CN108982452B - Multi-focus scanning three-dimensional imaging method and system based on double-spiral point spread function - Google Patents
Multi-focus scanning three-dimensional imaging method and system based on double-spiral point spread function Download PDFInfo
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Abstract
The invention discloses a multi-focus scanning three-dimensional imaging method and a system based on a double spiral point diffusion function, wherein laser beams irradiate a digital micromirror element at a preset angle; the digital micro-mirror element reflects the laser beam and then projects the laser beam onto the sample surface; switching the illumination mode of the digital micromirror element at equal intervals, exciting a periodically arranged dot matrix generated on the sample surface and moving along with the switching of the illumination mode; performing phase modulation on fluorescence generated by exciting a sample surface, converting a fluorescence signal with Gaussian distribution into a fluorescence signal in a double helix form, and acquiring the fluorescence signal in the double helix form by a detector to obtain a plurality of image data; and positioning all double helix points on each picture according to the acquired image data, intercepting to obtain a plurality of sub-regions, and performing wavefront reconstruction processing on all the sub-regions to obtain a three-dimensional reconstruction image of the sample. The samples are simultaneously excited through a plurality of focus points, the sample collection time is reduced, and the time resolution of the three-dimensional image scanning microscope system is greatly improved.
Description
Technical Field
The invention relates to the technical field of optical microscopy, in particular to a multi-focus scanning three-dimensional imaging method and system based on a double-spiral point spread function.
Background
LaserScanning confocal microscopes are an effective technical means for studying biological fine structures and are widely used in the biomedical field. In a confocal microscope system, a razor scanning mode of a pair of conjugate precise pinholes and a single focus point is adopted, so that the system can inhibit stray light from a non-focus plane, filter information out of the focus plane and obtain high image contrast. Although the confocal microscopy can realize super-resolution imaging, the resolution is influenced by the size of a pinhole, the smaller the pinhole is, the higher the resolution is, but the weaker the signal light which can be collected is, the resolution is directly improved, and the signal-to-noise ratio of a sample image is reduced. In recent years, in order to improve the resolution of a confocal microscope without reducing the signal-to-noise ratio, an image scanning microscope has been proposed in which a photomultiplier tube in a conventional confocal microscope is replaced with a CCD, and data processing is performed on the acquired signals to obtain a scanning image
The resolution is increased by times. When the thick sample is subjected to three-dimensional imaging, the system carries out tomography along the Z-axis direction, and information of each layer is spliced through an algorithm to obtain complete three-dimensional information of the sample.
Although the image scanning microscope has many advantages, the resolution can be improved and the signal-to-noise ratio is higher, but the CCD detector has weaker signal receiving capability and longer reading time, so the imaging speed of the image scanning microscope system is slower, and the scanning is carried out
The sample area of size takes 60s and, if the three-dimensional structure of the sample is to be imaged, a lot of time.
Thus, the prior art has yet to be improved and enhanced.
Disclosure of Invention
In view of the defects of the prior art, the present invention aims to provide a multi-focus scanning three-dimensional imaging method and system based on a double-spiral point spread function, which can excite a sample through a plurality of focus points simultaneously, improve the imaging range, reduce the sample acquisition time, and convert the acquired point spread function into a double-spiral form by performing phase modulation on fluorescence emitted by the sample, thereby realizing single two-dimensional scanning to obtain three-dimensional information of the sample, and greatly improving the time resolution of an image scanning microscope system.
In order to achieve the purpose, the invention adopts the following technical scheme:
a multi-focus scanning three-dimensional imaging method based on a double-spiral point spread function comprises the following steps:
irradiating the laser beam after beam expansion and collimation onto a digital micromirror element at a preset angle;
the digital micro-mirror element reflects the laser beam and then projects the laser beam onto the sample surface;
switching the illumination mode of the digital micromirror element at equal intervals, exciting a periodically arranged dot matrix on the sample surface and moving along with the switching of the illumination mode until the sample surface is completely excited to generate fluorescence;
carrying out phase modulation on fluorescence generated by exciting a sample surface, converting a fluorescence signal with Gaussian distribution into a fluorescence signal in a double-spiral form, and acquiring the fluorescence signal in the double-spiral form by a detector when an illumination mode is switched every time to obtain a plurality of image data;
and positioning and intercepting all double spiral points on each picture according to the acquired image data to obtain a plurality of sub-region image data, and performing wavefront reconstruction processing on all sub-regions to obtain a three-dimensional reconstruction image 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 laser beam is reflected by the digital micro-mirror element specifically comprises the following steps:
the laser beam enters a 4f system after being reflected by a digital micro-mirror element, and is projected onto a sample surface after reflected light of an unnecessary diffraction order is filtered by a diaphragm arranged on a 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 fluorescence generated by exciting a sample surface, fluorescence signals with Gaussian distribution are converted into fluorescence signals in a double-helix form, the fluorescence signals in the double-helix form are collected by a detector when an illumination mode is switched every time, and the step of obtaining a plurality of image data comprises the following steps:
fluorescence generated by exciting the sample surface is transmitted to the phase modulation unit;
loading a double helix point spread function phase on a phase modulation unit, carrying out phase modulation on the fluorescence signals with Gaussian distribution, and converting the fluorescence signals into double helix fluorescence signals;
and acquiring the fluorescent signal in the double-helix form by a detector when the illumination mode is switched every time to obtain a plurality of image data.
