CN115248498A - Structured light super-resolution self-adaptive microscope device based on LED light source and imaging method - Google Patents

Abstract

The invention discloses a structured light super-resolution self-adaptive microscope device and an imaging method based on an LED light source, relates to the field of microscopes, and has the advantages of simple structure, use of a DMD as a structured light generating device, modulation of different phases through a controller, capability of improving the resolution of an image acquired by a microscope by one time at most, no moving part, simplification of an adjusting process, convenience for stability and reliability of a system and reduction of the resolution of the image caused by imaging aberration of deep tissues.

Description

Structured light super-resolution self-adaptive microscope device based on LED light source and imaging method

Technical Field

The invention relates to the field of microscopes, in particular to a structured light super-resolution self-adaptive microscope device based on an LED light source and an imaging method.

Background

The super-resolution fluorescence microscope illuminated by structured light has the basic principle that: exciting fluorescence by using a light beam with a spatial structure, exciting a mixed frequency of a pattern and fluorophore density, and carrying high-frequency information which is usually invisible in a sample to a visible low-pass band of a microscope; by changing the direction and phase of the pattern, the fluorescence result is recorded, the obtained multiple image data sets are properly processed, the carried high-frequency information is extracted, and a super-resolution image of the detected object is reconstructed. The transverse resolution of SIM (Structure Illumination microscope) can theoretically reach twice that of the traditional fluorescence microscope, the imaging speed is high, and no special requirement is required for fluorescent labeling.

In addition, in the use of the structured light, the higher the axial resolution is, the higher the modulation frequency is required to be, so when the objective lenses with different parameters are used, the required modulation frequency is changed, and the spatial frequency of the grating is fixed, so when the objective lenses are switched, the grating with proper frequency needs to be manually replaced, and the operation and the use are troublesome.

The fluorescence microscope specifically labels biological tissues or cells and the like by means of a fluorescence probe and enables living body and high resolution imaging, but the conventional optical microscope is limited by a diffraction limit due to the fluctuating nature of light and it is difficult to achieve the diffraction limit resolution due to the presence of aberrations, which are mainly derived from the following three types: 1) Low order aberrations introduced by optical system tuning and optics imperfections; 2) Aberrations caused by non-uniform refractive index of the biological sample; 3) Aberration caused by mismatch of refractive index between the biological sample and the microscope immersion medium. The aberration causes the contrast and resolution of the image to be reduced, and the complexity of the biological tissue (such as the effect of reflecting and scattering light) affects the propagation of light, so that the fluorescence microscope cannot perform high-resolution imaging on the deep layer of the living biological tissue.

Therefore, when a living tissue or a tissue with a certain depth is imaged microscopically, the structured light is adjusted more complexly, the imaging speed is influenced, and when the tissue with the certain depth is imaged, the condition that the refractive index is uneven due to uneven thickness of the tissue occurs, so that aberration is difficult to correct, and the imaging definition is influenced.

When the fluorescence microscope is used, a xenon lamp or a halogen lamp is generally used as an illumination light source and illuminates a DMD chip to generate structured light, the emergent light of the DMD irradiates the surface of an object after passing through a filter, a spectroscope, an objective lens and other devices, and the service life of the halogen light source or the xenon lamp is shorter than that of an LED light source, so that the brightness of the light source is reduced after the fluorescence microscope is used for a certain time, and the light source needs to be replaced frequently; if the external voltage fluctuates, the light source intensity of the halogen light source or the xenon lamp will change, the change of the light intensity will cause the obtained fluorescence information of the object to be distorted or the effect to be poor, even the fluorescence signal of the object can not be excited due to the insufficient light intensity, so the change information of the light source intensity needs to be monitored in real time, and the stable and good illumination is the basic guarantee for the normal work of the fluorescence microscope.

Disclosure of Invention

The invention aims to provide a structured light super-resolution self-adaptive microscope device and an imaging method based on an LED light source, which can correct the aberration of microscope imaging in real time through the cooperation of structures so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme:

the structured light super-resolution self-adaptive microscope device based on the LED light source comprises a light source, wherein the light source is projected to the surface of a DMD device through Kohler illumination after being emitted; the DMD device generates cosine structured light to be illuminated, and the cosine structured light sequentially passes through the reflector and the focusing lens and enters the optical filter cube of the fluorescence module; the fluorescence module optical filter cube is used for reflecting the exciting light to the objective table and transmitting the fluorescence on the surface of the object on the objective table to the wavefront corrector; the wave-front corrector is connected with the wave-front controller and is used for correcting wave-front errors; and the camera is used for acquiring and shooting the fluorescence image after aberration correction.

