform parallel processing on all sub-regional image data, and obtain sub-regional image data through double Gaussian fitting Coordinates of the two intensity peaks of the middle double helix point , , attach the numerical pinhole of the Gaussian distribution and calculate the rotation angle of the double helix ;

S504. Perform deconvolution processing on the double helix points after adding digital pinholes to convert them into Gauss points, and locate the Gauss points after deconvolution, and calculate their Coordinate position (x, y) on , copy the intensity distribution of the Gaussian point to (a*x, a*y) position, where a is the preset multiple;

S505. Continue to read the n+1th picture in the image data and perform sub-region interception and data processing until all image data processing is completed, and the The image size is reduced to 1/a to obtain the three-dimensional information image of the sample, and according to the rotation angle of the double helix According to the corresponding relationship with the defocus distance of the sample, the depth of the sample at each scanning position is calculated, and the depth map of the sample is obtained by reconstruction.

In this embodiment, first, according to the size of the collected image data, a zero matrix with a preset multiple size is generated , the preset multiple size is preferably twice, and the data processing process is described below with twice the preset multiple size, and the nth image in the collected image data is read ,right Position and intercept the double helix point in the image to obtain image data of several sub-regions , there is only one double-helix point in each sub-region image data, by intercepting the double-helix point and surrounding pixels from the image, obtaining several sub-region image data can avoid multiple double-helix points in the region, reducing The positioning error of the two peaks of the double helix; then all sub-regional image data are processed in parallel, and sub-regional image data are obtained by double Gaussian fitting Coordinates of the two intensity peaks of the middle double helix point , , attach the numerical pinhole of the Gaussian distribution and calculate the rotation angle of the double helix , specifically the digital pinhole attached to the Gaussian distribution is passed according to the formula Generate a double Gaussian distributed digital pinhole and multiply it with the intensity peak coordinates of the double helix point to achieve, where c is a constant, and the noise and spurious signals can be filtered out by attaching a Gaussian distributed digital pinhole to further improve the reconstruction Accuracy; then deconvolute the double helix points after the additional digital pinholes, specifically use the Richardson-Lucy deconvolution algorithm to deconvolute the double helix points after the pinholes to convert them into Gaussian points, And locate the Gaussian point after deconvolution, and calculate its position in Coordinate position (x, y) on , copy the intensity distribution of the Gaussian point to (2*x, 2*y) position; then repeat the above process, continue to read the n+1th picture in the image data and perform sub-region interception and data processing, until all image data processing is completed, will The image size is reduced to 1/2 to obtain the three-dimensional information image of the sample, and according to the rotation angle of the double helix The corresponding relationship with the sample defocus distance calculates the sample depth at each scanning position, and reconstructs the depth map of the sample. The present invention simultaneously excites the sample through multiple focus points, improves the imaging range, reduces the sample acquisition time, and Through the phase modulation of the fluorescence emitted by the sample, the collected point spread function is converted into a double helix form, and then a single two-dimensional scan is achieved to obtain the three-dimensional information of the sample, which greatly improves the time resolution of the image scanning microscope system .

Below in conjunction with Figure 4 and Figure 5, the data processing process and effect of the multi-focus scanning three-dimensional imaging method based on the double helix point spread function provided by the present invention are described in detail:

After the laser beam is reflected by the digital micromirror elements under different illumination modes, the sample is excited to generate a fluorescent signal, and the fluorescent signal is phase-modulated to obtain a series of image data in the form of a double helix, and the acquired image data is processed. To achieve multi-focus scanning three-dimensional imaging, the specific data processing steps include:

S1. Generate a zero matrix I whose size is twice the size of the collected data image.

S2. Read the nth image I _n in the collected image stack, and perform denoising processing.

In step S2, the way of denoising processing _is to subtract the background noise from the gray value in In, and the acquisition of the background noise is to use the detector to collect 10 pieces of image data in the absence of fluorescent signals, and convert their gray values to The sum of the degree values is averaged as the background noise of the system.

S3. Locate each double helix point on I _n , and intercept these points from the image I _n , image data of several sub-regions, and only one double helix point exists in each intercepted sub-region.

