CN108121059A - A kind of parallel micro imaging systems of STED based on Structured Illumination - Google Patents

Abstract

The STED parallel microscopic imaging system based on structured light illumination provided by the present invention divides a beam of excitation light emitted by the excitation light laser into two beams of coherent light by designing the illumination module, and divides a beam of depleted light emitted by the depletion laser into two beams of coherent light. Two beams of coherent light are obtained by interference to obtain uniformly distributed excitation structured light and loss structured light respectively, and then excite and deplete the sample for STED parallel microscopic imaging, and then use the STED coordinate positioning method and The SIM frequency-domain spectrogram fusion method is used for image reconstruction to realize super-resolution imaging, which is helpful to expand the field of view and imaging speed of the STED microscope system.

Description

A STED Parallel Microscopic Imaging System Based on Structured Light Illumination

technical field

The invention relates to the field of design and manufacture of microscopic detection instruments, in particular to a STED parallel microscopic imaging system based on structured light illumination.

Background technique

Super-resolution microscopic imaging technology is a cutting-edge technology used in biological and other research fields. It is mainly divided into two types of microscopic imaging methods: coordinate positioning and coordinate random. Among them, the typical representative of the coordinate positioning microscopic imaging method is stimulated radiation depletion (STED) microscopic imaging. STED microscopic imaging directly uses optical methods to scan the sample point by point to achieve super-resolution imaging. However, limited by the single-point scanning method, the imaging speed of STED microscopic imaging is relatively slow.

In recent years, in order to improve the imaging speed of STED microscopic imaging technology, STED parallel microscopic imaging has been proposed. Among them, the better effect is the use of two-dimensional structured light illumination mode, which significantly improves the imaging speed. However, due to the addition of two-dimensional structured light, the complexity of the imaging system becomes higher, and the excitation light in this technology does not use structured light mode but wide-field excitation, which significantly reduces the signal-to-noise ratio of effective fluorescence and affects the imaging resolution.

Contents of the invention

The purpose of the present invention is:

A structured light illumination-based STED parallel microscopic imaging system with high imaging resolution and fast imaging speed is provided.

To achieve the above object, the present invention adopts the following technical solutions:

A STED parallel microscopic imaging system based on structured light illumination, including an illumination module, a detection module, a control module and an image reconstruction module;

The illumination module includes: an excitation light laser, a loss light laser, a liquid crystal spatial light modulator, a Wollaston prism, a first lens, a through-hole mask plate, a second lens, a first half-wave plate, a second half-wave sheet, reflecting mirror, first dichroic mirror, second dichroic mirror, tube lens, objective lens and three-dimensional nano-shift stage, the three-dimensional nano-shift stage can move in the XYZ three-dimensional direction, and the three-dimensional nano-shift stage carries the sample to be detected ;

The detection module includes an optical filter, a detection lens and an area array detector, and pixels in the area array detector can form a virtual pinhole;

The control module is electrically connected to the three-dimensional nano-shift stage, the area array detector and the liquid crystal spatial light modulator; the image reconstruction module is electrically connected to the control module; wherein:

The Gaussian beam emitted by the excitation light laser is used as the excitation light, and the excitation light is incident on the liquid crystal spatial light modulator, and the liquid crystal spatial light modulator modulates the excitation light to generate diffracted light, and the diffracted light includes ±1st-order diffracted light and 0-order diffracted light; the diffracted light is incident on the through-hole mask after being focused by the first lens, and the through-hole mask blocks the 0-order diffracted light, allowing only The ±1st-order diffracted light passes through, and the ±1st-order diffracted light passes through the first half-wave plate, the reflecting mirror, the dichromatic mirror, and the first half-wave plate sequentially after being collimated by the second lens. The dichroic mirror and tube lens enter the objective lens, and interfere at the front focal plane of the objective lens to generate interference fringe-shaped excitation structured light, and the excitation structured light illuminates and excites the sample;

The Gaussian beam emitted by the lost light laser is used as the lost light, and the lost light is divided into two beams of the first lost light and the second lost light whose polarization directions are perpendicular to each other through the Wollaston prism. The light and the second loss light have the same polarization direction after passing through the second half-wave plate, and the first loss light and the second loss light after passing through the second half-wave plate pass through the second dichroic mirror and the second loss light sequentially. After the tube lens enters the objective lens, and interferes at the front focal plane of the objective lens to generate interference fringe-shaped loss structured light, the loss structured light depletes the sample;