As a further scheme of the invention: the DMD device includes a plurality of mirrors and a micro-motor for controlling the mirrors, and the mirrors include a first mirror that is not angularly deflected, a second mirror that is deflected +12 degrees with respect to the first mirror, and a third mirror that is deflected-12 degrees with respect to the first mirror.

As a further scheme of the invention: and a light source detector is arranged at the position of +12 degrees or-12 degrees of deflection of the DMD device and is used for monitoring and recording the structural light intensity of the DMD device in real time.

As a further scheme of the invention: the fluorescence module filter cube comprises an excitation light filter, an emission light filter and a dichroic mirror.

As a further scheme of the invention: the wavefront corrector is provided with a spectroscope at one side of a light path, the wavefront sensor is arranged on a reflection light path of the spectroscope, and the camera is positioned on the light path of light transmitted by the spectroscope.

As a further scheme of the invention: a focusing objective lens is arranged between the fluorescence module optical filter cube and the objective table, and a focusing lens for focusing light is arranged between the camera and the spectroscope.

The imaging method of the structured light super-resolution self-adaptive microscope device based on the LED light source comprises the following steps:

s1, injecting fluorescent microspheres at the same depth of a tissue to be detected;

s2, an illumination light source is projected to the surface of the DMD device through Kohler illumination, the DMD device generates cosine structured light and projects the cosine structured light to a reflector, the reflector reflects the structured light to an objective lens to form parallel structured light which projects to a light filter cube of a fluorescence module and only allows light of a specific waveband to pass through;

s3, reflecting the light with the specific wave band to an objective table to excite the fluorescence of the object, wherein the fluorescence of the object passes through a fluorescence module optical filter cube;

s4, the wavefront corrector receives a fluorescence signal of a tissue to be detected, and the aberration condition from the fluorescent microsphere to the detector is obtained;

s5, taking the luminescence of the detected fluorescent microspheres as a reference light source to measure wavefront errors, generating control voltage of a wavefront corrector by using wavefront information in a reconstruction matrix mode, and applying current correction voltage to the wavefront corrector by the wavefront controller until the wavefront errors are minimum;

and S6, acquiring the fluorescence image after aberration correction by using a camera.

Compared with the prior art, the invention has the beneficial effects that: the invention has novel structure, uses DMD as a structured light generating device, modulates different phases through the controller, can improve the resolution of an image acquired by a microscope by one time at most, has no moving part, simplifies the adjustment process, is convenient for the stability and reliability of the system, solves the problem of image resolution reduction caused by deep tissue imaging aberration, corrects the aberration caused by non-uniform refractive index or change of a biological sample in real time by additionally arranging the self-adaptive optical device and the system at the front end of the detector, is convenient for a fluorescence microscope to carry out high resolution imaging on the deep part of a living biological tissue, is additionally provided with the light source intensity monitoring device by combining the characteristics of the DMD, is convenient for monitoring the change of the light source intensity in real time, provides real-time feedback for a user when the light intensity is changed, and thus carrying out light source replacement operation or checking the condition of voltage change and the like.

Drawings

FIG. 1 is a schematic diagram of the overall construction of a microscope system;

FIG. 2 is a side view of a DMD device;

FIG. 3 is a front view of a DMD device;

FIG. 4 is a schematic diagram of a deformable mirror in a wavefront corrector;

in the figure: 1-illumination light source, 2-first lens, 3-slit, 4-second lens, 5-DMD device, 6-reflector, 7-focusing lens, 8-excitation light filter, 9-dichroic mirror, 10-focusing objective lens, 11-objective table, 12-emission light filter, 13-wavefront corrector, 14-spectroscope, 15-wavefront sensor, 16-wavefront controller, 17-focusing mirror, 18-camera, 21-first reflector mirror, 22-second reflector, 23-third reflector, 31-thin reflecting mirror, 32-driver, 33-substrate, 34-wire.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the embodiment of the invention, the structured light super-resolution self-adaptive microscope system based on the LED light source comprises,

the light source emits light, and the light is projected to the surface of the DMD device 5 through the Kohler lighting system;

the DMD device 5 generates cosine structured light, and the cosine structured light enters a light filter cube of the fluorescence module after sequentially passing through a reflector 6 and a focusing lens 7;

the fluorescence module optical filter cube is used for reflecting exciting light to the objective table 11 and transmitting fluorescence on the surface of an object on the objective table 11 to the wavefront corrector 13;

the wavefront corrector 13 is connected with the wavefront controller 16 and is used for correcting the wavefront error;

and a camera 18, the camera 18 being configured to acquire and capture the aberration-corrected fluorescence image.

