Light section microscopic imaging device and method based on monochromatic broad structured light illumination

Abstract

The present invention discloses a light-sectioning microscopy imaging device and method based on monochromatic spread structured light illumination. The device includes a monochromatic spread light illumination generation module, a beam modulation module, an imaging module, and an image processing module. The monochromatic spread light generation module is used to generate monochromatic spread illumination light with a certain angular distribution; the beam modulation module is used to perform light modulation to generate stripe structured light with different phases; the imaging module is used to record intensity images of the sample at different axial positions under stripe structured light illumination with different phase shifts; and the image processing module is used to obtain a light-sectioning image at the current axial position based on multiple intensity images recorded at each axial position, and reconstruct a three-dimensional image of the sample based on the light-sectioning images at different axial positions. The present invention utilizes stripe structured light with different phase shifts to illuminate and image the sample layer by layer, and uses a phase shift algorithm to obtain a light-sectioning image at each thickness position, thereby achieving three-dimensional tomographic imaging of thick samples.

Description

Technical Field

[0001] The present invention belongs to the field of microscopic imaging technology, and in particular relates to a light-slicing microscopic imaging device and method based on monochromatic broad structured light illumination. Background Art

[0002] Structured illumination microscopy (SIM) is a wide-field super-resolution optical microscopy technique that uses structured light with varying phase shifts to illuminate a sample and sequentially records the resulting "moiré fringes." Finally, algorithms are used to reconstruct the sample's high-frequency information, resulting in a super-resolution image. SIM stands out among in vivo microscopy techniques for its low phototoxicity, fast imaging speed, and lack of special requirements for fluorescent samples.

[0003] SIM has super-resolution imaging and three-dimensional tomography capabilities. For example, Lei Ming et al. proposed a high-speed structured light illumination optical microscopy system and method based on digital micromirror device (DMD) projection. By recording nine raw structured light intensity images of the sample under three-dimensional, three-step phase-shifted structured light illumination, a sample image that exceeds the diffraction limit can be obtained. Furthermore, by recording intensity images with phase shifts of 0°, 120°, and 240°, a wide-field tomographic image at any axial position z can be calculated. Furthermore, by combining color imaging with three-dimensional SIM imaging, a three-dimensional color SIM microscopy method based on the Hilbert transform was proposed. This method only requires two raw images for each axial position to reconstruct a light slice image, effectively improving the speed and efficiency of three-dimensional color imaging. Kuang Cuifang et al. from Zhejiang University proposed a multi-color SIM super-resolution microscopy method based on a scanning galvanometer and DMD. This method uses a galvanometer to rapidly switch between different laser wavelengths and a DMD to generate striped structured light, achieving multi-color three-dimensional super-resolution SIM microscopy. The team led by Academician Dai Qionghai of Tsinghua University proposed expanding the imaging field of view by adding a relay lens behind the first image plane of the sample and a two-dimensional scanning galvanometer in the spectrum plane in the imaging optical path of the traditional SIM.

[0004] In current mainstream optical microscopy (SIM) methods, LED illumination and DMD / SLM (spatial light modulator) modulation are mostly used to generate stripe structured light. The pattern on the DMD is imaged and projected onto the sample along the directions of the 0th and ±1st order diffracted light. Due to the effects of distortion, the imaging system modulates the three diffracted light rays differently, resulting in a decrease in stripe contrast within the imaging field of view. Furthermore, due to the dispersion effect of the DMD, when using LED illumination and DMD to generate sinusoidal stripe structured light, the light intensity utilization rate of the illumination light reaching the sample plane is low. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, the present invention provides a light sectioning microscopy device and method based on monochromatic broad structured light illumination. The technical problems to be solved by the present invention are achieved through the following technical solutions:

[0006] The present invention provides a light sectioning microscopic imaging device based on monochromatic extended structured light illumination, comprising a monochromatic extended illumination generation module, a beam modulation module, an imaging module and an image processing module, wherein:

[0007] The monochromatic extended illumination generation module is used to generate partially coherent monochromatic extended illumination light with a certain angular distribution using a single-frequency laser;

[0008] The beam modulation module is used to modulate the monochromatic extended illumination light to generate stripe structured light with different phase shift amounts;

[0009] The imaging module is used to use the stripe structured light with different phase shift amounts to perform layer-by-layer imaging on different axial positions in the thickness direction of the sample, and obtain intensity images of the sample at each axial position under the illumination of different stripe structured light;

[0010] The image processing module is used to obtain a light slice image at a current axial position according to a plurality of intensity images at each axial position, and to reconstruct a three-dimensional image of the sample according to the light slice images at different axial positions.