In step S3, the double helix point and the surrounding pixels are intercepted from the image. A reasonable sub-region size can avoid multiple double helix points in the region and reduce the positioning error of the two peaks of the double helix. In this embodiment The crop size is 25X25 pixels.

S4. Perform parallel processing on all sub-regions.

S5. Obtain the precise positions of the two peaks of the double helix through double Gaussian fitting, and attach digital pinholes of Gaussian distribution, and calculate the rotation angle θ _n .

In step S5, the method of double Gaussian fitting is as follows: first find the positions of the two maximum points in the sub-region as the rough coordinates of the two Gaussian points in the double Gaussian fitting clock; then use the rough coordinates as the least squares The initial value of the multiplication, and select an appropriate standard deviation range as the upper and lower bounds of the standard deviation according to the size of the point; then fit the two Gaussian points to obtain the precise coordinates and standard deviation of each Gaussian point in the double helix.

S6. Use the RL algorithm to deconvolve the double helix points after the pinhole is added, and turn them into ordinary Gaussian points. The point spread function used for deconvolution is the theoretical double helix point spread function, and its two peaks The distance is constant and the angle is θ _n .

In step S6, the 100nm fluorescent beads are imaged in wide field, and the emitted fluorescence is phase-modulated to obtain double helix points, and a single double helix point is measured, and the standard deviation σ of the Gaussian distribution of its single peak is obtained, The theoretical point spread function is two opposite Gaussian points, the midpoint of their connecting line is located in the exact center of the image, the peak value of the Gaussian point is set to 1, and the standard deviation is σ.

S7. Calculate the position coordinates (x, y) of the unrolled Gaussian point in I _n , and copy the intensity distribution of the Gaussian point to the position (2*x, 2*y) of _I0 .

S8. Reduce the image size of I ₀ to one half of the original size to obtain a super-resolution three-dimensional information image of the sample.

In steps S7 and S8, the essence is the reallocation of pixels, copying the intensity distribution of Gaussian points to (2*x, 2*y) of I ₀ , and then reducing the image size of I ₀ to half of the original - This method is equivalent to moving the fluorescent signal near the excitation point detected by the CCD to the excitation point for a distance, the distance is half of the pixel position of the detector where the signal is located and the position of the excitation point. This method can effectively improve horizontal resolution.

S9. Calculate the depth of the sample at each scanning position through the relationship between the double helix rotation angle and the defocus distance of the sample, and reconstruct the depth map of the sample,

In step S9, the relationship between the rotation angle of the double helix and the defocus distance of the sample can be obtained by continuously moving the 100 nm fluorescent bead sample in the Z direction through the shift stage and collecting it by the detector after phase modulation, so as to obtain the individual fluorescent beads at different positions on the Z axis. The rotation angle of the double helix point spread function, by linearly fitting the angle data of the point spread function with the corresponding axial position, obtains the corresponding relationship between the rotation angle of the double helix point spread function and the position on the axis, in order to realize a single second The three-dimensional information of the sample is obtained by three-dimensional scanning, as shown in Figure 5(a) and Figure 5(b), which is the reconstruction of the kidney cell sample by using the multi-focus scanning three-dimensional imaging method based on the double helix point spread function provided by the present invention The image and the depth map greatly improve the time resolution of the image scanning microscope system without reducing the image resolution and signal-to-noise ratio, effectively broadening the application range of the image scanning microscope system.

The present invention also correspondingly provides a multi-focus scanning three-dimensional imaging system based on double helical point spread function, as shown in Figure 6, which includes a laser light source 1, a beam expanding collimation reflection module 21, a digital micro Mirror element 5, phase modulation acquisition module 30 and control terminal 20, the laser light source 1 is used to provide a continuous excitation beam; the beam expansion collimation reflection module 21 is used to expand and collimate the excitation beam, and to The laser beam after beam expansion and collimation is reflected to make it exit at a preset angle; the digital micromirror element 5 is used to reflect the laser beam and then project it onto the sample surface according to the imported lighting mode switched at equal intervals to excite A periodically arranged dot matrix is generated on the sample surface and moves with the switching of the illumination mode; the phase modulation acquisition module 30 is used to phase modulate the fluorescence generated by the excitation of the sample surface, and convert the fluorescence signal of the Gaussian distribution into a double helix form Fluorescent signal, and collect the fluorescent signal in the double helix form each time the lighting mode is switched, and obtain several pieces of image data; the control terminal 20 is used to import the preset lighting mode into the digital micromirror element, and According to the collected image data, all the double helix points on each picture are located and intercepted to obtain image data of several sub-regions, and the wavefront reconstruction processing is performed on all sub-regions to obtain the three-dimensional reconstruction map of the sample. For details, please refer to the corresponding embodiments of the above methods.