The Kohler lighting system comprises a first lens 2, a slit 3 and a second lens 4 which are sequentially arranged along a light path, wherein the first lens 2 is used for focusing an image of a light source at a crack, the slit 3 is used for shielding stray light, and the second lens 4 is used for converting the light source into parallel light to be emitted. The focusing lens 7 is used for converting light into parallel light, the fluorescence module filter cube comprises an excitation light filter 8, a dichroic mirror 9 and an emission light filter 12, and the excitation light filter 8 has selective light permeability and can permeate light of a specific wave band; the dichroic mirror 9 can selectively reflect and transmit light with a specific wave band according to the characteristics of surface coating; the emission light filter 12, like the excitation light filter 8, passes light of a specific wavelength band.

A focusing objective lens 10 is arranged between the cube of the fluorescence module optical filter and the objective table 11 and is used for focusing the excitation light reflected by the cube of the fluorescence module optical filter on an object on the objective table 11; the object stage 11 is used for placing an object to be detected; the wavefront corrector 13 is used to change the optical path lengths of the incident light at different positions.

The wavefront corrector 13 is provided with a spectroscope 14 at one side of the light path, the wavefront sensor 15 is arranged on the reflection light path of the spectroscope 14, the camera 18 is positioned on the light path of the light transmitted by the spectroscope 14, and a focusing lens 17 for focusing light is arranged between the camera 18 and the spectroscope 14; the beam splitter 14 has no selectivity to wavelength, and the beam splitter 14 generally divides the incident light into two paths according to the intensity, and the two paths of light are perpendicular to each other. The wavefront sensor 15 can acquire wavefront characteristics of different positions, and the wavefront controller 16 is used for controlling the wavefront corrector 13 to make a structure at different positions slightly change.

The DMD device 5 (digital micromirror device) is an array composed of a plurality of high-speed digital light-reflecting mirrors, the DMD device 5 is composed of a plurality of small aluminum mirror surfaces, the number of the mirror surfaces is determined by the display resolution, and one small mirror corresponds to one pixel. The DMD is an electronic-in, optical-out micro-electromechanical system (MEMS) by which developers can perform high-speed, efficient, and reliable spatial light modulation, each DMD containing millions of individually controlled micromirrors (built on corresponding CMOS memory cells). During operation, the DMD controller loads each elementary memory cell with a "1" or a "0", followed by the application of a mirror reset pulse, which causes each micromirror to electrostatically deflect about one hinge to reach the corresponding +/-12 ° state. The angle of divergence of these two effective states is repeatable due to the physical stop afforded by the two pogo pins.

The DMD is a method in which a computer controls the deflection of a small mirror on the DMD through a DMD drive board, adjusts the PWM value of the DMD to produce spatially uniform illumination in the +12 ° reflection direction of the DMD, and produces a modulation pattern required in the structured light method.

As shown in fig. 3, the DMD device 5 includes a plurality of mirrors 6 and a micro-motor for controlling the mirrors 6, and the mirrors 6 include a first mirror 21 that is not angularly deflected, a second mirror 22 that is deflected by +12 ° with respect to the first mirror 21, and a third mirror 23 that is deflected by-12 ° with respect to the first mirror 21. The size of the reflective mirror is about 10um square, the reflective mirror has three states, as shown in fig. 3, the first reflective mirror 21 is in a non-deflection state, the second reflective mirror 22 is in a state of deflecting by +12 degrees, the third reflective mirror 23 is in a state of deflecting by-12 degrees, the incident light on the surface of the DMD is generally parallel light, the directions of the reflected light are different in different deflection state reflective mirrors, as shown in fig. 2, the parallel light is incident on the surface of the DMD, the light reflection direction of the second reflective mirror 22 is vertically deviated by left 24 degrees, and the light reflection direction of the third reflective mirror 23 is vertically deviated by right 24 degrees.