[0011] In one embodiment of the present invention, the monochromatic extended illumination generation module includes a laser and a first microscope objective lens, a ground glass sheet, and a first lens sequentially arranged along the optical axis direction of the laser, wherein:

[0012] The laser is used to emit single-frequency laser;

[0013] The first microscope objective lens and the first lens are used to expand the single-frequency laser light generated by the laser to cover the digital projection device in the beam modulation module;

[0014] The frosted glass sheet is arranged perpendicular to the optical axis of the single-frequency laser and the axis of the frosted glass sheet deviates from the optical axis. The frosted glass sheet can rotate around the optical axis to generate dynamically scattered partially coherent light; the distance between the frosted glass sheet and the focus of the first lens is 7.5 to 15 mm to generate extended illumination light with a certain illumination angle distribution.

[0015] In one embodiment of the present invention, the beam modulation module includes a digital projection device, a second lens, a spectrum filter and a third lens arranged in sequence along the optical axis, wherein:

[0016] The digital projection device is used to perform light modulation on the monochromatic extended illumination light from the monochromatic extended illumination generation module to generate stripe structured light with different phase shift amounts;

[0017] The second lens is used to focus the stripe structured light on the spectrum filter;

[0018] The spectrum filter is used to perform spectrum selection on the stripe structured light modulated by the digital projection device, and only allows the spectrum of ±1 diffracted light to pass;

[0019] The third lens is used to expand and collimate the light beam spectrally selected by the spectrum filter into parallel illumination light.

[0020] In one embodiment of the present invention, the digital projection device is a digital micromirror array or a spatial light modulator, which is used to load binary structured light stripes and perform 0, 2π / 3, and 4π / 3 phase shift operations in sequence.

[0021] In one embodiment of the present invention, the beam modulation module further includes a first reflector and a second reflector obliquely disposed between the first lens and the digital projection device, for reflecting the monochromatic expanded illumination light from the first lens to a surface of the digital projection device.

[0022] In one embodiment of the present invention, the imaging module includes a third reflector, a dichroic mirror, a fourth lens, a fourth reflector, a piezoelectric shift stage, a second objective lens, a filter, and a camera, wherein:

[0023] The third reflector, the dichroic mirror, the fourth lens, the fourth reflector, the piezoelectric shift stage, and the second objective lens are sequentially arranged along the optical axis, the sample is arranged on a side of the second objective lens away from the piezoelectric shift stage, and the piezoelectric shift stage is capable of adjusting the axial distance between the sample and the second objective lens so that the stripe structured light from the second objective lens is focused on different layers in the thickness direction of the sample;

[0024] The filter and the camera are sequentially arranged on a side of the dichroic mirror away from the fourth lens, and are used to receive the light beam reflected back from the sample and obtain a single-layer light slice image.

[0025] In one embodiment of the present invention, the image processing module includes a data acquisition card and a computer, wherein:

[0026] The data acquisition card is electrically connected to the camera and is used to transmit the intensity images recorded by the camera under different stripe structured light illuminations and send them to the computer;

[0027] The computer is used to obtain an optical slice image at a current thickness position based on multiple intensity images at corresponding thickness positions of the sample, and to reconstruct a three-dimensional image of the sample based on the optical slice images at different thickness positions.

[0028] Another aspect of the present invention provides a light sectioning microscopic imaging method based on monochromatic broad structured light illumination, comprising:

[0029] S1: using the light sectioning microscopy imaging device based on structured light illumination according to any one of the above embodiments to obtain multiple intensity images of the sample under structured light illumination;

[0030] S2: obtaining a light slice image of the sample using the original intensity images under the multiple structured light illuminations;

[0031] S3: By moving the sample or objective lens along the axial direction and repeatedly obtaining multiple single-layer optical slice images of the sample at different thickness positions, plane images at different thickness positions are obtained to form a three-dimensional image of the sample.