Specifically, the phase modulation acquisition module includes a phase modulation unit 17 and a detector 19 arranged in sequence along the transmission direction of the optical path. The phase modulation unit 17 is used to phase modulate the fluorescence generated by the excitation of the sample surface, and convert the fluorescence signal of Gaussian distribution to converted into double-helix fluorescence signals; the detector 19 is used to collect the double-helix fluorescence signals each time the illumination mode is switched to obtain several pieces of image data. The phase modulation unit is a phase plate or a spatial light modulator. For details, please refer to the corresponding embodiments of the above method.

Specifically, the multi-focus scanning three-dimensional imaging system based on the double helical point spread function includes a laser light source 1, a first lens 2, a second lens 3, a first mirror 4, and a digital micromirror element 5 arranged in sequence along the optical path. , the third lens 6, the aperture 7, the fourth lens 8, the fifth lens 9, the dichroic plate 10, the objective lens 11, the sample 12, the tube lens 13, the linear polarizer 14, the sixth lens 15, the second mirror 16, The phase modulation unit 17 , the seventh lens 18 , the detector 19 and the control terminal 20 ; wherein the first lens 2 , the second lens 3 and the first mirror 4 form a beam expanding collimating reflection module 21 . Wherein the rear focal plane of the first lens 2 coincides with the front focal plane of the second lens 3, the digital micromirror element 5 is positioned at the position of the front focal plane of the third lens 6, and the diaphragm 7 is placed on the rear focal plane of the third lens 6 The position is used to block the reflected light of redundant diffraction orders, and the rear focal plane of the third lens 6 coincides with the front focal plane of the fourth lens 8 . The rear focal plane of the fourth lens 8 coincides with the front focal plane of the fifth lens 9, the front focal plane of the sixth lens 15 coincides with the position of the rear focal plane of the tube lens 13, and the phase modulation unit 17 is placed behind the sixth lens 15. The position of the focal plane, the position of the rear focal plane of the sixth lens 15 coincides with the position of the front focal plane of the seventh lens 18 . Wherein, the control terminal 20 is connected with the digital micromirror element 5 through a data line, and is used for importing the mode diagram to be displayed into the digital micromirror element 5, and the control terminal 20 is connected with the detector 19 through a data line, and is used for the detector The image collected by 19 is transmitted to the control terminal 20 , and the digital micromirror element 5 is connected to the detector 19 through a radio frequency line, which is used to transmit the external trigger signal of the digital micromirror element 5 to the detector 19 .

The laser light source 1 produces continuous laser light with a specific wavelength, which can be used to excite the sample to generate fluorescence, which is expanded and collimated after passing through the first lens 2 and the second lens 3, which can be changed by changing the first lens 2 and the second lens 3 The focal length is used to adjust the magnification of the beam expansion, and then, the laser light after the beam expansion and collimation is incident on the center of the digital micromirror element 5 display panel through the first reflector 4, and the incident angle is -24 degrees with the horizontal plane; the digital micromirror element The micro-mirror on 5 reflects the excitation light, and forms a sparse focusing lattice on the sample surface through the 4F system and the objective lens 11. After beam expansion, the laser beam is reflected by the digital micro-mirror element 5, and at the position of the back focal plane of the fourth lens 8 Form a plurality of focal points, then pass through the fifth lens 9 and the objective lens 11, form a sparse excitation dot matrix on the sample 12, the distribution of the dot matrix depends on the display mode loaded in the digital micromirror element 5 memory; The display mode of the mirror element 5, and send a trigger signal to the detector 19, the sparse focal points on the sample surface will be displaced with the mode change, and finally the entire sample will be fully illuminated, specifically through the control terminal 20. The sequence is imported into the memory of the digital micromirror device 5, and the switching interval of the mode can be set by the software of the digital micromirror device 5. A binary image with a mode of 1024X768, each pixel corresponds to a micromirror on the panel of the digital micromirror device, the pixel value 1 represents the 'on' state of the micromirror, and 0 represents the 'off' state of the micromirror; the sample sends The fluorescent light passes through the 4f system after the tube mirror 13 and is subjected to the phase modulation of the phase modulation unit, which is converted into a double helical form by the traditional point spread function; the detector 19 receives the trigger signal of the digital micromirror element 5 and simultaneously performs a process on the fluorescent signal Acquisition, the specific digital micromirror element 5 switches a new mode and sends a rising edge signal with a voltage of 3V at the same time, and the detector 19 starts to collect the fluorescent signal while receiving the signal, and the exposure time of the detector 19 is between two rising edge signals. interval to complete the acquisition of image data. For details, please refer to the corresponding embodiments of the above methods.