The sample is excited by the excited structured light and lost by the loss of the structured light to form a structured light field, and the structured light field sequentially passes through the objective lens, the tube lens, the second dichromatic mirror, and the second dichromatic mirror. A dichroic mirror, the filter and the detection lens form an image with uniform fringe distribution on the photosensitive surface of the area array detector, and the area array detector detects the image and passes through the virtual needle converting the image into an electrical signal after performing spatial filtering on the image;

The control module collects the electrical signal, and the image reconstruction module reconstructs the image of the electrical signal by using coordinate positioning and SIM frequency-domain spectrogram fusion to realize STED parallel microscopic imaging based on structured light illumination.

In some of the embodiments, the photosensitive surface of the liquid crystal spatial light modulator, the front focal plane of the objective lens, the light exit plane of the Wollaston prism and the photosensitive surface of the area array detector are in conjugate planes.

In some of the embodiments, the area detector is one of CCD or CMOS camera.

In some of these embodiments, by adjusting the phase distribution loaded on the liquid crystal spatial light modulator through the control module, the direction, period and initial phase of the excitation light field can be changed, so that the excitation light structured light field and loss The optical structured light field has the same direction and period, and the initial phase difference is half a period.

The present invention adopts the advantage of above-mentioned technical scheme to be:

The STED parallel microscopic imaging system based on structured light illumination provided by the present invention divides a beam of excitation light emitted by the excitation light laser into two beams of coherent light by designing the illumination module, and divides a beam of depleted light emitted by the depletion laser into two beams of coherent light. Two beams of coherent light are obtained by interference to obtain uniformly distributed excitation structured light and loss structured light respectively, and then excite and deplete the sample for STED parallel microscopic imaging, and then use the STED coordinate positioning method and The SIM frequency-domain spectrogram fusion method is used for image reconstruction to realize super-resolution imaging, which is helpful to expand the field of view and imaging speed of the STED microscope system.

Description of drawings

FIG. 1 is a schematic structural diagram of a STED parallel microscopic imaging system based on structured light illumination provided by an embodiment of the present invention.

Fig. 2 (a) is a schematic structural diagram of an excitation structured light light field provided by an embodiment of the present invention;

FIG. 2(b) is a schematic structural diagram of a lossy structured light field provided by an embodiment of the present invention;

Fig. 2(c) is a schematic structural diagram of an effective structured light field provided by an embodiment of the present invention;

Fig. 3 (a) is the striped image of structured light on the area array detector provided by the embodiment of the present invention;

FIG. 3(b) is a schematic structural diagram of a virtual pinhole provided by an embodiment of the present invention;

FIG. 4(a) is a schematic structural diagram of fusing frequency domain images of each two-dimensional image in the frequency domain provided by an embodiment of the present invention;

Fig. 4(b) is a schematic diagram of the structure of the final super-resolution image obtained through inverse Fourier transform transformation to the spatial domain provided by the embodiment of the present invention.

Among them: illumination module 110, detection module 120, control module 130, image reconstruction module 140, excitation light laser 111, loss light laser 112, liquid crystal spatial light modulator 113, Wollaston prism 114, first lens 115, through hole Mask plate 116, second lens 117, first half-wave plate 118, second half-wave plate 119, mirror 1110, first dichroic mirror 1111, second dichromatic mirror 1112, tube lens 1113, objective lens 1114, three-dimensional Nano displacement stage 1115 , optical filter 121 , detection lens 122 , area array detector 123 .

Detailed ways

Please refer to FIG. 1 , which is a STED parallel microscopic imaging system 100 based on structured light illumination provided by an embodiment of the present invention, an illumination module 110 , a detection module 120 , a control module 130 and an image reconstruction module 140 . in:

The illumination module 110 includes: an excitation light laser 111, a loss light laser 112, a liquid crystal spatial light modulator 113, a Wollaston prism 114, a first lens 115, a through-hole mask 116, a second lens 117, a first Half-wave plate 118, second half-wave plate 119, mirror 1110, first dichroic mirror 1111, second dichromatic mirror 1112, tube lens 1113, objective lens 1114 and three-dimensional nano-displacement stage 1115, described three-dimensional nano-displacement stage 1115 It can move in the XYZ three-dimensional direction, and the three-dimensional nano-displacement platform carries the sample to be detected.