[0032] In one embodiment of the present invention, the S2 includes:

[0033] Get three single-layer optical slice images of the sample at the current thickness position:

[0034] D m (x,y)=D in (x,y)·{1+cos[2πkx+2(m-1)π / 3]}+D out (x,y),

[0035] Among them, D in (x,y) represents the in-focus component of a single-layer light slice image, D out (x,y) represents the defocused component of a single-layer light slice, m represents the number of phase shifts, and k is the spatial frequency of the fringes;

[0036] Let m = 1, 2, 3 form a set of equations, and solve the coke component D according to the set of equations. in (x,y):

[0037]

[0038] In-focus component D in (x, y) performs deconvolution operation to obtain the plane image of the sample at the current thickness position:

[0039]

[0040] in, Indicates the coke component D in The Fourier spectrum of (x,y), represents the optical transfer function, represents the two-dimensional inverse Fourier transform, is the apodization function in the frequency domain, and w is the Wiener coefficient.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1. The illumination light of the present invention is generated by rotating frosted glass to decoherently use a single-frequency laser, which overcomes the dispersion effect of DMD on broadband illumination light; by adjusting the axial position between the frosted glass and the illumination light focus, the equivalent luminous point diameter and spatial coherence of the illumination can be changed, and the light slice thickness of the SIM can be reduced (improving the axial slice accuracy). As the distance between the frosted glass and the illumination light focus increases, the fringe contrast tends to decrease, while the slice accuracy (resolvable axial distance) tends to increase. When the distance between the two is 7.5-15mm (the effective scattered light spot diameter is 2mm), both high fringe contrast and slice accuracy can be taken into account (such as Figure 4 shown).

[0043] 2. This invention generates high-contrast fringe structured light across the entire imaging field of view by applying binary structured light stripes to a digital projection device (digital micromirror array or spatial light modulator) and filtering the illumination light spectrum (blocking the 0th-order light spectrum and allowing only the ±1st diffracted light spectrum to pass). This method achieves a fringe period twice as long as conventional methods on the DMD, allowing more pixels (twice as many as conventional direct fringe projection methods) to produce fringe structured light with the same period, further facilitating fringe acquisition and phase shifting operations.

[0044] 3. The light-sectioning microscopy device based on monochromatic broad-stranded structured light illumination proposed in this invention can be applied to various inverted microscopes. By replacing the third reflector in the optical path with a dichroic mirror, the device integrates the entire illumination and fluorescence imaging unit behind the microscope body, minimizing the requirements for the microscope body. When the third reflector is replaced with a dichroic mirror, dual-mode imaging (e.g., fluorescence / phase) can be achieved with other imaging components within the microscope body.

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 1 is a schematic structural diagram of a light sectioning microscopy imaging device based on monochromatic broad structured light illumination provided by an embodiment of the present invention;

[0047] Figure 2 The embodiment of the present invention provides a stripe structured light generated on a sample before and after blocking the zero-order spectrum of the illumination light;

[0048] Figure 3Schematic diagram of stripe structured light loaded on a DMD in an embodiment of the present invention (simultaneous phase shift operations of 0, 2π / 3, and 4π / 3);

[0049] Figure 4 is a schematic diagram showing how the optical slice thickness (tomographic imaging accuracy) and fringe contrast vary with the distance between the ground glass and the focus of the first objective lens in an embodiment of the present invention;

[0050] Figure 5 Schematic diagram of the combination of a light sectioning microscopy imaging device based on structured light illumination and a mainstream microscope according to an embodiment of the present invention;

[0051] Figure 6 Schematic diagram of the experimental results of agar ball samples in wide field mode and OS-SIM mode of the embodiment of the present invention;

[0052] Figure 7 Schematic diagram of the experimental results of optical slice imaging of a cross-section sample of a lily stigma in wide-field mode and in the OS-SIM mode of an embodiment of the present invention, respectively.