In the multi-focus scanning three-dimensional imaging method based on the double-spiral point spread function, all double-spiral points on each picture are positioned and intercepted according to the collected image data to obtain a plurality of sub-region image data, and the steps of performing wavefront reconstruction processing on all sub-regions to obtain the three-dimensional reconstruction graph of the sample comprise:
generating a zero matrix with preset multiple size according to the size of the collected image data
；
Reading the nth image in the acquired image data
To, for
Positioning and intercepting double spiral points in the image acquisition system to obtain a plurality of sub-region image data
Only one double helix point exists in each sub-region image data;
parallel processing of all sub-region image dataObtaining sub-region image data by double-Gaussian fitting
Two intensity peak coordinates of middle double helix point
，
Attaching Gaussian distributed digital pinholes and calculating the rotation angle of the double helix
；
Deconvoluting the double helix point after the addition of the digital pinhole to convert the double helix point into a Gaussian point, positioning the Gaussian point after deconvolution, and calculating the position of the Gaussian point
Coordinate position (x, y) of the point, copying the intensity distribution of the gaussian point to
At the (a x, a y) position of (a), wherein a is a predetermined multiple;
continuously reading the (n + 1) th image in the image data, performing sub-region interception and data processing until all the image data are processed, and performing image processing on the image data
The size of the image is reduced to 1/a to obtain a three-dimensional information image of the sample, and the three-dimensional information image is obtained according to the rotation angle of the double helix
And calculating the sample depth of each scanning position according to the corresponding relation with the defocusing distance of the sample, and reconstructing to obtain a depth map of the sample.
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
Generating double-Gaussian distributed digital pinholes and multiplying the digital pinholes by the intensity peak coordinates of the double-helix points, wherein c is a constant.
In the multi-focus scanning three-dimensional imaging method based on the double-spiral point spread function, the preset multiple is two times.
In the multi-focus scanning three-dimensional imaging method based on the double spiral 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-spiral point spread function comprises the following components which are sequentially arranged along the transmission direction of an optical path:
a laser light source for providing a continuous beam of excitation light;
the beam expanding collimation reflection module is used for expanding and collimating the excitation beam and reflecting the laser beam subjected to beam expanding and collimation to enable the laser beam to be emitted at a preset angle;
the digital micro-mirror element is used for reflecting the laser beam and then projecting the laser beam onto the sample surface according to the introduced lighting mode switched at equal intervals, and exciting a periodically arranged dot matrix generated on the sample surface and moving along with the switching of the lighting mode;
the phase modulation acquisition module is used for carrying out phase modulation on fluorescence generated by exciting a sample surface, converting fluorescence signals with Gaussian distribution into fluorescence signals in a double-helix form, and acquiring the fluorescence signals in the double-helix form when an illumination mode is switched every time to obtain a plurality of image data;
and the control terminal is used for guiding a preset illumination mode into the digital micromirror element, positioning and intercepting all double helix points on each picture according to the acquired image data to obtain a plurality of sub-region image data, and performing wavefront reconstruction processing on all sub-regions to obtain a three-dimensional reconstruction image 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 comprises:
the phase modulation unit is used for carrying out phase modulation on fluorescence generated by exciting the sample surface and converting a fluorescence signal with Gaussian distribution into a fluorescence signal in a double helix form;
and the detector is used for collecting the double-helix fluorescence signal when the illumination mode is switched every time to obtain a plurality of image data.
In the multi-focus scanning three-dimensional imaging system based on the double spiral 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-spiral point diffusion function, the laser beam after beam expanding and collimating irradiates the digital micromirror element at a preset angle; the digital micro-mirror element reflects the laser beam and then projects the laser beam onto the sample surface; switching the illumination mode of the digital micromirror element at equal intervals, exciting a periodically arranged dot matrix on the sample surface and moving along with the switching of the illumination mode until the sample surface is completely excited to generate fluorescence; carrying out phase modulation on fluorescence generated by exciting a sample surface, converting a fluorescence signal with Gaussian distribution into a fluorescence signal in a double-spiral form, and acquiring the fluorescence signal in the double-spiral form by a detector when an illumination mode is switched every time to obtain a plurality of image data; and positioning and intercepting all double spiral points on each picture according to the acquired image data to obtain a plurality of sub-region image data, and performing wavefront reconstruction processing on all sub-regions to obtain a three-dimensional reconstruction image of the sample. The sample can be excited by a plurality of focusing points simultaneously, the imaging range is improved, the sample collecting time is reduced, the phase modulation is carried out on the fluorescence emitted by the sample, the collected point spread function is converted into a double-helix form, the three-dimensional information of the sample is obtained by single two-dimensional scanning, and the time resolution of the image scanning microscope system is greatly improved.
Drawings
Fig. 1 is a flowchart of a multi-focus scanning three-dimensional imaging method based on a double-helix point spread function provided by the invention.
