CN114002836A - Adaptive optics-based multifocal structure light super-resolution imaging method and system - Google Patents
Adaptive optics-based multifocal structure light super-resolution imaging method and system Download PDFInfo
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Abstract
The invention discloses a multifocal structure light super-resolution imaging method and a system based on adaptive optics, wherein the method comprises the following steps: irradiating the laser beam after beam expansion and collimation on a spatial light modulator; modulating the laser by a spatial light modulator and transmitting the laser to the surface of the sample; transmitting single-photon or multi-photon fluorescence wavefront information generated by a single excitation point to a Hartmann sensor; controlling the spatial light modulator to preload a corresponding correction phase diagram, and controlling the deformable mirror to load a corresponding correction voltage matrix; and controlling the spatial light modulator to continuously load the multi-focus excitation dot matrix phase diagram, and sequentially receiving the sample fluorescence information after self-adaptive optical correction through the detector. The method utilizes the characteristics of high modulation precision of the spatial light modulator and high energy utilization rate of the deformable mirror, realizes self-adaptive optical correction of a laser excitation light path by using the spatial light modulator, and improves the imaging depth; and the deformable mirror is used for realizing the self-adaptive optical correction of the fluorescence light path, improving the imaging resolution and reducing the fluorescence energy loss.
Description
Technical Field
The invention relates to the field of application of optical microscopic imaging technology, in particular to a multifocal structured light super-resolution imaging method and system based on adaptive optics.
Background
Optical microscopy imaging has become an important tool for studying biological microstructures. In 2010, Muller and Enderlein realized Image Scanning Microscopy for the first time [ Muller, et al, "Image Scanning Microscopy", Phy. Rew.Let.,198101-198014(2010) ], which can realize about 2 times of improvement of lateral resolution, and is also called structured light super-resolution imaging technology. In 2012, a multi-focus structured light super-resolution imaging technology is proposed, which adopts a multi-point parallel scanning mode, greatly improves the imaging speed of a structured light super-resolution microscope, and provides a favorable tool for researching biological structures, information transduction and disease diagnosis and treatment in living body samples.
When microscopic imaging is performed on a biological sample, the effect of aberrations on the imaging results is very significant. For practical imaging systems, the aberrations originate mainly from three aspects: one is the inherent aberration of the system; secondly, aberration caused by mismatching of refractive indexes of the objective lens immersion medium and the sample; and the third is the aberration caused by the non-uniform refractive index inside the sample. The aberrations cause the focused spot to become diffuse. The concrete expression is as follows: the excitation spot becomes dispersed resulting in a decrease in the excitation efficiency of the sample. The fluorescent light spot emitted by the sample becomes diffuse causing the imaging of the sample to become blurred. The effect of aberrations on imaging depth and imaging resolution is particularly pronounced when imaging samples at large depths.
Therefore, the prior art has yet to be improved.
Disclosure of Invention
The invention provides a multifocal structural light super-resolution imaging method and system based on adaptive optics, aiming at solving the technical problem of large aberration of the conventional multifocal structural light super-resolution imaging technology.
The technical scheme adopted by the invention for solving the technical problem is as follows:
in a first aspect, the present invention provides a multifocal structural light super-resolution imaging method based on adaptive optics, including the following steps:
generating laser by a laser, and irradiating the laser on a spatial light modulator after beam expanding and collimating;
modulating the laser light through the spatial light modulator, and transmitting the modulated laser light to a sample surface to form a single excitation point on the sample surface;
transmitting single-photon or multi-photon fluorescence wavefront information generated by the single excitation point to a Hartmann sensor;
controlling the spatial light modulator to preload a corresponding correction phase diagram according to the wave front aberration detected by the Hartmann sensor, and controlling the deformable mirror to load a corresponding correction voltage matrix;
and controlling the spatial light modulator to continuously load the generated multifocal excitation dot matrix phase diagram, and sequentially receiving the sample fluorescence information after self-adaptive optical correction through a detector until scanning of the multifocal excitation light array is completed.
In one implementation, the generating laser light by a laser, expanding the beam and collimating the laser light, and then irradiating the laser light on a spatial light modulator includes:
generating laser with a preset wavelength by the laser;
performing beam expansion and collimation on the laser through a first lens group to obtain a laser beam with a preset radius;
the laser beam is irradiated on the spatial light modulator by a first mirror.
In one implementation, the modulating the laser light by the spatial light modulator and transmitting the modulated laser light to the surface of the sample includes:
phase modulating the laser irradiated on the spatial light modulator according to the phase modulation parameters to form a modulated excitation light spot at a Fourier surface of a first lens behind the spatial light modulator;
filtering redundant diffraction orders in the excitation light spots through a diaphragm to obtain target excitation light spots;
and projecting the target excitation light spot to the surface of the sample through a second lens group and an objective lens.