Fig. 3 is a simplified diagram of a small mirror array on the surface of the DMD, for example, a few columns of mirrors are deflected by +12 ° or-12 °, as shown in fig. 3, the 1 st column indicates that the mirrors are deflected by +12 °, the 2 nd column does not deflect or deflects by-12 °, and then the 3 rd column deflects by +12 °, so as to form a loop, if the deflection of +12 ° is the required structured light pattern, the remaining small mirrors can deflect by-12 °, and the light is projected onto a light source detector, thereby completing the detection of the light intensity change of the light source by using the remaining small mirrors without affecting the structured light illumination.

The adaptive microscope system further comprises a light source detector, wherein the light source detector is located at a position where the DMD device 5 deflects by +12 degrees or-12 degrees and is used for monitoring and recording the structural light intensity of the DMD device 5 in real time. Incident light irradiates the surface of the DMD after passing through the Kohler illumination dodging system, the surface of the DMD is already provided with input fixed patterns, for example, some columns of small reflectors 6 deflect by +12 degrees to form structured light patterns, and the rest reflectors 6 deflecting by-12 degrees are unnecessary positions, so that the light source detectors can be reasonably arranged, the light reflected by the reflectors 6 deflecting by-12 degrees is on the light source detectors, and the light source detectors can monitor and record the change of the incident light intensity in real time on the premise of not influencing the structured light illumination.

The optical filter cube of the fluorescence module is composed of an excitation optical filter 8, an emission optical filter 12 and a dichroic mirror 9, cosine-structured light is illuminated on a focusing objective lens 10 through reflection of the dichroic mirror 9 and then focused on the surface of a sample, emission light on the surface of the object passes through a focusing lens 7 and then reaches the dichroic mirror 9, the wavelength can penetrate through the dichroic mirror 9 due to the fact that the wavelength of the excited fluorescence is long, the wavelength enters the emission optical filter 12, the emission optical filter 12 further filters the waveband of the excitation light, only fluorescence signals pass through, and the contrast of images collected by a camera 18 is improved conveniently.

A structured light super-resolution self-adaptive microscope imaging method based on an LED light source comprises,

s1, injecting fluorescent microspheres at the same depth of a tissue to be detected;

s2, the illumination light source 1 is projected to the surface of a DMD device 5 through Kohler illumination, the DMD device 5 generates cosine structured light and projects the cosine structured light to a reflector 6, the reflector 6 reflects the structured light to an objective lens to form parallel structured light, the parallel structured light is projected to a fluorescence module optical filter cube, and only light with a specific waveband passes through the parallel structured light;

s3, reflecting the light with the specific wave band to an objective table to excite the fluorescence of the object, wherein the fluorescence of the object passes through a fluorescence module optical filter cube;

s4, the wavefront corrector 13 receives a fluorescence signal of a tissue to be detected, and the aberration condition from the fluorescent microsphere to the detector is obtained;

s5, taking the luminescence of the detected fluorescent microspheres as a reference light source to measure wavefront errors, generating control voltage of the wavefront corrector 13 by using wavefront information in a reconstruction matrix mode, and applying a certain current correction voltage to the wavefront corrector 13 by the wavefront controller 16 until the wavefront errors are minimum;

s6, the camera 18 collects the fluorescence image after aberration correction.

The principle on which the adaptive microscope imaging method in the application is based is as follows:

self-adaptive optics comes from astronomical observation, and because the influence of the dynamic wavefront error that atmospheric turbulence produced, when telescope magnification is great, the formation of image facula can take place fuzzy and shake, and this very big influence the resolution ratio of observing. The optical path of the internal information in the deep tissue layer at different imaging positions on the image plane of the camera 18 is different due to the difference of the refractive index and even the refractive index change caused by the tiny movement of the tissue, so that aberration is generated, and the image is blurred and unclear, therefore, the aberration condition from the microsphere to the detector can be obtained by injecting the fluorescent microspheres in the depth direction of the tissue, the image of the fluorescent microspheres is adjusted to be clearest through the adjustment of the wavefront corrector 13, and the image of the object tissue at the same depth is also clear due to the correction of the aberration. The fluorescence signal passes through a wave-front corrector 13 of an adaptive optics system, a mode of injecting fluorescence microspheres at the same depth of a tissue to be detected is adopted, a wave-front error is measured by detecting the light emission of the fluorescence microspheres as a reference light source, then a matrix reconstruction mode is adopted, wave-front corrector 13 control voltage is generated by utilizing wave-front information, finally a certain current correction voltage is applied to the wave-front corrector 13 until the wave-front error is minimum, the adaptive correction system is closed, as shown in figure 1, the object fluorescence signal passing through a light emission filter 12 passes through the wave-front corrector 13, a spectroscope 14, a wave-front sensor 15 and a wave-front controller 16 system, the aberration of a fluorescence image is corrected, and the other path passes through the focusing of a focusing mirror 17 and is imaged on the surface of a detector of a scientific camera 18, namely, the aberration of the fluorescence image received by the camera 18 is corrected, so the definition of the fluorescence image is better than that before correction.