In summary, in the multi-focus scanning three-dimensional imaging method and system based on the double helix point spread function provided by the present invention, the laser beam after beam expansion and collimation is irradiated on the digital micromirror element at a preset angle; The micromirror element reflects the laser beam and projects it onto the sample surface; the illumination mode of the digital micromirror element is switched at equal intervals to excite the periodically arranged dot matrix on the sample surface and move with the illumination mode switch until the sample surface All are excited to generate fluorescence; phase modulation is performed on the fluorescence generated by the excitation of the sample surface, the fluorescence signal of Gaussian distribution is converted into a fluorescence signal in the form of a double helix, and the fluorescence signal in the form of a double helix is collected by the detector every time the illumination mode is switched Fluorescence signal, to obtain several pieces of image data; according to the collected image data, all the double helix points on each picture are located and intercepted to obtain several sub-regional image data, and the wavefront reconstruction processing is performed on all sub-regions to obtain 3D reconstruction of the sample. The sample can be excited at the same time through multiple focal points, which improves the imaging range and reduces the sample acquisition time, and through the phase modulation of the fluorescence emitted by the sample, the collected point spread function is converted into a double helix form, thereby achieving a single shot Two-dimensional scanning obtains three-dimensional information of the sample, which greatly improves the time resolution of the image scanning microscope system.

It can be understood that those skilled in the art can make equivalent replacements or changes according to the technical solutions and inventive concepts of the present invention, and all these changes or replacements should belong to the protection scope of the appended claims of the present invention.

Claims (10)

1. a kind of multifocal spot scan three-D imaging method based on double helix point spread function, which is characterized in that including walking as follows It is rapid:

Laser beam after beam-expanding collimation is exposed in digital micromirror elements with predetermined angle；

The digital micromirror elements are projected on sample surface after reflecting laser beam；

Switch the light illumination mode of the digital micromirror elements at equal intervals, excite the dot matrix that periodic arrangement is generated on sample surface and with Light illumination mode switching movement, generate fluorescence until sample surface is all excited；

To sample surface be excited generation fluorescence carry out phase-modulation, the fluorescence signal of Gaussian Profile is converted into duplex form Fluorescence signal, the fluorescence signal of the duplex form is acquired in switching light illumination mode every time by detector, if obtaining Dry width image data；

Double helix point all on every width picture is positioned and intercepted according to acquired image data, obtains several Asias Region image data carries out wavefront reconstruction processing to all subprovince domains, obtains the Three-dimensional Gravity composition of sample.

2. the multifocal spot scan three-D imaging method according to claim 1 based on double helix point spread function, feature It is, the step that the digital micromirror elements are projected on sample surface after reflecting laser beam specifically includes:

Laser beam enters 4f system after digital micromirror elements reflect, and is filtered out by the diaphragm that is arranged in Fourier plane more It is projected on sample surface after the reflected light of the remaining order of diffraction.

3. the multifocal spot scan three-D imaging method according to claim 1 based on double helix point spread function, feature It is, it is described that phase-modulation is carried out to the be excited fluorescence of generation of sample surface, the fluorescence signal of Gaussian Profile is converted into double spiral shells The fluorescence signal of rotation form acquires the fluorescence signal of the duplex form by detector in switching light illumination mode every time, The step of obtaining several width image datas include:

The be excited fluorescence of generation of sample surface is transmitted to phase modulation unit；

It is loaded into double helix point spread function phase in phase modulation unit, phase tune is carried out to the fluorescence signal of Gaussian Profile System, is converted into the fluorescence signal of duplex form；

The fluorescence signal for acquiring the duplex form in switching light illumination mode every time by detector, obtains several width images Data.