In one implementation, the transmitting the single-photon or multi-photon fluorescence wavefront information generated by the single excitation point to the hartmann sensor includes:
setting the deformable mirror to be in a plane state;
loading a phase map of the single excitation point by the spatial light modulator;
transmitting the single-photon or multi-photon fluorescence wavefront information generated by the single excitation point to the Hartmann sensor, and acquiring the fluorescence wavefront information through the Hartmann sensor.
In one implementation, the controlling the spatial light modulator to preload a corresponding correction phase map and controlling the deformable mirror to load a corresponding correction voltage matrix according to the wavefront aberration detected by the hartmann sensor includes:
calculating the wavefront aberration according to the collected fluorescence wavefront information;
calculating an aberration correction phase diagram of a laser light path according to the wavefront aberration;
combining the Hartmann sensor and the deformable mirror into closed-loop correction, and obtaining a voltage distribution matrix of a correction surface type of the deformable mirror through iterative calculation;
and performing self-adaptive optical correction on the fluorescence light path according to the voltage distribution matrix to obtain corrected wavefront aberration.
In one implementation, the calculating an aberration-corrected phase map of a laser light path from wavefront aberrations includes:
calculating a PV surface type in the wavefront aberration, and performing interpolation processing on the PV surface type to obtain a PV coordinate value;
and calculating the aberration correction phase diagram according to the PV coordinate value and a preset formula.
In one implementation, the preset formula is:
mod(K*PV(x1,y1)/λ,2π)；
λ represents the wavelength of the probe wavefront;
k denotes a response factor.
In one implementation, the combining the hartmann sensor and the deformable mirror into a closed-loop correction, and obtaining a voltage distribution matrix of a correction surface type of the deformable mirror through iterative calculation includes:
sequentially adjusting the voltage value of the brake of the deformable mirror according to a preset voltage, and judging whether the detected wavefront aberration tends to a preset value;
and if the detected wavefront aberration tends to a preset value, setting the current voltage value of the brake of the deformable mirror as a voltage distribution matrix of the correction surface type of the deformable mirror.
In one implementation, the controlling the spatial light modulator to continuously load the generated multi-focus excitation dot matrix phase map and sequentially receive the fluorescence information of the sample after adaptive optical correction through the detector includes:
controlling the deformable mirror to load the correction surface shape;
controlling the spatial light modulator to load the aberration-corrected phase map;
sequentially loading the generated multifocal excitation dot matrix phase diagrams to scan a sample;
and sequentially acquiring fluorescent images generated by the corresponding multifocal dot matrix excitation samples through a camera to obtain the multifocal structural light super-resolution imaging.
In a second aspect, the present invention provides a multifocal structured light super-resolution imaging system based on adaptive optics, including: the system comprises an illuminating device, an adaptive optical correction device, a fluorescence detection imaging device and a terminal; the lighting device, the adaptive optical correction device and the fluorescence detection imaging device are respectively connected with the terminal;
the lighting device includes: a laser and a spatial light modulator; the spatial light modulator is used for modulating incident laser emitted by the laser to generate single-point exciting light, multi-focus lattice exciting light and multi-focus lattice exciting light corrected by the adaptive optical correction device;
the adaptive optical correction device includes: a Hartmann sensor and a deformable mirror; the Hartmann sensor is used for detecting wavefront information, and the deformable mirror is used for correcting system aberration during imaging and image difference caused by sample aberration on imaging;
the fluorescence detection imaging device includes: a camera; the camera is used for detecting a fluorescence signal emitted by the sample;
the terminal is used for controlling the laser to generate laser and controlling the spatial light modulator to modulate the laser;
the device comprises a Hartmann sensor, a spatial light modulator, a deformable mirror, a phase correction image sensor and a phase correction image sensor, wherein the phase correction image sensor is used for detecting the wave front aberration;
and the multi-focus excitation dot matrix phase diagram is used for controlling the spatial light modulator to continuously load the generated multi-focus excitation dot matrix phase diagram, and the sample fluorescence information after self-adaptive optical correction is sequentially received through the detector until the scanning of the multi-focus excitation dot matrix is finished, so that the multi-focus structure light super-resolution imaging is realized.