Fig. 2a is a schematic diagram of an illumination mode in the multi-focus scanning three-dimensional imaging method based on the double helix point spread function provided by the invention.
Fig. 2b is a laser intensity distribution diagram generated on a sample in the multi-focus scanning three-dimensional imaging method based on the double helix point spread function provided by the invention.
Fig. 2c is an intensity distribution diagram of a fluorescence signal generated by exciting a sample on a detector after phase modulation in the multi-focus scanning three-dimensional imaging method based on the double helix point spread function provided by the invention.
FIG. 3 is a graph comparing dual spiral point spread function and standard point spread function imaging at different depths.
Fig. 4 is a flowchart of image data processing in an application embodiment of the multi-focus scanning three-dimensional imaging method based on the double spiral point spread function provided by the invention.
Fig. 5 (a) is a diagram of a renal cell sample reconstructed by using the multi-focus scanning three-dimensional imaging method based on the double spiral point spread function provided by the invention.
FIG. 5 (b) is a depth map of the sample corresponding to the map (a).
FIG. 6 is a light path diagram of a multi-focus scanning three-dimensional imaging system based on a double spiral point spread function provided by the invention.
Detailed Description
In view of the defects of slow imaging speed of a three-dimensional image scanning phase system and the like in the prior art, the invention aims to provide a multi-focus scanning three-dimensional imaging method and system based on a double-spiral point spread function.
In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Referring to fig. 1, the multi-focus scanning three-dimensional imaging method based on the double spiral point spread function provided by the present invention includes the following steps:
s10, irradiating the laser beam after beam expansion and collimation onto a digital micro-mirror element at a preset angle;
s20, the digital micro-mirror element reflects the laser beam and then projects the laser beam onto the sample surface;
s30, switching the illumination mode of the digital micro-mirror element at equal intervals, exciting the periodically arranged dot matrix on the sample surface and moving along with the switching of the illumination mode until the sample surface is completely excited to generate fluorescence;
s40, carrying out phase modulation on fluorescence generated by exciting the sample surface, converting the fluorescence signals with Gaussian distribution into fluorescence signals in a double-helix form, and acquiring the fluorescence signals in the double-helix form by a detector when an illumination mode is switched every time to obtain a plurality of image data;
s50, positioning and intercepting all double spiral points on each picture according to the acquired image data to obtain a plurality of sub-region image data, and performing wavefront reconstruction processing on all sub-regions to obtain a three-dimensional reconstruction image of the sample.
The invention provides a multi-focus scanning three-dimensional imaging method based on a double helix point diffusion function, which comprises the steps of firstly expanding and collimating laser beams generated by a laser source and then irradiating the laser beams onto a digital micro-mirror element at a preset angle, wherein the preset angle is preferably-24 degrees, although other incident angles are adopted in other embodiments, the method is not limited by the invention, then reflecting the laser beams by the digital micro-mirror element and then projecting the laser beams onto a sample surface, integrating a reflective micro-mirror array and a Static Random-Access Memory (SRAM) by the digital micro-mirror element, corresponding to each pixel to a rotatable micro-mirror, controlling the emergent angle of the reflected light by rotating the position of the micro-mirror, enabling each micro-mirror to be used as an optical switch, and further controlling the on and off of each micro-mirror according to the requirement to further control the bright and dark position of the reflected light, therefore, after the digital micromirror element reflects the laser beam, the illumination mode of the digital micromirror element is switched at equal intervals, the switching schematic diagram of the illumination mode is shown as fig. 2a, the illumination mode of the digital element is switched, so that the laser beam is reflected by the illumination mode to excite a periodically arranged dot matrix on the sample surface and move along with the switching of the illumination mode until the sample surface is completely excited to generate fluorescence, as shown in fig. 2a, wherein the illumination mode is a binary image which is pre-led into the memory of the digital micromirror element, each pixel value corresponds to the switching state of each micromirror, and the periodically arranged dot matrix can be generated on the sample surface through different illumination modes and move according to the switching of the illumination mode. As shown in fig. 2b, after the sample surface is completely excited to generate fluorescence, firstly, the fluorescence is subjected to phase modulation, and is converted into a fluorescence signal in a double-spiral form, as shown in fig. 2c, the fluorescence signal in the double-spiral form is synchronously acquired by a detector when the illumination mode is switched each time, a plurality of image data are obtained, then, all the acquired image data are processed, all double-spiral points on each picture are positioned and intercepted according to the acquired image data, a plurality of sub-region image data are obtained, therefore, wave front reconstruction processing is performed on all the sub-regions, a three-dimensional reconstruction image of the sample is obtained, as the point spread function excited by the sample is converted into a double-spiral point spread function form after phase modulation, as shown in fig. 3, the light intensity distribution is two opposite gaussian points, and the double-spiral point rotates along with the change of the defocus distance, the rotation angle is in direct proportion to the defocusing distance, so that three-dimensional nanometer positioning can be realized through a double-helix point diffusion function, the positioning precision is extremely high, and a high-resolution sample three-dimensional reconstruction image is obtained. In this embodiment, the predetermined angle is preferably-24 degrees, although other incident angles may be used in other embodiments, which is not limited by the present invention.