4. the multifocal spot scan three-D imaging method according to claim 1 based on double helix point spread function, feature It is, double helix point all on every width picture is positioned and intercepted according to acquired image data, obtains several Sub- region image data carries out wavefront reconstruction processing to all subprovince domains, and the step of obtaining the Three-dimensional Gravity composition of sample includes:

According to acquired image data size, the null matrix of a presupposition multiple size is generated；

Read the n-th width figure in acquired image data, rightIn double helix point positioned and intercepted, obtain it is several A Asia region image data, it is each Asia region image data in there is only a double helix points；

Parallel processing is carried out to all sub- region image datas, is fitted by double gauss and obtains sub- region image dataIn double spiral shells Revolve two intensity peak coordinates of point,, enclose the digital pin hole of Gaussian Profile and calculate double helix Rotation angle；

To after additional character pin hole double helix click through deconvolution processing, so that it is converted to Gauss point, and to deconvolution after Gauss point is positioned, calculate itsOn coordinate position (x, y), the intensity distribution of Gauss point is copied to(a*x, A*y) on position, wherein a is presupposition multiple；

Continue the (n+1)th width figure read in image data and carry out the interception of subprovince domain and data processing, until all picture numbers It is completed according to processing, it willPicture size be reduced into 1/a and obtain the three-dimensional information image of sample, and according to the rotation angle of double helix DegreeWith the corresponding relationship of sample defocus distance, the sample depth of each scan position is calculated, reconstruct obtains the depth of sample Figure.

5. the multifocal spot scan three-D imaging method according to claim 4 based on double helix point spread function, feature It is, the step of digital pin hole for enclosing Gaussian Profile specifically includes:

According to formulaGenerate double gauss distribution digital pin hole and with it is double The intensity peak coordinate of spiral points is multiplied, and wherein c is constant.

6. the multifocal spot scan three-D imaging method according to claim 4 based on double helix point spread function, feature It is, the presupposition multiple is twice.

7. the multifocal spot scan three-D imaging method according to claim 3 based on double helix point spread function, feature It is, the phase modulation unit is phase-plate or spatial light modulator.

8. a kind of multifocal spot scan 3-D imaging system based on double helix point spread function, which is characterized in that including along optical path What transmission direction was set gradually:

Laser light source, for providing continuous excitation beam；

Beam-expanding collimation reflecting module, for being expanded and being collimated to excitation beam, and to the laser beam after beam-expanding collimation Carrying out reflection is emitted its predetermined angle；

Digital micromirror elements project after reflecting laser beam for the light illumination mode switched at equal intervals according to importing To sample surface, excites the dot matrix for generating periodic arrangement on sample surface and moved as light illumination mode switches；

Phase-modulation acquisition module, for sample surface be excited generation fluorescence carry out phase-modulation, by the glimmering of Gaussian Profile Optical signal is converted to the fluorescence signal of duplex form, and acquires the duplex form in switching light illumination mode every time Fluorescence signal obtains several width image datas；

Controlling terminal, for default light illumination mode to be directed into the digital micromirror elements, and according to acquired image number It is positioned and is intercepted according to double helix point all on every width picture, several sub- region image datas are obtained, to all Asias Region carries out wavefront reconstruction processing, obtains the Three-dimensional Gravity composition of sample.

9. the multifocal spot scan 3-D imaging system according to claim 8 based on double helix point spread function, feature It is, the phase-modulation acquisition module includes setting gradually along optic path direction:

Phase modulation unit believes the fluorescence of Gaussian Profile for carrying out phase-modulation to the be excited fluorescence of generation of sample surface Number be converted to the fluorescence signal of duplex form；

Detector obtains several width figures for acquiring the fluorescence signal of the duplex form in switching light illumination mode every time As data.

10. the multifocal spot scan 3-D imaging system according to claim 9 based on double helix point spread function, feature It is, the phase modulation unit is phase-plate or spatial light modulator.