The invention adopts the technical scheme and has the following effects:
the invention utilizes the characteristics of high modulation precision of the spatial light modulator and high energy utilization rate of the deformable mirror, realizes self-adaptive optical correction of a laser light path by using the spatial light modulator in a laser excitation light path, and further improves the imaging depth; in addition, the self-adaptive optical correction of the fluorescence light path is realized by using the deformable mirror in the fluorescence light path, so that the imaging resolution is improved, and the fluorescence energy loss is reduced. Compared with the existing imaging mode, the method can realize dot matrix generation, scanning and correction only by the spatial light modulator through a device with simple structure and simple and convenient operation under the condition of not adopting galvanometer scanning, thereby improving the depth and resolution of multifocal structured light super-resolution imaging, and eliminating the influence of system aberration and sample aberration on the imaging depth and imaging resolution during large-depth imaging through self-adaptive optical correction.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
Fig. 1 is a flow chart of a multifocal structured light super-resolution imaging method based on adaptive optics in one implementation of the invention.
FIG. 2 is a phase diagram of spatial light modulator loading when measuring wavefront information in one implementation of the invention.
FIG. 3 is a schematic diagram of a single point excitation spot generated when a spatial light modulator is loaded with a phase map in one implementation of the invention.
FIG. 4 is a schematic diagram of a voltage distribution matrix when the deformable mirror is in a planar state in one implementation of the invention.
FIG. 5 is a schematic illustration of wavefront aberrations detected by a Hartmann sensor when measuring wavefront information in one implementation of the present invention.
FIG. 6 is a phase diagram of an aberration corrected phase map loaded by an adaptive optical timing spatial light modulator and a resulting multi-focal spot matrix in one implementation of the invention.
FIG. 7 is a schematic view of a multifocal lattice after optical correction in one implementation of the invention.
FIG. 8 is a schematic voltage distribution matrix diagram of an anamorphic mirror loaded aberration correcting surface in one implementation of the present invention.
FIG. 9 is a schematic representation of sample imaging after adaptive optical correction in one implementation of the invention.
Fig. 10 is a schematic structural diagram of a multi-focus structured light super-resolution imaging device based on adaptive optics in an implementation manner of the present invention.
In the figure: 1. a laser; 2. a half-wave plate; 3. a first lens; 4. a second lens; 5. a first reflector; 6. a spatial light modulator; 7. a third lens; 8. a second reflector; 9. a diaphragm; 10. a dichromatic mirror; 11. a fourth lens; 12. a third reflector; 13. an objective lens; 14. a sample; 15. a fifth lens; 16. a deformable mirror; 17. a sixth lens; 18. a seventh lens; 19. a Hartmann sensor; 20. a mirror can be turned over; 21. a detector; 22. a computer.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
In order to make the objects, technical solutions and advantages 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.
Exemplary method
As shown in fig. 1, an embodiment of the present invention provides a multifocal structured light super-resolution imaging method based on adaptive optics, including the following steps:
and S100, generating laser by a laser, expanding the beam, collimating and irradiating the laser on a spatial light modulator.
In this embodiment, the adaptive optics-based multifocal structural optical super-resolution imaging method is implemented by an adaptive optics-based multifocal structural optical super-resolution imaging system; the system makes full use of the characteristics of high modulation precision of the spatial light modulator and high energy utilization rate of the deformable mirror. The spatial light modulator is used in the laser excitation light path to realize self-adaptive optical correction of the laser excitation light path, so that the imaging depth is further improved; the self-adaptive optical correction of the fluorescence light path is realized by using the deformable mirror in the fluorescence light path, and the fluorescence energy loss is reduced while the imaging resolution is improved.
As shown in fig. 10, the adaptive optics based multi-focus structured light super-resolution imaging system includes: the device comprises a laser, a half-wave plate, a first lens, a second lens, a first reflector, a spatial light modulator, a third lens, a second reflector, a diaphragm, a dichroscope, a fourth lens, a third reflector, an objective lens, a sample, a fifth lens, a deformable mirror, a sixth lens, a seventh lens, a Hartmann sensor, a reversible reflector, a detector, a computer and the like.
In the system, the laser, the spatial light modulator, the deformable mirror, the hartmann sensor and the detector are respectively connected with the computer to transmit corresponding detection data to the computer, or realize adaptive optical correction of a laser excitation light path and adaptive optical correction of a fluorescence light path under the control of the computer.
When the adaptive optics-based multifocal structural light super-resolution imaging method is implemented, the computer is used for controlling the laser to emit laser with a preset wavelength; the laser with the preset wavelength can be femtosecond laser with the wavelength of 920nm and used for multiphoton imaging, can be laser with the wavelength of 640nm and used for single photon imaging, and can also be laser with other wavelengths; because the size of the laser emitted by the laser is the designated radius, the laser emitted by the laser needs to be subjected to beam expanding and collimating treatment to obtain a laser beam with a preset radius; for example, the preset radius is 2 mm.