Further, the step S20 is followed by:
and S21, the laser beam enters a 4f system after being reflected by the digital micro-mirror element, and is projected onto the sample surface after the reflected light of the redundant diffraction orders is filtered by a diaphragm arranged on the Fourier surface.
In the embodiment, after being reflected by the digital micro-mirror element, the laser beam enters the 4f system, light of redundant diffraction orders is filtered by the diaphragm on the Fourier surface, and then the laser beam is projected onto the sample surface, and the light of the redundant diffraction orders is filtered by the diaphragm, so that the beam quality can be effectively improved, and the imaging resolution of the system is improved.
Specifically, the step S40 includes:
s401, transmitting fluorescence generated by exciting the sample surface to a phase modulation unit;
s402, loading a double-helix point spread function phase on a phase modulation unit, carrying out phase modulation on the fluorescence signals with Gaussian distribution, and converting the fluorescence signals into double-helix fluorescence signals;
and S403, acquiring the double-helix fluorescence signal through a detector when the illumination mode is switched every time, and acquiring a plurality of image data.
In this embodiment, the fluorescence generated after the sample surface is excited by the laser beam is transmitted to the phase modulation unit, the phase modulation unit is loaded with a double helix point spread function phase, specifically, the phase modulation unit is a phase plate or a spatial light modulator, when the spatial light modulator is adopted, a double helix phase plate is imaged on a liquid crystal panel of the spatial light modulator, wherein the double helix phase plate is formed by coherent superposition of a group of Laguerre-Gauss (LG) mode light beams satisfying a specific rule, the point spread function excited by the sample is changed into a double helix point spread function form after phase modulation, so that the fluorescence signal with gaussian distribution is subjected to phase modulation and converted into a double helix fluorescence signal, and then the double helix fluorescence signal is collected by the detector when the illumination mode is switched each time, obtaining a plurality of image data, i.e. the digital micromirror element sends a trigger signal to the detector while switching the illumination mode, e.g. a rising edge signal with a voltage of 3V at the colleague switching the new mode, and the detection is performedThe device synchronously acquires after receiving the trigger signal to obtain a series of image data I1，I2…InImage data acquisition is completed for subsequent three-dimensional reconstruction.
Further, after acquiring a series of image data, performing data processing on the acquired image data to obtain a three-dimensional reconstruction map of the sample, specifically, step S50 includes:
s501, generating a zero matrix with preset multiple size according to the size of the collected image data
；
S502, reading the nth picture in the collected image data
To, for
Positioning and intercepting double spiral points in the image acquisition system to obtain a plurality of sub-region image data
Only one double helix point exists in each sub-region image data;
s503, performing parallel processing on all the sub-region image data, and obtaining the sub-region image data through double-Gaussian fitting
Two intensity peak coordinates of middle double helix point
，
Attaching Gaussian distributed digital pinholes and calculating the rotation angle of the double helix
；
S504, performing deconvolution processing on the double-spiral point with the digital pinhole, converting the double-spiral point into a Gaussian point, positioning the Gaussian point after deconvolution, and calculating the position of the Gaussian point
Coordinate position (x, y) of the point, copying the intensity distribution of the gaussian point to
At the (a x, a y) position of (a), wherein a is a predetermined multiple;
s505, continuously reading the (n + 1) th image in the image data, performing sub-region interception and data processing until all the image data are processed, and performing image processing on the image data
The size of the image is reduced to 1/a to obtain a three-dimensional information image of the sample, and the three-dimensional information image is obtained according to the rotation angle of the double helix
And calculating the sample depth of each scanning position according to the corresponding relation with the defocusing distance of the sample, and reconstructing to obtain a depth map of the sample.
In this embodiment, a zero matrix with a preset multiple size is generated according to the size of the acquired image data
The preset multiple size is preferably two times, the data processing process is explained by the double preset multiple size in the following, and the nth graph in the acquired image data is read
To, for
Positioning and intercepting double spiral points in the image acquisition system to obtain a plurality of sub-region image data
Only one double-spiral point exists in each sub-region image data, and a plurality of sub-region image data can be obtained by intercepting the double-spiral points and surrounding pixels from the image, so that a plurality of double-spiral points in the region can be avoided, and the positioning error of two peaks of double spiral is reduced; then, all the sub-region image data are processed in parallel, and the sub-region image data are obtained through double-Gaussian fitting
Two intensity peak coordinates of middle double helix point
，
Attaching Gaussian distributed digital pinholes and calculating the rotation angle of the double helix
Specifically, the Gaussian distribution attached digital pinhole is obtained by using a formula
Generating double-Gaussian-distribution digital pinholes, and multiplying the double-Gaussian-distribution digital pinholes by the intensity peak coordinates of double-helix points, wherein c is a constant, noise and stray signals can be filtered by attaching the Gaussian-distribution digital pinholes, and the accuracy of reconstruction is further improved; and then carrying out deconvolution processing on the double helix points after the addition of the digital pinhole, specifically carrying out deconvolution operation on the double helix points after the pinhole by using a Richardson-Lucy deconvolution algorithm to convert the double helix points into Gaussian points, positioning the Gaussian points after the deconvolution, and calculating the positions of the Gaussian points after the deconvolution
Coordinate position (x, y) of the point, copying the intensity distribution of the gaussian point to
At the (2 x, 2 y) position of (a); then repeating the above process, continuously reading the (n + 1) th image in the image data, performing sub-region interception and data processing until all the image data are processed, and performing image processing
Is reduced to 1/2 to obtain a three-dimensional information image of the sample, and is based on the rotation angle of the double helix
The invention simultaneously excites the sample through a plurality of focus points, improves the imaging range, reduces the sample acquisition time, and converts the acquired point spread function into a double helix form by carrying out phase modulation on fluorescence emitted by the sample, thereby realizing single two-dimensional scanning to obtain the three-dimensional information of the sample and greatly improving the time resolution of an image scanning microscope system.