The aberration can cause the broadening of a focusing light spot, the reduction of imaging resolution, brightness and contrast, and the error source of the microscope mainly has two aspects: 1. the design and installation and adjustment errors of the microscope optical system; 2. the error caused by the non-uniformity of the refractive index of the sample generally increases with the imaging depth, and the aberration at different positions within the sample differs more.

The wavefront distortion is directly measured by using fluorescence emitted by a guide star with a known diameter, photoprotein and the like or using backscattered light of a sample as beacon light of a WFS (WFS), wherein the common WFS comprises a shack-Hartmann wavefront sensor 15 (SHWS), a shearing interferometer, a rectangular pyramid wavefront sensor 15 and the like, wherein the SHWS is most widely applied, and the Hartmann sensor divides an incident wavefront into a series of small regions, namely sub-apertures, by adopting an aperture division method and then focuses each sub-aperture on a detection surface. The signals of each sub-aperture are recorded by a photoelectric detector, and then the wave front phase average slope of each sub-aperture area is respectively calculated, the wave front phase of the input wave front is reconstructed by a certain recovery algorithm, and the wave front slope detected by the Hartmann sensor is the first derivative of the incident wave front phase, so that the recovery from the wave front slope to the wave front phase can be realized by a certain algorithm. In astronomy, the actual aberration is usually represented by a point diagram of ray tracing, the aberration can also be represented by wave aberration, and the wave surface is generally not spherical after the light wave emitted from an object point passes through an optical system, so the aberration is usually represented by the point diagram or the wave front difference.

The wavefront corrector 13 is an active optical device capable of rapidly changing the phase of a wavefront, and is also a core device of an AO system, and can correct a distorted wavefront by changing the path through a transmission medium or the refractive index of the transmission medium. The wavefront corrector 13 includes a deformable mirror, which is composed of a thin reflective mirror adhered to a driver, and the driver generates positive and negative deformation when positive and negative external voltages are applied, so as to drive the thin mirror to deform and change the wavefront of the system, thereby achieving the function of correcting wavefront errors.

The illumination light source 1 in the imaging method S2 of the microscope device generally uses a xenon lamp or a halogen light source, has a wide spectral range and is convenient for selecting a specific waveband light source for use subsequently; the first lens 22 focuses the image of the light source at the slit 3; the slit 3 can block stray light; the second lens 4 can convert the light source into parallel light to be emitted; the first lens 2, the slit 3 and the second lens 4 form a Kohler illumination system, so that emergent light is uniform and then is projected onto the surface of the DMD device 5.

The light source generates exciting light with a specific spectrum band by a scheme of performing spectrum filtering on a wide-spectrum light source, and the purpose of being matched with various fluorescent probes is achieved. The wide-spectrum light source can be a high-power xenon lamp, halogen lamp light source or mercury lamp light source, and the emergent spectrum section of the wide-spectrum light source covers the range from visible light to near infrared wavelength, and is about 400 nm-2500 nm. The broad spectrum light source can generate white light with uniform light intensity distribution in the wavelength range from visible light to near infrared. Different exciting light filters 8 can be installed at the positions of the exciting light filters 8 in a switching mode, and a user can select a proper exciting light filter 8 according to a used fluorescent probe, so that emergent light is narrow-spectrum exciting light which can fully excite the fluorescent probe and does not introduce light interference of other spectrums. Wherein the exciting light filter 8 is a small-diameter band-pass interference filter, and the band-pass spectrum band of the filter is matched with the absorption spectrum of the used fluorescent probe.

The light emitted by the light source is firstly homogenized by the Kohler lighting system, then is reflected to the reflective mirror 6 by the surface of the DMD, and then reaches the surface of the sample to be detected and the structured light on the surface of the object by the condensing lens, the dichroic mirror 9, the objective lens and the like.