Specifically, as shown in fig. 10, when the laser of the laser is expanded and collimated, the laser can be expanded and collimated through the first lens and the second lens, so as to obtain a required laser size; and irradiating the laser beam after beam expansion and collimation on the spatial light modulator through a fixed first reflecting mirror so as to perform phase modulation on the laser beam after beam expansion and collimation through the spatial light modulator.
That is, in an implementation manner of this embodiment, step S100 specifically includes the following steps:
step S110, generating laser with preset wavelength by the laser;
step S120, performing beam expanding and collimation on the laser through a first lens group to obtain a laser beam with a preset radius;
step S130, the laser beam is irradiated on the spatial light modulator through a first mirror.
In this embodiment, the laser emitted by the laser is irradiated on the spatial light modulator, and the spatial light modulator can be used to modulate the laser, so that the modulation process of the laser and the laser path is realized without adopting galvanometer scanning.
As shown in fig. 1, in an implementation manner of the embodiment of the present invention, the adaptive optics based multi-focus structured light super-resolution imaging method further includes the following steps:
and S200, modulating the laser through the spatial light modulator, and transmitting the modulated laser to the surface of the sample to form a single excitation point on the surface of the sample.
In this embodiment, after the incident laser light is irradiated on the spatial light modulator, the computer performs phase modulation on the laser light irradiated on the spatial light modulator according to the phase modulation parameter, so as to form a modulated excitation spot at the fourier plane of the first lens (i.e., the third lens in fig. 10) behind the spatial light modulator.
Further, the modulated excitation light spot is reflected by the second reflecting mirror and enters the diaphragm; redundant diffraction orders in the excitation light spot can be filtered through the diaphragm, so that a target excitation light spot is obtained; and then, the target excitation light spot is projected to the sample surface through the second lens group and the objective lens, that is, through the dichroic mirror, the fourth lens, the third reflector and the objective lens in fig. 10.
That is, in an implementation manner of this embodiment, the step S200 specifically includes the following steps:
step S210, performing phase modulation on the laser irradiated on the spatial light modulator according to the phase modulation parameters to form a modulated excitation light spot on a Fourier surface of a first lens behind the spatial light modulator;
step S220, filtering redundant diffraction orders in the excitation light spot through a diaphragm to obtain a target excitation light spot;
and step S230, projecting the target excitation light spot to the surface of the sample through a second lens group and an objective lens.
In the embodiment, the spatial light modulator modulates the incident laser to obtain the target excitation light spot, so that a single-point excitation effect is formed on the surface of the sample, and the wave front information of the fluorescence information generated by the single-point excitation of the sample is detected by the Hartmann sensor.
As shown in fig. 1, in an implementation manner of the embodiment of the present invention, the adaptive optics based multi-focus structured light super-resolution imaging method further includes the following steps:
and step S300, transmitting the fluorescence wavefront information generated by the single excitation point to a Hartmann sensor.
In this embodiment, after the target excitation light spot is projected onto the surface of the sample, the fluorescence emitted by the sample after being irradiated is reflected by the deformable mirror and then detected by the hartmann sensor or the camera, so as to transmit the detected fluorescence wavefront information to the computer.
Specifically, as shown in fig. 10, the fluorescence of the sample passes through the objective lens, the third reflector, the fourth lens, the dichroic mirror, the fifth lens, the anamorphic lens, the sixth lens, the reversible reflector and the seventh lens in sequence, and is transmitted to the hartmann sensor, so as to be detected by the hartmann sensor.
Further, in order to obtain the fluorescence wavefront information of the sample, it is necessary to set the deformable mirror to a planar state according to an initial voltage distribution matrix (or a default voltage distribution matrix), wherein the initial voltage distribution matrix is as shown in fig. 4; then, loading a phase map of the single excitation point through the spatial light modulator, wherein the phase map is as shown in fig. 2; correspondingly, a single-point light spot generated by a single excitation point is shown in fig. 3; and finally, transmitting the single-photon or multi-photon fluorescence wavefront information generated by a single excitation point to the Hartmann sensor, and acquiring the fluorescence wavefront information of the sample.
That is, in an implementation manner of this embodiment, the step S300 specifically includes the following steps:
step S310, setting the deformable mirror to be in a plane state;
step S320, loading the phase map of the single excitation point through the spatial light modulator;
step S330, transmitting the single-photon or multi-photon fluorescence wavefront information generated by the single excitation point to the Hartmann sensor, and acquiring the fluorescence wavefront information through the Hartmann sensor.
In the embodiment, by controlling the state of the deformable mirror and using the phase diagram of the single excitation point loaded by the spatial light modulator, the fluorescence wavefront information of the sample can be detected in the hartmann sensor, so that corresponding optical correction can be performed according to the detected information.