[0053] Description of reference numerals:

[0054] 1-Laser; 2-First objective lens; 3-Frosted glass; 4-First lens; 5-First reflector; 6-Second reflector; 7-Spatial light modulator; 8-Second lens; 9-Spectral filter; 10-Third lens; 11-Third reflector; 12-Dichroic mirror; 13-Fourth lens; 14-Fourth reflector; 15-Piezoelectric shift stage; 16-Second objective lens; 17-Sample; 18-Filter; 19-Camera; 20-Data acquisition card; 21-Computer; 22-Epi-illumination light source; 23-Bright field imaging module; 24-Microscope main body; 25-Microscope eyepiece. DETAILED DESCRIPTION

[0055] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the following is a detailed description of a light sectioning microscopy imaging device and method based on monochromatic broad structured light illumination proposed in accordance with the present invention, in combination with the accompanying drawings and specific embodiments.

[0056] The aforementioned and other technical contents, features, and effects of the present invention are clearly presented in the following detailed description of the specific embodiments in conjunction with the accompanying drawings. Through the description of the specific embodiments, a deeper and more specific understanding of the technical means and effects adopted by the present invention to achieve the intended purpose can be obtained. However, the accompanying drawings are provided for reference and illustration purposes only and are not intended to limit the technical solutions of the present invention.

[0057] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations are intended to cover non-exclusive inclusion, such that an article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of additional identical elements in the article or device comprising the element.

[0058] Example 1

[0059] See Figure 1 , Figure 1 This is a schematic diagram of the structure of a light-sectioning microscopy imaging device based on monochromatic broad structured light illumination provided by an embodiment of the present invention. The light-sectioning microscopy imaging device includes a monochromatic broad structured light generation module, a light beam modulation module, an imaging module, and an image processing module, wherein the monochromatic broad structured light generation module is used to generate partially coherent, monochromatic broad structured light with a certain angular distribution using a single-frequency laser; the light beam modulation module is used to modulate the monochromatic broad structured light to generate stripe structured light with different phase shifts; the imaging module is used to use stripe structured light with different phase shifts to perform layer-by-layer imaging of different axial positions in the thickness direction of the sample, and obtain intensity images of the sample under different stripe structured light illumination at each axial position; the image processing module is used to obtain a light-section image of the current axial position based on multiple intensity images of each axial position, and reconstruct a three-dimensional image of the sample based on the light-section images at different axial positions.

[0060] Furthermore, the monochromatic extended illumination generation module includes a laser 1 and a first microscope objective lens 2, a frosted glass sheet 3 and a first lens 4 arranged in sequence along the optical axis direction of the laser 1, wherein the laser 1 is used to emit single-frequency laser light; the first microscope objective lens 2 and the first lens 4 are used to expand the single-frequency laser light generated by the laser 1 to cover the digital projection device 7 in the beam modulation module; the frosted glass sheet 3 is arranged perpendicular to the optical axis of the single-frequency laser light and the axis of the frosted glass sheet 3 deviates from the optical axis, and the frosted glass sheet 3 can rotate around the optical axis to generate dynamically scattered partially coherent light. The distance between the frosted glass sheet 3 and the focus of the first lens 4 is 7.5 to 15 mm to generate extended illumination light with a certain illumination angle distribution, which helps to generate stripe structured light with high stripe contrast and high axial slicing accuracy.

[0061] The beam modulation module includes a digital projection device 7, a second lens 8, a spectrum filter 9 and a third lens 10 arranged in sequence along the optical axis, wherein the digital projection device 7 is used to modulate the monochromatic extended illumination light from the monochromatic extended illumination generation module to generate stripe structured light with different phase shift amounts; the second lens 8 is used to focus the stripe structured light on the spectrum filter 9; the spectrum filter 9 is used to perform spectrum selection on the stripe structured light modulated by the digital projection device 7, and only allows the spectrum of ±1 diffracted light to pass through; the third lens 10 is used to expand and collimate the light beam spectrally selected by the spectrum filter into parallel illumination light.

[0062] In this embodiment, the digital projection device 7 is a digital micromirror array (DMD) or a spatial light modulator (SLM), which is used to load binary structured light stripes and perform phase shift operations of 0, 2π / 3, and 4π / 3 in sequence.

[0063] Furthermore, the beam modulation module further includes a first reflector 5 and a second reflector 6 tiltedly arranged between the first lens 4 and the digital projection device 7 , for reflecting the monochromatic expanded illumination light from the first lens 4 to the surface of the digital projection device 7 .