The optical signal conduction collection portion conducts the excitation light to a detection area on the stage 11, an object to be detected is on the detection area, and the collection portion can collect an optical signal of interest from the detection area. As shown in fig. 1, the filter cube of the fluorescence module includes an excitation light filter 8, a dichroic mirror 9, and an emission light filter 12, wherein cosine-structured light is illuminated on the front focal plane of the focusing objective lens 10 by reflection of the dichroic mirror 9, and then the light is illuminated on the object surface on the objective stage 11 by penetrating through the objective lens, the position plane of the object surface is the focal plane of the objective lens, and the reflection band and the transmission band of the dichroic mirror 9 can be selected as required.

The magnifying objective is a finite far field achromatic objective with RMS external threads, the magnification times can be generally 4X,10X,20X and 40X, the objectives are in parfocal, the fluorescent image can be magnified, a magnified real image is formed on the conjugate point of the magnifying objective, and the magnified real image passes through various optical devices and is finally projected onto a detection chip of the scientific camera 18.

As shown in fig. 1, different optical filters are selected to enter corresponding light paths, so that different fluorescence probes can be excited and fluorescence images of the corresponding fluorescence probes after being excited are collected, multispectral imaging of different fluorescence probes is conveniently achieved, the fluorescence images of an object on an object stage 11 after being excited are firstly received by an objective lens and then pass through a dichroic mirror 9, the dichroic mirror 9 has selective reflection and transmission characteristics on wavelength, the excited fluorescence can pass through the dichroic mirror 9 and then be projected onto an emission light filter 12, the emission light filter 12 has selective transmission on wavelength, stray light except the fluorescence can be filtered, interference is reduced, light is continuously transmitted, the surface of an adaptive optical corrector is reflected onto a spectroscope 14, the spectroscope 14 does not have wavelength selectivity, the fluorescence is generally divided into two paths according to light intensity, one path passes through a wavefront corrector 1313, the spectroscope 14, a wavefront sensor 15 and a wavefront controller 16, the closed loop forms an adaptive optical aberration correction system, aberration caused by uneven refractive index at the position of the object to be detected can be corrected in real time, aberration of the fluorescence images is corrected, the other path passes through a wavefront sensor 17, the imaging of a scientific camera 18, and the aberration of the fluorescence images can be corrected better than before the fluorescence images are focused by a focus camera 18.

The camera 18 generally uses a Scientific Complementary Metal Oxide Semiconductor (SCMOS) camera for receiving the light signal with weak imaging intensity and converting the light signal into a digital image by sampling, and the digital image effectively images the fluorescence signal with weak imaging intensity to obtain an original fluorescence image and also can collect an excitation light and a white light image.

The wavefront phase error is corrected by a certain deformation generated by a deformable mirror, the driving voltage of a driver is directly related to the wavefront phase error, and the general control method is to firstly obtain the wavefront phase error at the position of a driving point and then calculate the control error voltage of the position through a proportional algorithm.

The method comprises the following specific steps:

1. calculating the wavefront

The Hartmann sensor adopts an aperture division method to divide an incident wave surface into a series of small areas, namely sub-apertures, and then focuses each sub-aperture on a detection surface respectively. And recording signals of each sub-aperture through a photoelectric detector, calculating the wave front phase average slope of each sub-aperture area, and reconstructing the wave front phase of the input wave front by a certain recovery algorithm.

The wave aberration in the sub-aperture has the following relationship with the wavefront slope measured by the detector:

wherein:

jth sub-aperture centroid coordinate

I _i : signal intensity of ith pixel point on CCD

(X _i ，Y _i ): coordinate position of ith pixel on CCD

λ: incident beam wavelength

f: focal length of imaging system

S _j : jth sub-aperture entrance pupil area

Φ (, y): wave aberration at (x, y) position on entrance pupil plane

X-direction slope of j-th sub-aperture incident wave front

Y-direction slope of j-th sub-aperture incident wavefront

It can be seen that the wavefront slope detected by the hartmann sensor is the first derivative of the phase of the incident wavefront, and therefore the reconstruction of the wavefront slope into the wavefront phase can be achieved by a certain algorithm.

2. Wavefront restoration

Since the measured quantity of the H-S sensor is the average wavefront slope within the sub-aperture, an algorithm is required to find the wavefront error and the correction voltage that each driver should apply, a process called wavefront reconstruction. The current wave-front restoration algorithm used for the Hartmann wave-front sensor mainly comprises a Zernike mode method.

Mode wavefront reconstruction is to decompose the wavefront phase into a series of orthogonal modes and then to derive the partial derivatives for these modes for x and y, respectively. The signal detected by the CCD is essentially these partial derivatives. For a circular pupil, the analytical formula for the current relatively common wavefront phase decomposition is the Zernike mode decomposition:

wherein:

in the formula, m and n are respectively axial order and radial order.