As shown in fig. 1, in an implementation manner of the embodiment of the present invention, the adaptive optics based multi-focus structured light super-resolution imaging method further includes the following steps:
and S400, controlling the spatial light modulator to preload a corresponding correction phase diagram according to the wave front aberration detected by the Hartmann sensor, and controlling the deformable mirror to load a corresponding correction voltage matrix.
In the present embodiment, the wavefront aberration is calculated according to the fluorescence wavefront information detected by the hartmann sensor, wherein the fluorescence wavefront information and the wavefront aberration are shown in fig. 5; the specific calculation method is as follows: acquiring the wavefront slope of each sub-aperture according to the offset between the image point corresponding to each sub-aperture of the micro-lens array in the Hartmann sensor and a reference point (the coordinate position of the reference point needs to be determined in advance); on the basis, the wave-front reconstruction algorithm (such as a region method wave-front reconstruction algorithm, a mode wave-front reconstruction algorithm and the like) of the shack-Hartmann wave-front sensor can be selected, and the wave-front aberration can be obtained through reconstruction; the wavefront reconstruction algorithm is the prior art, and is not described in detail.
Further, according to the detected wavefront aberration, calculating an aberration correction phase diagram of the laser optical path to use the aberration correction phase diagram as an adaptive optical correction diagram of the subsequent laser optical path, wherein the aberration correction phase diagram is shown in fig. 6; and then, forming closed-loop correction by the Hartmann sensor and the deformable mirror, and obtaining a voltage distribution matrix of a correction surface type of the deformable mirror through iterative calculation in about 3 seconds, thereby realizing the self-adaptive correction process of a fluorescence light path and obtaining corrected wavefront aberration.
That is, in an implementation manner of this embodiment, the step S400 specifically includes the following steps:
step S410, calculating the wavefront aberration according to the collected fluorescence wavefront information;
step S420, calculating an aberration correction phase diagram of a laser light path according to the wavefront aberration;
step S430, forming closed-loop correction on the Hartmann sensor and the deformable mirror, and obtaining a voltage distribution matrix of a correction surface type of the deformable mirror through iterative calculation;
step S440, performing self-adaptive optical correction on the fluorescence light path according to the voltage distribution matrix to obtain corrected wavefront aberration.
In this embodiment, in the process of calculating the aberration-corrected phase map, the calculation method is specifically as follows:
firstly, determining the PV (x, y) surface type according to the calculated wavefront aberration; then, this PV pattern is interpolated to obtain a PV coordinate value (x)1,y2) So that its surface shape size matches the mirror surface size of the spatial light modulator; finally, calculating the aberration correction phase diagram according to the PV coordinate value and a preset formula; wherein the preset formula is as follows:
mod(K*PV(x1,y1)/λ,2π)；
λ represents the wavelength of the probe wavefront;
k denotes a response factor.
In the above formula, the response factor can be obtained by testing in the following way:
firstly, placing a uniform solution on a sample table;
then, loading and generating a phase diagram for single-point detection and a superposed phase diagram of a zero phase difference phase on the spatial light modulator, and setting the wave front aberration detected by the Hartmann sensor at the moment as a reference value;
finally, the zero phase-difference phase on the spatial light modulator is replaced with some known phase-difference phase map P (x1, y1), and the aberration value PV (x, y) is detected on the hartmann sensor. Wherein, the ratio of the phase difference diagram P (x1, y1) and the PV (x, y) detected on the Hartmann sensor is the response factor K.
That is, in an implementation manner of this embodiment, step S420 specifically includes the following steps:
step S421, calculating a PV surface type in the wavefront aberration, and performing interpolation processing on the PV surface type to obtain a PV coordinate value;
step S422, calculating the aberration correction phase diagram according to the PV coordinate value and a preset formula.
In this embodiment, in the process of calculating the voltage distribution matrix of the correction surface type of the deformable mirror, the voltage of the actuator of the deformable mirror needs to be sequentially changed according to a preset voltage (for example, starting from 0V, changing the voltage value according to an increment of 0.5V), so that the measured wavefront aberration tends to 0 change (i.e., tends to the preset value); when the measured wave front aberration is gradually reduced and tends to be stable, the voltage value of the brake of the deformable mirror is the voltage distribution matrix of the correction surface type of the deformable mirror; wherein, the voltage distribution matrix of the correction surface type is shown in fig. 8; in the calculation process, the voltage value of the brake of the deformable mirror needs to be adjusted in sequence, and the change of the wavefront aberration tending to 0 is determined in real time.
That is, in an implementation manner of this embodiment, the step S430 specifically includes the following steps:
step S431, sequentially adjusting the voltage value of the brake of the deformable mirror according to a preset voltage, and judging whether the detected wavefront aberration tends to the preset value;
step S432, if the detected wavefront aberration tends to a preset value, setting the current voltage value of the actuator of the deformable mirror as a voltage distribution matrix of the correction surface type of the deformable mirror.