[0064] Continue to see Figure 1 The imaging module includes a third reflector 11, a dichroic mirror 12, a fourth lens 13, a fourth reflector 14, a piezoelectric shift stage 15, a second objective lens 16, a filter 18, and a camera 19. The third reflector 11, the dichroic mirror 12, the fourth lens 13, the fourth reflector 14, the piezoelectric shift stage 15, and the second objective lens 16 are sequentially arranged along the optical axis. The sample 17 is disposed on the side of the second objective lens 16 away from the piezoelectric shift stage 15. The piezoelectric shift stage 15 is capable of adjusting the axial distance between the sample 17 and the second objective lens 16 so that the stripe structured light from the second objective lens 16 is focused on different layers along the thickness direction of the sample 17. The filter 18 and the camera 19 are sequentially disposed on the side of the dichroic mirror 12 away from the fourth lens 13, and are configured to receive the light beam reflected from the sample 17 and obtain an original intensity image under the illumination of the single-layer structured light. The camera 19 in this embodiment is a scientific research-grade sCMOS camera.

[0065] The image processing module of this embodiment includes a data acquisition card 20 and a computer 21, wherein the data acquisition card 20 is electrically connected to the camera 19 and is used to transmit the intensity image recorded by the camera 19 under different stripe structured light illumination and send it to the computer 21; the computer 21 is used to obtain the light slice image at the current thickness position based on multiple intensity images at the corresponding thickness positions of the sample, and reconstruct the three-dimensional image of the sample based on the light slice images at different thickness positions.

[0066] During use, the single-frequency laser light emitted by the laser 1 passes through the first microscope objective lens 2, the ground glass sheet 3 and the first lens 4 in sequence to form a monochromatic expanded illumination light after beam expansion and collimation. The monochromatic expanded illumination light passes through the first reflector 5 and the second reflector 6 and is incident on the spatial light modulator 7. The spatial light modulator 7 loads binary structured light stripes and performs phase shift operations of 0, 2π / 3, and 4π / 3 in sequence to modulate the light beam to produce striped structured light with different phases. The light beam then passes through the second lens 8, the spectrum filter 9, and the third lens 10. The spectrum filter 9 is used to perform spectrum selective filtering (blocking the zero order) on the light beam modulated by the spatial light modulator, allowing only the spectrum of ±1 diffracted light to pass through. Finally, the illumination beam is imaged onto the sample 17 through the imaging module. The ±1 diffracted light interferes in the focal plane of the sample 17 and produces two-dimensional binary structured illumination on the sample 17. The fluorescence excited after illuminating the sample returns along the original light path (still passing through the second objective lens 16, the reflector 14, and the fourth lens 13 in sequence), passes through the dichroic mirror 12 and the filter 18, and is received by the sCMOS camera 19.

[0067] That is to say, at a thickness position of the sample, the stripes loaded on the spatial light modulator need to be phase-shifted in three steps of 0, 2π / 3, and 4π / 3, and three sets of original intensity images need to be recorded using an sCMOS camera respectively.

[0068] See Figure 2 , Figure 2 It is a stripe structured light generated on a sample before and after blocking the zero-order spectrum of the illumination light, provided by an embodiment of the present invention, wherein (a) shows the stripe structured light generated when the zero-order spectrum of the illumination light is not blocked, and (b) shows the stripe structured light generated after blocking the zero-order spectrum of the illumination light. The stripe periods loaded by the DMD in (a) and (b) are 6 pixels and 12 pixels respectively; (c) shows the intensity distribution along the two white line segments 1 and 2 in (a); and (d) shows the intensity distribution along line segment 3 in (b). Figure 2 It can be seen that in the traditional structured light generation method, when the stripes with a period of 6 pixels loaded on the DMD are directly projected onto the sample, the 0-order and ±1-order light of the structured light undergo three-beam interference to form striped structured light. The distortion of the system introduces different phase distributions to the three beams of light, causing the three sets of interference fringes formed by the three-beam interference to be superimposed together in space. In some areas, the stripes have high contrast, while in other areas, the contrast is low. In contrast, the embodiment of the present invention uses a spectral filter to block the zero order, and only the ±1-order light interference is used to form the striped structured light. High-contrast stripes appear in the entire area of the sample, thereby obtaining a high imaging signal-to-noise ratio and a clearer image.