Thus, the wavefront phase can be decomposed with Zernike modes:

wherein:

a _k : zernike polynomial coefficient of k-th order

Z _k (x, y): zernike polynomials of order k

Thus, the wavefront slope detected by the Hartmann sensor is related to the wavefront phase of the incident beam by:

is recorded as:

G＝DA+ε

due to the incomplete orthogonality of the partial derivatives of the Zernike functions and the non-orthogonality of the functions at limited sampling points, the rank of the matrix D may be incomplete and the condition numbers of the equations may vary. For arbitrary 2m and n, the least squares solution of the above equation can be used with the generalized inverse D ⁺ Represents:

A＝D ⁺ G+(1-D ⁺ D)Y

y is an arbitrary vector having a square equation of least squares | D when Y =0 ⁺ The solution in the sense of D-a | = min and minimum norm | a | = min is:

A＝D ⁺ G

thus, the inverse matrix D of D is obtained ⁺ That is, the Zernike coefficient a can be obtained _k A is to _k The wavefront phase can be calculated by substituting (2-4).

3. Wavefront correction

The wave-front corrector is a key device for realizing the dynamic error correction of the adaptive optical system, and comprises two main devices, namely a high-speed tilting mirror and a deformable mirror.

Fig. 4 is a schematic view showing the structure of the deformable mirror, which is composed of a thin reflective mirror 31 bonded to the actuator, and a substrate 33 and a wire 34 are sequentially provided. When positive and negative external voltages are applied to the driver 32, positive and negative deformations are generated, so that the thin mirror is driven to deform to change the wavefront of the system, and the function of correcting wavefront errors is achieved.

Using Gram-Schmidt orthogonalization method or singular value decompositionMoore-Penrose type generalized inverse matrix D of D matrix calculated by method ⁺ Then, the least squares minimum norm solution a = D of the equation can be given ⁺ G. The wavefront phase error is then given by:

Φ(x，y)＝∑a _k Z _k (x，y)

the wavefront phase error is corrected by a certain deformation of the deformable mirror, the driving voltage of the driver is directly related to the wavefront phase error, and the deformable mirror driving voltage required for generating the wavefront phase error needs to be calculated from phi (x, y), and can be generally calculated by using a direct proportional control method.

The principle of the direct proportional control method is that the wavefront phase error of the position of a driving point is firstly obtained, and then the control error voltage of the position is calculated through a proportional algorithm. Because the gluing surface between the mirror surface of the deformable mirror and the driver has a certain size, high-order deformation cannot be generated in the gluing area, otherwise, the deformable mirror is degummed. Therefore, the average phase of the mirror error in the mirror glue area should be calculated, and the wavefront phase of the jth glue area is obtained by the equation (2-4):

can be simplified as follows:

t is a corrective element, a ₀ For the phase shift constant of the wavefront to be determined, when a ₀ Where 0, the mode of the wavefront phase Φ (j) is nearly minimal, the above equation can be written as:

Φ＝T _Z A＝(T _Z D ⁺ )G

the control voltage is as follows:

V＝PΦ＝(PT _Z D ⁺ )G＝V _p G

matrix P _t*t Is a phase ratio matrix which converts the wave front phase phi into the driving voltage V, V of the deformable mirror _p It is a proportional control matrix that converts the slope G into a control voltage V.

By the control method, the aberration caused by the uneven or changed refractive index of the biological sample can be corrected in real time, and the high-resolution imaging of the deep part of the living biological tissue by the fluorescence microscope is effectively improved.

Example 2:

in S2, structured light with different phases is generated by adjusting the DMD, and high-frequency information can be reversely derived through an algorithm, i.e., resolution of the acquired image is improved, and theoretically, the resolution can be improved by one time at most, specifically:

by designing stripe patterns with different inclination directions, generally three inclination directions, with 120 DEG direction difference, stripe patterns with different phases, generally three phases and 2 pi/3 phase difference, and projecting the stripe patterns onto an object to be detected, wherein 9 patterns are obtained in total, the obtained image also consists of three kinds of frequency information, including low-frequency information (or called zero-frequency information), and high-frequency and low-frequency information obtained by translating the zero-frequency information to the left and right sides, the low-frequency information and the high-frequency information mixed together in the obtained image can be separated by a proper algorithm, and are respectively moved to the original correct positions and then superposed, thereby realizing the expansion of sample information in a frequency domain, greatly improving the resolution level after reconstruction, generally representing the low-resolution information by the low-frequency component of the object to be detected, and representing the high-resolution information by the high-frequency component, and obtaining the high-frequency component by a series of data inversion, thereby improving the resolution of the object to be detected.