The following describes in detail the data processing procedure and effect of the multi-focus scanning three-dimensional imaging method based on the double helix point spread function, with reference to fig. 4 and 5:
after laser beams are reflected by digital micro-mirror elements in different illumination modes, a sample is excited to generate fluorescence signals, the fluorescence signals are subjected to phase modulation to obtain a series of double-spiral image data, the obtained image data are processed to realize multi-focus scanning three-dimensional imaging, and the specific data processing steps comprise:
and S1, generating a zero matrix I with the size being twice of the size of the acquired data image.
S2, reading the nth picture I in the collected image stacknAnd denoising.
In step S2, the denoising process is performed by using InThe background noise is obtained by acquiring 10 image data by using a detector under the condition of no fluorescence signal, and the gray value of the image data is subtracted from the gray value of the background noiseThe sum is averaged as the background noise of the system.
S3, pair InLocate each double helix point and remove these points from image InAnd (4) intercepting a plurality of sub-region image data, wherein each intercepted sub-region only has one double spiral point.
In step S3, the double-helix points and the surrounding pixels are extracted from the image, and the reasonable size of the sub-region can avoid multiple double-helix points appearing in the region, thereby reducing the positioning error of two peaks of the double-helix, in this embodiment, the extracted size is 25X25 pixels.
And S4, performing parallel processing on all the subregions.
S5, obtaining the accurate positions of two peaks of the double helix by double-Gaussian fitting, attaching Gaussian distributed digital pinholes, and calculating the rotation angle thetan。
In step S5, the double-gaussian fitting method includes: firstly, finding out the positions of two maximum value points in a subregion as rough coordinates of two Gaussian points of a double Gaussian fitting clock; then, using the rough coordinates as an initial value of a least square method, and selecting a proper standard deviation range as an upper and lower definite limits of the standard deviation according to the size of the point; and then fitting the two Gaussian points to obtain the accurate coordinate and standard deviation of each Gaussian point in the double helix.
S6, deconvoluting the double-spiral point with the pinhole by using an RL algorithm to change the point into a common Gaussian point, wherein the point spread function used by deconvolution is a theoretical double-spiral point spread function, the distance between two peaks is constant, and the angle is thetan。
In step S6, a 100nm fluorescent bead wide field is imaged, and the phase modulation is performed on the emitted fluorescence to obtain double spiral points, a single double spiral point is measured, and the standard deviation σ of the gaussian distribution of a single peak is obtained, the theoretical point spread function is two opposite gaussian points, the midpoint of the connecting line of the two opposite gaussian points is located at the center of the image, the peak value of the gaussian point is set to 1, and the standard deviation is σ.
S7, calculating the Gaussian point after the back rolling is InPosition coordinates (x, y) of (1), willCopying the intensity distribution of Gaussian points to I0At the position of (2 x, 2 y).
S8, mixing I0The size of the image is reduced to one half of the original size, and a three-dimensional information image of the sample with super-resolution is obtained.
In steps S7 and S8, which are essentially the redistribution of pixels, the intensity distribution of Gaussian points is copied to I0(2 x, 2 y), and mixing I0The method of reducing the image size to one half of the original size is equivalent to moving a fluorescence signal near an excitation point detected by the CCD to the position of the excitation point for a certain distance, and the distance is half of the pixel position of a detector where the signal is located and the position of the excitation point.
S9, calculating the sample depth of each scanning position according to the relation between the double helix rotation angle and the sample defocusing distance, reconstructing a depth map of the sample,
In step S9, the relationship between the double helix rotation angle and the defocus distance of the sample can be obtained by continuously moving a 100nm fluorescent bead sample in the Z direction by a displacement stage, phase-modulating the sample, and collecting the result by a detector, to obtain the double helix point spread function rotation angle of a single fluorescent bead at different positions of the Z axis, and by linearly fitting the angle data of the point spread function with the corresponding axial position, to obtain the corresponding relationship between the double helix point spread function rotation angle and the axial position, to further obtain three-dimensional information of the sample by single two-dimensional scanning, as shown in fig. 5 (a) and 5 (b), the invention provides a method for reconstructing a image and a depth map of a renal cell sample by using the multi-focus scanning three-dimensional imaging method based on the double helix point spread function, which can greatly improve the time resolution of an image scanning microscopy system without reducing the image resolution and the signal-to noise ratio, effectively broadens the application range of the image scanning microscope system.