The detection module 120 includes a filter 121 , a detection lens 122 and an area array detector 123 , and pixels in the area array detector 123 can form a virtual pinhole.

The control module 130 is electrically connected to the three-dimensional nano-shift stage 1115 , the area array detector 123 and the liquid crystal spatial light modulator 113 ; the image reconstruction module 140 is electrically connected to the control module 130 .

The working principle of the STED parallel microscopic imaging system 100 based on structured light illumination provided by the present invention is described in detail below:

The Gaussian beam emitted by the excitation light laser 111 is used as the excitation light, and the excitation light 111 is incident on the liquid crystal spatial light modulator 113, and the liquid crystal spatial light modulator 113 modulates the excitation light to generate diffracted light, so The diffracted light includes ±1st-order diffracted light and 0-order diffracted light; the diffracted light enters the through-hole mask 116 after being focused by the first lens 115, and the through-hole mask 116 blocks the The 0-order diffracted light only allows the ±1-order diffracted light to pass through, and the ±1-order diffracted light is collimated by the second lens 117 and then passes through the first half-wave plate 118 and the mirror 1110 in turn. , the first dichroic mirror 1111, the second dichroic mirror 1112 and the tube lens 1113 enter the objective lens 1114, and interfere at the front focal plane of the objective lens 1114 to generate interference fringe-like excited structured light , the excited structured light illuminates and excites the sample;

The Gaussian beam emitted by the lost light laser 112 is used as the lost light, and the lost light is divided into two beams of the first lost light and the second lost light whose polarization directions are perpendicular to each other through the Wollaston prism 114. The first lost light and the second lost light have the same polarization direction after passing through the second half-wave plate 119, and the first lost light and the second lost light after passing through the second half-wave plate 119 pass through the second and second half-wave plates in turn. The color mirror 1112 and the tube lens 1113 enter the objective lens 1114, and interfere at the front focal plane of the objective lens 1114 to generate interference fringe-shaped loss structured light, and the loss structured light depletes the sample;

The sample is excited by the excitation structured light and lost by the loss of the structured light to form a structured light field, and the structured light field passes through the objective lens 1114, the tube lens 1113, the second dichroic mirror 1112, The first dichroic mirror 1111, the filter 121 and the detection lens 122 form an image with uniform fringe distribution on the photosensitive surface of the area array detector 122, and the area array detector 122 detects the converting the image into an electrical signal after performing spatial filtering on the image through the virtual pinhole;

The control module 130 collects the electrical signal, and the image reconstruction module 140 reconstructs the image of the electrical signal by using coordinate positioning and SIM frequency-domain spectrogram fusion to realize STED parallel microscopic imaging based on structured light illumination.

Preferably, the photosensitive surface P1 of the liquid crystal spatial light modulator 113 , the front focal plane P3 of the objective lens 1114 , the light exit plane P4 of the Wollaston prism 114 and the photosensitive surface P5 of the area array detector 123 are in conjugate planes.

Further, the area array detector 123 can be a CCD or CMOS camera, which can receive the fluorescence signal of effective structured light in the fluorescent sample and convert it into an electrical signal; and the pixels in the area array detector 123 can form a virtual pinhole, Spatial filtering is performed on the detected structured light.

Further, since the control module 140 is electrically connected to the three-dimensional nano-displacement stage 1115, the area array detector 123 and the liquid crystal spatial light modulator 113, the three-dimensional nano-displacement stage 1115 can be controlled by the control module 140. , the scanning control of the area array detector 123 and the liquid crystal spatial light modulator 113, and the control module 130 transmits the collected electrical signal to the image reconstruction module 140, and the image reconstruction module 140 uses the SIM frequency domain spectrogram fusion method Image reconstruction is performed on the electrical signal to realize STED parallel microscopic imaging based on structured light illumination.