The invention has novel structure and stable operation, when in use, the DMD is used as a structured light generating device, different phases are modulated by the controller, the resolution of an image acquired by a microscope can be improved by one time at most, no moving part is arranged, the adjustment process is simplified, the system is convenient to be stable and reliable, the problem of image resolution reduction caused by deep tissue imaging aberration is solved, the self-adaptive optical device and the system are additionally arranged at the front end of the detector, the aberration caused by non-uniform or changed refractive index of a biological sample is corrected in real time, the fluorescence microscope is convenient to carry out high-resolution imaging on deep parts of living biological tissues, a light source intensity monitoring device is additionally arranged by combining the characteristics of the DMD, the change of the light source intensity is convenient to monitor in real time, and when the light intensity is changed, a real-time feedback is provided for a user, so that the light source replacement operation or the condition of voltage change is checked, and the like.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (7)

1. Structured light super-resolution self-adaptive microscope device based on LED light source, which is characterized by comprising:

the light source is projected to the surface of the DMD device through Kohler illumination after being emitted;

the DMD device generates cosine structured light to be illuminated, and the cosine structured light sequentially passes through the reflector and the focusing lens and enters the optical filter cube of the fluorescence module;

the fluorescence module optical filter cube is used for reflecting exciting light to the objective table and transmitting fluorescence on the surface of an object on the objective table to the wavefront corrector;

the wave-front corrector is connected with the wave-front controller and is used for correcting wave-front errors;

and the camera is used for acquiring and shooting the fluorescence image after aberration correction.

2. The LED light source based structured light super-resolution adaptive microscope device according to claim 1, wherein the DMD device comprises a plurality of mirrors and a micro-motor for controlling the mirrors, the mirrors comprising a first mirror that is not angularly deflected, a second mirror that is deflected +12 ° with respect to the first mirror, and a third mirror that is deflected-12 ° with respect to the first mirror.

3. The structured light super-resolution adaptive microscope device based on the LED light source as claimed in claim 2, wherein a light source detector is arranged at a position where the DMD device deflects by +12 ° or-12 °, and is used for monitoring and recording the structured light intensity of the DMD device in real time.

4. The LED light source-based structured light super-resolution adaptive microscope device according to claim 1, wherein the fluorescence module filter cube is comprised of a piece of excitation light filter, a piece of emission light filter, and a piece of dichroic mirror.

5. The LED light source based structured light super-resolution adaptive microscope device according to claim 1, wherein the wavefront corrector is provided with a beam splitter on one side of the optical path, the wavefront sensor is provided on the reflected optical path of the beam splitter, and the camera is located on the optical path of the transmitted light of the beam splitter.

6. The LED light source based structured light super-resolution adaptive microscope device according to claim 5, wherein a focusing objective lens is arranged between the fluorescence module filter cube and the objective table, and a focusing lens for focusing light is arranged between the camera and the spectroscope.

7. The imaging method of the LED light source based structured light super-resolution adaptive microscope device according to any one of claims 1 to 6, comprising:

s1, injecting fluorescent microspheres at the same depth of a tissue to be detected;

s2, an illumination light source is projected to the surface of a DMD device through Kohler illumination, the DMD device generates cosine structured light and projects the cosine structured light to a reflector, the reflector reflects the structured light to an objective lens to form parallel structured light, the parallel structured light is projected to a light filter cube of a fluorescence module, and only light with a specific waveband passes through the light filter cube;

s3, reflecting the light with the specific wave band to an objective table to excite the fluorescence of the object, wherein the fluorescence of the object passes through a fluorescence module optical filter cube;

s4, the wavefront corrector receives a fluorescence signal of a tissue to be detected, and the aberration condition from the fluorescent microsphere to the detector is obtained;

s5, taking the luminescence of the detected fluorescent microspheres as a reference light source to measure wavefront errors, generating control voltage of a wavefront corrector by using wavefront information in a reconstruction matrix mode, and applying current correction voltage to the wavefront corrector by the wavefront controller until the wavefront errors are minimum;

and S6, acquiring the fluorescence image after aberration correction by using a camera.

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