The invention also correspondingly provides a multi-focus scanning three-dimensional imaging system based on the double spiral point spread function, as shown in fig. 6, which comprises a laser light source 1, a beam expanding collimation reflection module 21, a digital micro-mirror element 5, a phase modulation acquisition module 30 and a control terminal 20, which are sequentially arranged along the transmission direction of a light path, wherein the laser light source 1 is used for providing continuous excitation light beams; the beam expanding collimation reflection module 21 is used for expanding and collimating the excitation light beam, and reflecting the laser light beam after expanding and collimating to enable the laser light beam to be emitted at a preset angle; the digital micro-mirror element 5 is used for reflecting the laser beam according to the introduced lighting mode switched at equal intervals and then projecting the laser beam onto the sample surface, and exciting a periodically arranged dot matrix generated on the sample surface and moving along with the switching of the lighting mode; the phase modulation acquisition module 30 is configured to perform phase modulation on fluorescence generated by exciting a sample surface, convert a fluorescence signal with gaussian distribution into a fluorescence signal in a double-helix form, and acquire the fluorescence signal in the double-helix form when an illumination mode is switched each time, so as to obtain a plurality of image data; the control terminal 20 is configured to introduce a preset illumination mode into the digital micromirror element, position and intercept all double helix points on each picture according to the acquired image data, obtain a plurality of sub-region image data, and perform wavefront reconstruction processing on all sub-regions to obtain a three-dimensional reconstruction image of the sample. Please refer to the corresponding embodiments of the above methods.
The phase modulation acquisition module comprises a phase modulation unit 17 and a detector 19 which are sequentially arranged along the transmission direction of an optical path, wherein the phase modulation unit 17 is used for carrying out phase modulation on fluorescence generated by exciting a sample surface and converting a fluorescence signal with Gaussian distribution into a fluorescence signal in a double helix form; the detector 19 is used for collecting the fluorescence signal in the form of double helix when the illumination mode is switched each time, and obtaining a plurality of image data. The phase modulation unit is a phase plate or a spatial light modulator. Please refer to the corresponding embodiments of the above methods.
Specifically, the multi-focus scanning three-dimensional imaging system based on the double-spiral point spread function comprises a laser light source 1, a first lens 2, a second lens 3, a first reflector 4, a digital micro-mirror element 5, a third lens 6, a diaphragm 7, a fourth lens 8, a fifth lens 9, a dichroic filter 10, an objective lens 11, a sample 12, a tube mirror 13, a linear polarizer 14, a sixth lens 15, a second reflector 16, a phase modulation unit 17, a seventh lens 18, a detector 19 and a control terminal 20 which are sequentially arranged along an optical path; the first lens 2, the second lens 3 and the first reflector 4 form a beam expanding collimation reflection module 21. The back focal plane of the first lens 2 coincides with the front focal plane of the second lens 3, the digital micromirror element 5 is located at the position of the front focal plane of the third lens 6, the diaphragm 7 is placed at the position of the back focal plane of the third lens 6 and is used for blocking the reflected light of the redundant diffraction orders, and the back focal plane of the third lens 6 coincides with the front focal plane of the fourth lens 8. The back 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 back focal plane of the tube mirror 13, the phase modulation unit 17 is placed at the back focal plane of the sixth lens 15, and the back focal plane of the sixth lens 15 coincides with the front focal plane of the seventh lens 18. The control terminal 20 is connected with the digital micromirror element 5 through a data line for guiding a pattern diagram to be displayed into the digital micromirror element 5, the control terminal 20 is connected with the detector 19 through a data line for transmitting an image acquired by the detector 19 into the control terminal 20, and the digital micromirror element 5 is connected with the detector 19 through a radio frequency line for transmitting an external trigger signal of the digital micromirror element 5 into the detector 19.