In the embodiment, the aberration correction phase diagram of the laser excitation light path is calculated through wavefront aberration calculation, so that the laser excitation light path can be corrected, and the voltage distribution matrix of the correction surface type is calculated through wavefront aberration, so that the fluorescence light path can be corrected.
As shown in fig. 1, in an implementation manner of the embodiment of the present invention, the adaptive optics based multi-focus structured light super-resolution imaging method further includes the following steps:
and S500, controlling the spatial light modulator to continuously load the generated multifocal excitation dot matrix phase diagram, and sequentially receiving the sample fluorescence information corrected by the self-adaptive optical system through the detector until the multifocal excitation light array is scanned.
In this embodiment, after obtaining the calibration parameters of the laser light path and the fluorescence light path, the deformable mirror may be controlled to load the calibration surface type, that is, the actuator of the deformable mirror is controlled by the voltage distribution matrix of the calibration surface type, so that the deformable mirror is deformed correspondingly from a planar state, and the adaptive calibration of the fluorescence light path is realized.
Meanwhile, the aberration correction phase diagram can be loaded by the spatial light modulator, and then the phase diagrams of the multifocal dot matrixes generated under the modulation of the spatial light modulator are sequentially loaded so as to scan the sample; wherein, the phase diagram of the multifocal lattice is shown in fig. 7.
Finally, a camera is used for sequentially collecting fluorescence images generated by corresponding multi-focus point lattice excited samples, and the obtained fluorescence image sequences of the samples are subjected to superposition calculation of corresponding pixel gray values, so that wide-field fluorescence images with wide-field imaging resolution can be obtained; and after the sample fluorescence image sequence is subjected to pixel repositioning processing and deconvolution processing, a super-resolution image with resolution improved by about 2 times can be obtained.
That is, in an implementation manner of this embodiment, the step S500 specifically includes the following steps:
step S510, controlling the deformable mirror to load the correction surface type;
step S520, controlling the spatial light modulator to load the aberration correction phase map;
step S530, sequentially loading the generated multi-focus excitation dot matrix phase diagram to scan a sample;
and S540, sequentially collecting the fluorescence images generated by the corresponding multifocal lattice excitation samples through a camera to obtain the multifocal structural light super-resolution imaging.
It is worth mentioning that when a series of phase diagrams of the multifocal excitation dot matrix are continuously loaded through the spatial light modulator, the sample fluorescence information after adaptive optical correction can be sequentially received through the detector until multifocal excitation is achieved, and the light array scans the whole field of view. The multi-focus excitation lattice after adaptive optical correction is shown in fig. 7; the imaging result of the multifocal structured light after adaptive optical correction on the sample (mouse cerebral blood vessel) is shown in fig. 9.
The embodiment utilizes the characteristics of high modulation precision of the spatial light modulator and high energy utilization rate of the deformable mirror, realizes self-adaptive optical correction of the laser excitation light path by using the spatial light modulator in the laser excitation light path, and further improves the imaging depth; in addition, the self-adaptive optical correction of the fluorescence light path is realized by using the deformable mirror in the fluorescence light path, so that the imaging resolution is improved, and the fluorescence energy loss is reduced. Compared with the existing imaging mode, the method can realize dot matrix generation, scanning and correction only by the spatial light modulator through a device with simple structure and simple and convenient operation under the condition of not adopting galvanometer scanning, thereby improving the depth and resolution of multifocal structured light super-resolution imaging, and eliminating the influence of system aberration and sample aberration on the imaging depth and imaging resolution during large-depth imaging through self-adaptive optical correction.
Exemplary device
Based on the above embodiment, the present invention further provides a multifocal structured light super-resolution imaging device based on adaptive optics, including: the system comprises an illuminating device, an adaptive optical correction device, a fluorescence detection imaging device and a terminal; the lighting device, the adaptive optical correction device and the fluorescence detection imaging device are respectively connected with the terminal;
as shown in fig. 10, the lighting device includes: a laser 1 and a spatial light modulator 6; the spatial light modulator 6 is used for modulating incident laser emitted by the laser 1 to generate single-point excitation light, multi-focus lattice excitation light and multi-focus lattice excitation light corrected by the adaptive optical correction device;
the adaptive optical correction device includes: a hartmann sensor 19 and a deformable mirror 16; the Hartmann sensor 19 is used for detecting wave front information, and the deformable mirror 16 is used for correcting system aberration during imaging and image difference caused by sample aberration to imaging;
the fluorescence detection imaging device includes: a camera (i.e., detector 21 in the figure); the camera is used for detecting a fluorescence signal emitted by the sample;
the terminal (i.e. the computer 22 in the figure) is used for controlling the laser 1 to generate laser and controlling the spatial light modulator 6 to modulate the laser; and is used for controlling the spatial light modulator 6 to pre-load the corresponding correction phase diagram and controlling the deformable mirror 16 to load the corresponding correction voltage matrix according to the wave front aberration detected by the Hartmann sensor 19; and the controller is used for controlling the spatial light modulator 6 to continuously load the generated multifocal excitation dot matrix phase diagram, and sequentially receiving the sample fluorescence information after the self-adaptive optical correction through the detector 21 until the multifocal excitation light array is scanned, so as to realize the multifocal structure light super-resolution imaging.