[0069] See Figure 3 , Figure 3Schematic diagram of stripe structured light loaded on a DMD in an embodiment of the present invention (simultaneously performing three-step phase shift operations of 0, 2π / 3, and 4π / 3). Figure 3 It is demonstrated that by translating the stripe image along the normal direction of the stripes on the DMD, stripe structured light with different phase shift amounts can be generated. By loading binary stripes with a stripe period of 12 pixels on the DMD and blocking the zero-order spectrum of the illumination light, stripe structured light with a period of 0.324μm (in order to achieve better tomographic imaging effects, the period of the stripe structured light is generally selected to be twice the theoretical diffraction limit resolution of the optical microscope system) is generated on the sample plane. By moving four pixels in sequence along the normal direction of the stripes on the DMD, the stripes can be phase shifted in three steps of 0, 2π / 3, and 4π / 3 in sequence. It should be noted that in order to generate stripe structured light with a period of 0.324μm on the sample plane, the traditional method requires the projection of stripes with a period of 6 pixels. The embodiment of the present invention only requires the projection of stripes with a period of 12, which is more conducive to the spatial sampling of the stripes and improving the phase shift accuracy.

[0070] See Figure 4 , Figure 4 Schematic diagrams showing how light slice thickness (a) and fringe contrast (b) vary with the defocus distance between the ground glass and the objective lens focal point. Figures (a) and (b) show that as the defocus distance between the ground glass and the objective lens increases (forming a monochromatic extended light source), the light slice thickness decreases, but the fringe contrast decreases. When the defocus distance between the two is 4.5 to 15 mm, both a thin light slice thickness and high fringe contrast are achieved.

[0071] Furthermore, after the camera 19 acquires a single-layer optical slice image, the computer 21 is used to obtain a planar image at the current thickness position based on multiple single-layer optical slice images at corresponding thickness positions of the sample, and reconstruct a three-dimensional image of the sample based on multiple planar images at different thickness positions. Specifically, the distribution of the sine / cosine fringe light field on the sample plane is expressed as:

[0072]

[0073] Where I is the average intensity of the illumination light, △ is the fringe modulation, k is the spatial frequency of the fringe, is the phase shift of the structured light; for simplicity, I = 1 and Δ = 1. During imaging, the structured light fringes only exist in an area with an axial distance of approximately 500 nm near the focal plane (the fringe contrast decreases with increasing defocus distance).

[0074] Structured illumination light-section microscopy allows for the distinction between in-focus and out-of-focus components within the depth of field of the objective lens. Specifically, the components with structural fringes are in-focus, while the components without structural fringes are out-of-focus. Therefore, the wide-field two-dimensional image recorded by the sCMOS camera 19 can be divided into two components: one in-focus and the other out-of-focus. The mathematical expression for this is as follows:

[0075] D(x,y)=D in (x,y)+D out (x,y) (1)

[0076] Where D(x,y) represents the wide-field two-dimensional image recorded by the camera under uniform wide-field illumination, and D in (x,y) represents the in-focus component of the wide-field two-dimensional image, D out (x,y) represents the defocus component of the wide-field two-dimensional image. Under structured illumination, the distribution expression of the sine / cosine light field is:

[0077]

[0078] For simplicity, let I = 1, △ = 1. The structural fringes will only modulate the focal component D in (x, y) and the defocus component D out (x,y) has no effect. Therefore, the imaging result under structured light illumination can be rewritten as (1):

[0079] D m (x,y)=D in (x,y)·{1+cos[2πkx+2(m-1)π / 3]}+D out (x,y) (2)

[0080] Here, m=1, 2, and 3 represent the number of phase shifts. The above formula assumes that the increment of the phase shift amount in each phase shift operation is 2π / 3, and the corresponding phase shift amounts are 0, 2π / 3, and 4π / 3, respectively.