The laser light source 1 generates continuous laser with a specific wavelength, can be used for exciting a sample to generate fluorescence, expands beam and collimates after passing through the first lens 2 and the second lens 3, can adjust the multiplying power of the expanded beam by changing the focal lengths of the first lens 2 and the second lens 3, then, the laser after expanding beam and collimating enters the center of a display panel of the digital micromirror element 5 through the first reflector 4, and the incident angle forms-24 degrees with the horizontal plane; the micro-reflector on the digital micro-mirror element 5 reflects the excitation light, a sparse focusing dot matrix is formed on the sample surface through the 4F system and the objective lens 11, the laser light after beam expansion is reflected by the digital micro-mirror element 5, a plurality of focusing points are formed at the position of the back focal plane of the fourth lens 8, the sparse excitation dot matrix is formed on the sample 12 through the fifth lens 9 and the objective lens 11, and the distribution of the dot matrix depends on the display mode loaded in the memory of the digital micro-mirror element 5; the display mode of the digital micromirror device 5 is continuously switched, and a trigger signal is sent to the detector 19, a sparse focusing point on the sample surface is displaced along with the mode change, and finally the whole sample is completely illuminated, specifically, a series of display modes are sequentially introduced into the memory of the digital micromirror device 5 through the control terminal 20, and the switching interval of the modes can be set by the software of the digital micromirror device 5. A binary image with a mode of 1024X768, wherein each pixel corresponds to a micro-mirror on a digital micro-mirror element panel, the pixel value 1 represents the 'on' state of the micro-mirror, and 0 represents the 'off' state of the micro-mirror; fluorescence emitted by the sample passes through a 4f system behind the tube lens 13 and is subjected to phase modulation of the phase modulation unit, and the fluorescence is converted into a double-helix form by a traditional point spread function; the detector 19 collects the fluorescent signal while receiving the trigger signal of the digital micromirror element 5, specifically, the digital micromirror element 5 switches a new mode and simultaneously sends a rising edge signal with a voltage of 3V, the detector 19 starts to collect the fluorescent signal while receiving the signal, the exposure time of the detector 19 is the interval between the two rising edge signals, and the collection of the image data is completed. 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 invention, the laser beam after beam expanding and collimating irradiates onto the digital micromirror element at a preset angle; the digital micro-mirror element reflects the laser beam and then projects the laser beam onto the sample surface; switching the illumination mode of the digital micromirror element at equal intervals, exciting a periodically arranged dot matrix on the sample surface and moving along with the switching of the illumination mode until the sample surface is completely excited to generate fluorescence; carrying out phase modulation on fluorescence generated by exciting a sample surface, converting a fluorescence signal with Gaussian distribution into a fluorescence signal in a double-spiral form, and acquiring the fluorescence signal in the double-spiral form by a detector when an illumination mode is switched every time to obtain a plurality of image data; and positioning and intercepting all double spiral points on each picture according to the acquired image data to obtain a plurality of sub-region image data, and performing wavefront reconstruction processing on all sub-regions to obtain a three-dimensional reconstruction image of the sample. The sample can be excited by a plurality of focusing points simultaneously, the imaging range is improved, the sample collecting time is reduced, the phase modulation is carried out on the fluorescence emitted by the sample, the collected point spread function is converted into a double-helix form, the three-dimensional information of the sample is obtained by single two-dimensional scanning, and the time resolution of the image scanning microscope system is greatly improved.
It should be understood that equivalents and modifications of the technical solution and inventive concept thereof may occur to those skilled in the art, and all such modifications and alterations should fall within the scope of the appended claims.
Claims (9)
1. A multi-focus scanning three-dimensional imaging method based on a double-spiral point spread function is characterized by comprising the following steps:
irradiating the laser beam after beam expansion and collimation onto a digital micromirror element at a preset angle;
the digital micro-mirror element reflects the laser beam and then projects the laser beam onto the sample surface;
switching the illumination mode of the digital micromirror element at equal intervals, exciting a periodically arranged dot matrix on the sample surface and moving along with the switching of the illumination mode until the sample surface is completely excited to generate fluorescence;
carrying out phase modulation on fluorescence generated by exciting a sample surface, converting a fluorescence signal with Gaussian distribution into a fluorescence signal in a double-spiral form, and acquiring the fluorescence signal in the double-spiral form by a detector when an illumination mode is switched every time to obtain a plurality of image data;
positioning and intercepting all double helix points on each picture according to the acquired image data to obtain a plurality of sub-region image data, and performing wavefront reconstruction processing on all sub-regions to obtain a three-dimensional reconstruction image of the sample;
the method comprises the following steps of positioning and intercepting all double spiral points on each picture according to acquired image data to obtain a plurality of sub-region image data, and performing wavefront reconstruction processing on all sub-regions to obtain a three-dimensional reconstruction image of a sample:
generating a zero matrix I with preset multiple size according to the size of the collected image data0；
Reading acquired imagesN-th drawing I in datanTo 1, pairnPositioning and intercepting double spiral points to obtain a plurality of sub-region image data S1，S2…SnOnly one double helix point exists in each sub-region image data;
all the sub-region image data are processed in parallel, and the sub-region image data S is obtained through double-Gaussian fittingnTwo intensity peak coordinates (x) of the middle double helix pointn，1，yn，1)，(xn，2，yn，2) Attaching Gaussian-distributed digital pinholes and calculating the rotation angle theta of the double helixn；
Deconvoluting the double helix point after the addition of the digital pinhole to convert the double helix point into a Gaussian point, positioning the Gaussian point after deconvolution, and calculating the position of the Gaussian point InCoordinate position (x, y) of the point, copying the intensity distribution of the gaussian point to I0At the (a x, a y) position of (a), wherein a is a predetermined multiple;
continuously reading the (n + 1) th image in the image data, carrying out sub-region interception and data processing until all the image data are processed, and processing I0The size of the image is reduced to 1/a to obtain a three-dimensional information image of the sample, and the three-dimensional information image is obtained according to the rotation angle theta of the double helixnAnd calculating the sample depth of each scanning position according to the corresponding relation with the defocusing distance of the sample, and reconstructing to obtain a depth map of the sample.