The image reconstruction module 140 uses coordinate positioning and SIM frequency-domain spectrogram fusion to reconstruct the image of the electrical signal, and realizes the working process of STED parallel microscopic imaging based on structured light illumination in detail below:

First, Parallel Detection of Effective Structured Light Fields

The phase distribution loaded on the liquid crystal spatial light modulator 113 is adjusted by the control module 130, and the direction, period, and initial phase of the excitation light field are changed, so that the excitation light structured light field and the loss light structured light field have the same direction and period, the initial phase difference is half a period; the sample forms an effective structured light field after the excitation of the excited structured light and the loss of the loss structured light, and the effective structured light field passes through the objective lens 1114, the tube lens 1113, The second dichroic mirror 1112, the first dichroic mirror 1111, the filter 121 and the detection lens 122 form an image with uniform fringe distribution on the photosensitive surface of the area array detector 123, In order to realize the detection and collection of parallel optical signals, please refer to Fig. 2(a), Fig. 2(b) and Fig. 2(c) respectively representing the excited structured light field, the loss structured light field and the effective structured light field.

Second, obtain the STED image of a given structured light direction and a given sample position

It can be understood that when the direction of the structured light and the position of the sample are given, the image that forms a uniform stripe distribution on the photosensitive surface of the area array detector 123 contains many light signal stripes, and each light signal stripe corresponds to a row of virtual pinholes , Fig. 3(a) and Fig. 3(b) respectively show the striped image of structured light on the area array detector and the virtual pinhole, where the long striped rectangle represents the light intensity distribution of a strip, and the small square grid represents the surface The pixels of the array detector, the larger square grid represents a virtual pinhole (including multiple pixels), the arrangement of the virtual pinhole is located in the center of the light signal stripes, suppressing stray light interference, and detecting each pixel in the virtual pinhole The obtained light intensity _i is superimposed, and the pixel value of the STED image corresponding to a point in the sample can be obtained Perform the same processing on all the virtual pinholes corresponding to each optical signal stripe to obtain the STED image pixel value corresponding to one row in the sample, and perform the same processing on the virtual pinholes in all optical signal stripes to obtain a pixel value STED image of the distribution of values in striped intervals.

Third, obtain the STED image of the sample after scanning in a given structured light direction

Keeping the direction of the structured light (excited structured light and depleted structured light) unchanged, the three-dimensional nano-shift stage 1115 is used to drive the sample to move step by step along the direction perpendicular to the structured light stripe, and the total number of times of movement in each structured light stripe direction is d _p /d _s -1, d _p is the fringe spatial period, d _s is the single moving step distance, each time the translation stage stays at a position, the area array detector 123 is used to record the sample stripe-shaped fluorescent image at this position, and the above-mentioned first The two-step processing method will obtain a STED image with pixel values distributed in strips and intervals. In this way, d _p /d _s STED images with pixel values distributed in strips and intervals will be obtained in each structured light stripe direction. These images are superimposed to generate an image corresponding to the direction of a structured light stripe. This image reconstruction method is recorded as a coordinate positioning method. The image realizes super-resolution imaging in a direction perpendicular to the structured light stripe.

Fourth, obtain the STED image of the sample scanned under structured light in all directions

Rotate the Wollaston prism 114, and correspondingly rotate the phase diagram loaded on the liquid crystal spatial light modulator 113, so that the structured light field fringes of the excitation light and loss light at the focal plane of the objective lens 1114 can be located in different directions, and generate different directions The effective fluorescent structured light field of each direction, the third step and the second step are performed on the effective fluorescent structured light field in all directions, and a two-dimensional image containing one-dimensional super-resolution information in each direction of the structured light field can be obtained.

Fifth, the super-resolution image is obtained through SIM frequency domain spectrum fusion

The SIM frequency-domain spectrogram fusion method is used for image reconstruction. First, the multiple two-dimensional images obtained in the fourth step are transformed into the frequency domain by Fourier transform, as shown in Figure 4(a). In the frequency domain, each The frequency domain images of the two-dimensional images are fused, as shown in Figure 4(b), and finally converted to the spatial domain through inverse Fourier transform to obtain the final super-resolution image.

In the STED parallel microscopic imaging system based on structured light illumination provided by the present invention, by designing the illumination module 110, one beam of excitation light emitted by the excitation light laser 111 is divided into two beams of coherent light, and one beam emitted by the lossy light laser 112 is The loss light is divided into two beams of coherent light, and uniformly distributed excitation structured light and loss structure light are obtained by interference, respectively, and the excitation and loss structure light are excited and lost to the sample, and STED parallel microscopic imaging is carried out, and then the coordinates of STED are used The positioning method and the SIM frequency-domain spectrogram fusion method are used for image reconstruction to realize super-resolution imaging, which is helpful to expand the field of view and imaging speed of the STED microscope system.