[0081] In order to understand the in-focus component D of the imaging result under structured light illumination in (x,y), usually requires three phase shifts with an interval of 2π / 3 to form the equation system:

[0082]

[0083] Therefore, D in (x,y) can be solved as:

[0084]

[0085] Furthermore, in order to supplement the influence of the system point spread function on the imaging resolution, the in-focus component Din (x, y) is deconvolved to obtain the in-focus component after deconvolution, that is, the plane image D of the sample at the current thickness position SIM (x,y):

[0086]

[0087] in, Indicates the coke component D in The Fourier spectrum of (x,y), represents the Optical Transfer Function (OTF), which is mathematically the Fourier transform of the imaging system point spread function (PSF). represents the two-dimensional inverse Fourier transform, is the frequency domain apodization function (used to reduce artifacts in the image), w is the Wiener coefficient, which is selected as 0.2 based on experience. In order to achieve better reconstruction results, a Gaussian apodization function is used. In the experiment, 240nm fluorescent microspheres (RF240C, excitation wavelength 532nm, radiation wavelength 610nm) were imaged. Since the diameter of the microspheres is much smaller than the spatial resolution of the system, the two-dimensional intensity distribution of the microsphere image is used as the PSF of the system.

[0088] In the experiment, by recording three intensity patterns of the sample under structured light illumination with phase shifts of 0, 2π / 3, and 4π / 3, we can use formulas (4) and (5) to solve the focused image of the sample after removing the out-of-focus background. In addition, for the convenience of comparison, the wide-field mode image under uniform illumination can be obtained by averaging D1(x,y), D2(x,y), and D3(x,y):

[0089] D wide-field (x,y)=[D1(x,y)+D2(x,y)+D3(x,y)] / 3 (6)

[0090] Subsequently, the axial distance between the sample 17 and the second objective lens 16 is adjusted by the piezoelectric shift stage 15, so that the second objective lens 16 is focused on different layers in the thickness direction of the sample 17 and multiple wide-field two-dimensional imaging images of the sample at different axial positions are repeatedly obtained, and plane images at different axial positions are obtained to form a three-dimensional image of the sample.

[0091] Further, see Figure 5 , Figure 5Schematic diagram of the combination of the light sectioning microscopy imaging device based on structured light illumination and a mainstream microscope according to an embodiment of the present invention. The light sectioning microscopy imaging device proposed in the embodiment of the present invention can be directly used in the current mainstream inverted microscope. The structure of the inverted microscope mainly includes an epi-illumination light source 22, a bright field imaging module 23, a microscope body 24, and an eyepiece 25. By replacing the third reflector 14 in the light sectioning microscopy imaging device proposed in the embodiment of the present invention with a dichroic mirror, a dual-mode (fluorescence / bright field) imaging system can be formed with other imaging modules of the microscope body (such as the bright field imaging module 23).

[0092] In summary, the illumination light of the present invention utilizes a single-frequency laser generated by rotating frosted glass to decoherently generate the illumination light, overcoming the dispersion effect of the DMD on broadband illumination light. By adjusting the axial position between the frosted glass and the illumination light focal point, the equivalent luminous spot diameter and spatial coherence of the illumination can be changed, reducing the SIM optical slice thickness (improving axial slice accuracy). As the distance between the frosted glass and the illumination light focal point increases, the fringe contrast decreases, while the slice accuracy (resolvable axial distance) increases. When the distance between the frosted glass and the illumination light focal point is 7.5-15 mm (effective scattered light spot diameter of 2 mm), both high fringe contrast and slice accuracy can be achieved.

[0093] This method generates high-contrast fringe structured light across the entire imaging field of view by applying binary structured light stripes to a digital projection device (digital micromirror array or spatial light modulator) and filtering the illumination spectrum (blocking the 0th-order light spectrum and allowing only the ±1st diffracted light spectrum to pass). This method achieves double the fringe period on the DMD compared to traditional methods, allowing more pixels (twice as many as in traditional direct fringe projection methods) to produce fringe structured light with the same period, further facilitating fringe acquisition and phase shifting.

[0094] The light-sectioning microscopy device based on structured light illumination proposed in this invention can be applied to various inverted microscopes. By replacing the third reflector in the optical path with a dichroic mirror, the device integrates the entire illumination and fluorescence imaging unit behind the microscope body, minimizing the requirements for the microscope body. When the third reflector is replaced with a dichroic mirror, dual-mode imaging (e.g., fluorescence / phase) can be achieved with other imaging components of the microscope body.