As shown in fig. 10, the system further includes: a half-wave plate 2, a first lens 3, a second lens 4 and a first reflector 5 connected with the laser 1; a third lens 7, a second mirror 8 and a diaphragm 9 connected to the spatial light modulator 6; a dichroic mirror 10, a fourth lens 11, a third reflecting mirror 12, an objective lens 13, and a fifth lens 15 connected to the anamorphic mirror 16; a sixth lens 17 and a seventh lens 18 connected to the hartmann sensor 19; and a turnable mirror 20 connected to said detector 21.
In practical applications, the laser optical path and the calibration method of the system are specifically as described above, and the fluorescence optical path and the calibration method thereof are also specifically as described above.
It will be understood by those skilled in the art that all or part of the processes of the methods of the above embodiments may be implemented by hardware related to instructions of a computer program, which may be stored in a non-volatile computer readable storage medium, and when executed, may include the processes of the embodiments of the methods described above. Any reference to memory, storage, databases, or other media used in embodiments provided herein may include non-volatile and/or volatile memory.
In summary, the present invention provides a method and a system for super-resolution imaging of multi-focus structured light based on adaptive optics, wherein the method comprises: irradiating the laser beam after beam expansion and collimation on a spatial light modulator; modulating the laser by a spatial light modulator and transmitting the laser to the surface of the sample; transmitting single-photon or multi-photon fluorescence wavefront information generated by a single excitation point to a Hartmann sensor; controlling the spatial light modulator to preload a corresponding correction phase diagram, and controlling the deformable mirror to load a corresponding correction voltage matrix; and controlling the spatial light modulator to continuously load the multi-focus excitation dot matrix phase diagram, and sequentially receiving the sample fluorescence information after self-adaptive optical correction through the detector. The method utilizes the characteristics of high modulation precision of the spatial light modulator and high energy utilization rate of the deformable mirror, realizes self-adaptive optical correction of a laser excitation light path by using the spatial light modulator, and improves the imaging depth; and the self-adaptive optical correction of a fluorescence light path is realized by using the deformable mirror, so that the imaging resolution is improved, and the fluorescence energy loss is reduced.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.
Claims (10)
1. A multifocal structure light super-resolution imaging method based on adaptive optics is characterized by comprising the following steps:
generating laser by a laser, and irradiating the laser on a spatial light modulator after beam expanding and collimating;
modulating the laser light through the spatial light modulator, and transmitting the modulated laser light to a sample surface to form a single excitation point on the sample surface;
transmitting single-photon or multi-photon fluorescence wavefront information generated by the single excitation point to a Hartmann sensor;
controlling the spatial light modulator to preload a corresponding correction phase diagram according to the wave front aberration detected by the Hartmann sensor, and controlling the deformable mirror to load a corresponding correction voltage matrix;
and controlling the spatial light modulator to continuously load the generated multifocal excitation dot matrix phase diagram, and sequentially receiving the sample fluorescence information after self-adaptive optical correction through a detector until scanning of the multifocal excitation light array is completed.
2. The adaptive optics based multifocal structured light super-resolution imaging method according to claim 1, wherein the laser generates laser light through a laser, and irradiates the laser light on a spatial light modulator after beam expanding and collimating, and the method comprises the following steps:
generating laser with a preset wavelength by the laser;
performing beam expansion and collimation on the laser through a first lens group to obtain a laser beam with a preset radius;
the laser beam is irradiated on the spatial light modulator by a first mirror.
3. The adaptive optics based multifocal structured light super-resolution imaging method according to claim 1, wherein the modulating the laser light by a spatial light modulator and transmitting the modulated laser light to the sample surface comprises:
phase modulating the laser irradiated on the spatial light modulator according to the phase modulation parameters to form a modulated excitation light spot at a Fourier surface of a first lens behind the spatial light modulator;
filtering redundant diffraction orders in the excitation light spots through a diaphragm to obtain target excitation light spots;
and projecting the target excitation light spot to the surface of the sample through a second lens group and an objective lens.