2. The multi-focus scanning three-dimensional imaging method based on the double spiral point spread function according to claim 1, wherein the step of projecting the laser beam reflected by the digital micro-mirror element onto the sample surface specifically comprises:
the laser beam enters a 4f system after being reflected by a digital micro-mirror element, and is projected onto a sample surface after reflected light of an unnecessary diffraction order is filtered by a diaphragm arranged on a Fourier surface.
3. The method of claim 1, wherein the step of performing phase modulation on fluorescence generated by exciting a sample surface, converting a fluorescence signal with Gaussian distribution into a fluorescence signal in a double helix form, and acquiring the fluorescence signal in the double helix form by a detector each time an illumination mode is switched to obtain a plurality of image data comprises:
fluorescence generated by exciting the sample surface is transmitted to the phase modulation unit;
loading a double helix point spread function phase on a phase modulation unit, carrying out phase modulation on the fluorescence signals with Gaussian distribution, and converting the fluorescence signals into double helix fluorescence signals;
and acquiring the fluorescent signal in the double-helix form by a detector when the illumination mode is switched every time to obtain a plurality of image data.
4. The multi-focus scanning three-dimensional imaging method based on the double spiral point spread function according to claim 1, wherein the step of attaching the Gaussian distributed digital pinholes specifically comprises:
according to the formula
Generating double-Gaussian distributed digital pinholes and multiplying the digital pinholes by the intensity peak coordinates of the double-helix points, wherein c is a constant.
5. The method of claim 1, wherein the preset multiple is two times.
6. The method according to claim 3, wherein the phase modulation unit is a phase plate or a spatial light modulator.
7. The utility model provides a three-dimensional imaging system of multifocal scanning based on double helix point spread function which characterized in that, includes that along the light path transmission direction sets gradually:
a laser light source for providing a continuous beam of excitation light;
the beam expanding collimation reflection module is used for expanding and collimating the excitation beam and reflecting the laser beam subjected to beam expanding and collimation to enable the laser beam to be emitted at a preset angle;
the digital micro-mirror element is used for reflecting the laser beam and then projecting the laser beam onto the sample surface according to the introduced lighting mode switched at equal intervals, and exciting a periodically arranged dot matrix generated on the sample surface and moving along with the switching of the lighting mode;
the phase modulation acquisition module is used for carrying out phase modulation on fluorescence generated by exciting a sample surface, converting fluorescence signals with Gaussian distribution into fluorescence signals in a double-helix form, and acquiring the fluorescence signals in the double-helix form when an illumination mode is switched every time to obtain a plurality of image data;
the control terminal is used for leading a preset illumination mode into the digital micromirror element, positioning and intercepting all double helix points on each picture according to the acquired image data to obtain a plurality of sub-region image data, and performing wavefront reconstruction processing on all sub-regions to obtain a three-dimensional reconstruction image of the sample;
the control terminal is specifically configured to:
positioning and intercepting all double spiral points on each picture according to the acquired image data to obtain a plurality of sub-region image data, and performing wavefront reconstruction processing on all sub-regions to obtain a three-dimensional reconstruction image of the sample, wherein the step of obtaining the three-dimensional reconstruction image of the sample comprises the following steps:
generating a zero matrix I with preset multiple size according to the size of the collected image data0；
Reading the nth picture I in the acquired image datanTo 1, pairnPositioning and intercepting double spiral points to obtain a plurality of sub-region image data S1，S2…SnOnly one double helix point exists in each sub-region image data;
all the sub-region image data are processed in parallel, and the sub-region image data S is obtained through double-Gaussian fittingnTwo intensity peak coordinates (x) of the middle double helix pointn，1，yn，1)，(xn，2，yn，2) Attaching Gaussian-distributed digital pinholes and calculating the rotation angle theta of the double helixn；
Deconvoluting the double helix point after the addition of the digital pinhole to convert the double helix point into a Gaussian point, positioning the Gaussian point after deconvolution, and calculating the position of the Gaussian point InCoordinate position (x, y) of the point, copying the intensity distribution of the gaussian point to I0At the (a x, a y) position of (a), wherein a is a predetermined multiple;
continuously reading the (n + 1) th image in the image data, carrying out sub-region interception and data processing until all the image data are processed, and processing I0The size of the image is reduced to 1/a to obtain a three-dimensional information image of the sample, and the three-dimensional information image is obtained according to the rotation angle theta of the double helixnAnd calculating the sample depth of each scanning position according to the corresponding relation with the defocusing distance of the sample, and reconstructing to obtain a depth map of the sample.
8. The system of claim 7, wherein the phase modulation acquisition module comprises, in order along the optical path transmission direction:
the phase modulation unit is used for carrying out phase modulation on fluorescence generated by exciting the sample surface and converting a fluorescence signal with Gaussian distribution into a fluorescence signal in a double helix form;
and the detector is used for collecting the double-helix fluorescence signal when the illumination mode is switched every time to obtain a plurality of image data.
9. The multi-focus scanning three-dimensional imaging system based on the double spiral point spread function of claim 8, wherein the phase modulation unit is a phase plate or a spatial light modulator.
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