Of course, the structured light illumination-based STED parallel microscopic imaging system of the present invention can also have various transformations and modifications, and is not limited to the specific structure of the above-mentioned embodiment. In a word, the protection scope of the present invention shall include those transformations, substitutions and modifications obvious to those skilled in the art.

Claims (4)

1. the parallel micro imaging systems of a kind of STED based on Structured Illumination, which is characterized in that including lighting module, detection mould Block, control module and image reconstruction module；

The lighting module includes：Excite light laser, loss light laser, LCD space light modulator, Wollaston prism, First lens, vias masks plate, the second lens, the first half-wave plate, the second half-wave plate, speculum, the first dichroic mirror, the two or two color Mirror, cylinder mirror, object lens and three-dimensional manometer displacement platform, the three-dimensional manometer displacement platform can be in XYZ three-dimensional direction movings, three wieners Rice displacement platform carries detected sample；

The detecting module includes optical filter, finder lens and planar array detector, and the pixel in the planar array detector can shape Into dummy pinhole；

The control module is electrically connected at the three-dimensional manometer displacement platform, planar array detector and LCD space light modulator； Described image rebuilds module and is electrically connected at the control module；Wherein：

For the Gaussian beam that the excitation light laser is sent as exciting light, the exciting light incides into the liquid crystal spatial light tune Device processed, the LCD space light modulator are modulated the exciting light generation diffraction light, and the diffraction light spreads out including ± 1 grade Penetrate light and 0 order diffraction light；The diffraction light is incident after first lens focus to enter the vias masks plate, the through hole 0 order diffraction light described in mask plate shielding only allows ± 1 order diffraction light by the way that ± 1 order diffraction light is saturating through described second It is laggard through first half-wave plate, the speculum, the dichroic mirror, second dichroic mirror and cylinder mirror successively again after mirror collimation Enter the object lens, and interfered at the object lens front focal plane, generate the excitation structure light of interference fringe shape, the excitation knot Structure light is illuminated sample excitation；

The Gaussian beam that the loss light laser is sent is lost as loss light, the loss light through the Wollaston prism Light is divided into the mutually perpendicular first loss light in two beam polarization directions and the second loss light, the first loss light and the second loss light It is identical through the second half-wave plate rear polarizer direction, after second half-wave plate first loss light and second loss light again according to It is secondary to enter the object lens after second dichroic mirror and the cylinder mirror, and interfered at the front focal plane of the object lens, it is raw Into the loss structure light of interference fringe shape, sample is lost in the loss structure light；

The loss of excitation and loss structure light of the sample through the excitation structure light, forms structure light light field, the structure light light Field is successively through the object lens, the cylinder mirror, second dichroic mirror, first dichroic mirror, the optical filter and the detection Lens, then form the image of uniform stripe distribution in the photosurface of the planar array detector, described in planar array detector detection Described image is simultaneously converted into electric signal after carrying out space filtering to described image by the dummy pinhole by image；

The control module gathers the electric signal, and described image rebuilds module and coordinate setting and SIM frequency-domain spectrum figures is used to melt Image reconstruction is carried out to the electric signal, realizes the parallel micro-imagings of STED based on Structured Illumination.

2. the parallel micro imaging systems of the STED according to claim 1 based on Structured Illumination, which is characterized in that described The photosurface of LCD space light modulator, the front focal plane of the object lens, the exit plane of the Wollaston prism and the face Array detector photosurface is in conjugate planes.

3. the parallel micro imaging systems of the STED according to claim 1 based on Structured Illumination, which is characterized in that described Planar array detector is the magazine one kind of CCD or CMOS.

4. the parallel micro imaging systems of the STED according to claim 1 based on Structured Illumination, which is characterized in that pass through Phase distribution of the control module adjustment loading on LCD space light modulator can change direction, the week of exciting light light field Phase and initial phase so that the exciting light structure light light field and loss photo structure light light field have identical direction and cycle, just Phase differs half period.