4. The adaptive optics based multifocal structured light super-resolution imaging method according to claim 1, wherein the transmitting of the single-photon or multi-photon fluorescence wavefront information generated by the single excitation point to the Hartmann sensor comprises:
setting the deformable mirror to be in a plane state;
loading a phase map of the single excitation point by the spatial light modulator;
transmitting the single-photon or multi-photon fluorescence wavefront information generated by the single excitation point to the Hartmann sensor, and acquiring the fluorescence wavefront information through the Hartmann sensor.
5. The adaptive optics based multifocal structured light super-resolution imaging method according to claim 1, wherein the controlling the spatial light modulator to preload the corresponding correction phase map and the deformable mirror to load the corresponding correction voltage matrix according to the wavefront aberration detected by the Hartmann sensor comprises:
calculating the wavefront aberration according to the collected fluorescence wavefront information;
calculating an aberration correction phase diagram of a laser light path according to the wavefront aberration;
combining the Hartmann sensor and the deformable mirror into closed-loop correction, and obtaining a voltage distribution matrix of a correction surface type of the deformable mirror through iterative calculation;
and performing self-adaptive optical correction on the fluorescence light path according to the voltage distribution matrix to obtain corrected wavefront aberration.
6. The adaptive optics based multifocal structured light super-resolution imaging method according to claim 5, wherein said calculating an aberration-corrected phase map of the laser light path from wavefront aberrations comprises:
calculating a PV surface type in the wavefront aberration, and performing interpolation processing on the PV surface type to obtain a PV coordinate value;
and calculating the aberration correction phase diagram according to the PV coordinate value and a preset formula.
7. The adaptive optics based multifocal structured light super-resolution imaging method according to claim 6, characterized in that the preset formula is:
mod(K*PV(x1,y1)/λ,2π)；
λ represents the wavelength of the probe wavefront;
k denotes a response factor.
8. The adaptive optics based multifocal structured light super-resolution imaging method according to claim 5, wherein the Hartmann sensor and the deformable mirror are combined into a closed loop correction, and a voltage distribution matrix of a correction surface type of the deformable mirror is obtained through iterative calculation, and the method comprises the following steps:
sequentially adjusting the voltage value of the brake of the deformable mirror according to a preset voltage, and judging whether the detected wavefront aberration tends to a preset value;
and if the detected wavefront aberration tends to a preset value, setting the current voltage value of the brake of the deformable mirror as a voltage distribution matrix of the correction surface type of the deformable mirror.
9. The adaptive optics based multifocal structural light super-resolution imaging method according to claim 8, wherein the controlling the spatial light modulator continuously loads the generated multifocal excitation dot matrix phase diagram, and sequentially receives sample fluorescence information after adaptive optics correction through a detector, and the method comprises the following steps:
controlling the deformable mirror to load the correction surface shape;
controlling the spatial light modulator to load the aberration-corrected phase map;
sequentially loading the generated multifocal excitation dot matrix phase diagrams to scan a sample;
and sequentially acquiring fluorescent images generated by the corresponding multifocal dot matrix excitation samples through a camera to obtain the multifocal structural light super-resolution imaging.
10. A multifocal structured light super-resolution imaging system based on adaptive optics is characterized by comprising: the system comprises an illuminating device, an adaptive optical correction device, a fluorescence detection imaging device and a terminal; the lighting device, the adaptive optical correction device and the fluorescence detection imaging device are respectively connected with the terminal;
the lighting device includes: a laser and a spatial light modulator; the spatial light modulator is used for modulating incident laser emitted by the laser to generate single-point exciting light, multi-focus lattice exciting light and multi-focus lattice exciting light corrected by the adaptive optical correction device;
the adaptive optical correction device includes: a Hartmann sensor and a deformable mirror; the Hartmann sensor is used for detecting wavefront information, and the deformable mirror is used for correcting system aberration during imaging and image difference caused by sample aberration on imaging;
the fluorescence detection imaging device includes: a camera; the camera is used for detecting a fluorescence signal emitted by the sample;
the terminal is used for controlling the laser to generate laser and controlling the spatial light modulator to modulate the laser;
the device comprises a Hartmann sensor, a spatial light modulator, a deformable mirror, a phase correction image sensor and a phase correction image sensor, wherein the phase correction image sensor is used for detecting the wave front aberration;
and the multi-focus excitation dot matrix phase diagram is used for controlling the spatial light modulator to continuously load the generated multi-focus excitation dot matrix phase diagram, and the sample fluorescence information after self-adaptive optical correction is sequentially received through the detector until the scanning of the multi-focus excitation dot matrix is finished, so that the multi-focus structure light super-resolution imaging is realized.
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