[0095] Example 2

[0096] Based on the above embodiment 1, this embodiment provides a light sectioning microscopy imaging method based on structured light illumination, the method comprising:

[0097] S1: Obtain multiple wide-field two-dimensional images of the sample using the light sectioning microscopy imaging device based on structured light illumination described in Example 1;

[0098] S2: obtaining a planar image of the sample according to the plurality of single-layer optical slice images;

[0099] Specifically, obtain three single-layer optical slice images of the sample at the current thickness position:

[0100] D m (x,y)=D in (x,y)·{1+cos[2πkx+2(m-1)π / 3]}+D out (x,y),

[0101] Among them, D in (x,y) represents the in-focus component of a single-layer light slice image, D out (x,y) represents the defocused component of a single-layer light slice, m represents the number of phase shifts, and k is the spatial frequency of the fringes;

[0102] Let m = 1, 2, 3 form a set of equations, and solve the coke component D according to the set of equations. in (x,y):

[0103]

[0104] In-focus component D in (x, y) performs deconvolution operation to obtain the plane image of the sample at the current thickness position:

[0105]

[0106] in, Indicates the coke component D in The Fourier spectrum of (x,y), represents the optical transfer function, represents the two-dimensional inverse Fourier transform, is the apodization function in the frequency domain, and w is the Wiener coefficient.

[0107] S3: By moving the sample or objective lens along the axial direction and repeatedly obtaining multiple single-layer optical slice images of the sample at different thickness positions, plane images at different thickness positions are obtained to form a three-dimensional image of the sample.

[0108] The following experiments are conducted to verify the effectiveness of the light sectioning microscopy imaging device and method based on structured light illumination of the present invention.

[0109] (Experiment 1) A sample of fluorescent beads dissolved in agar was used as the test sample to verify the tomographic imaging capability of the optical sectioning microscopy method proposed in the present invention. In the experiment, the agar fluorescent bead sample was placed on the stage and imaged in widefield mode and in the OS-SIM (Optical Sectioning SIM) mode proposed in the present invention. Figure 6 , Figure 6 Schematic diagram of the experimental results of agar ball samples in wide field mode and OS-SIM mode of the embodiment of the present invention, wherein: Figure 6 (a) Experimental results of imaging the agar pellet sample at different depths in wide-field mode and OS-SIM mode respectively; Figure 6 (b) is the lateral resolution of imaging in OS-SIM mode; Figure 6 (c) is a comparison of the signal-to-noise ratio at different depths in widefield mode and OS-SIM mode. A widefield illumination image is generated by loading a picture with uniform grayscale distribution on the DMD, and the widefield illumination images of different samples at different axial planes (z = 15 μm, 30 μm, 45 μm, 60 μm, 75 μm) are recorded by moving the objective lens, as shown in Figure 2. Figure 6 (a) is shown. Secondly, for each axial plane position, three grayscale images with phase shifts of 0, 2π / 3, and 4π / 3 are loaded on the DMD in sequence, and three intensity images D1(x, y), D2(x, y), and D3(x, y) of the sample under different phase-shifted structured light illumination are recorded. Subsequently, the OS-SIM images of different axial planes are obtained by the corresponding OS-SIM algorithm of formulas (4) and (5). The imaging results are shown in FIG. Figure 6 (b) is shown. Figure 6 Compared with the wide-field illumination image in (a), the OS-SIM optical slice image of the sample at different depth positions effectively suppresses the out-of-focus background and realizes the tomographic function. At the same time, in the wide-field mode, the lateral resolution of the imaging is 335±5nm and the longitudinal resolution is 920±58nm. Using the experimental device (OS-SIM) in the present invention, the lateral resolution of the imaging is 292±10nm and the longitudinal resolution is 554±98nm. Figure 6 As shown in (b), the axial resolution and the ability to suppress out-of-focus background in OS-SIM mode are significantly improved compared to wide-field mode. The signal-to-noise ratio of wide-field illumination and OS-SIM imaging modes at different imaging depths was measured, as shown in Figure 2. Figure 6 As shown in (c), the imaging signal-to-noise ratio (SNR) of both imaging modes decreases with increasing imaging depth. When the imaging depth increases from 15 μm to 75 μm, the imaging SNR of the OS-SIM mode decreases from 6 times to 2 times compared to the widefield mode.

[0110] (Experiment 2) Please see Figure 7 , Figure 7 Schematic diagram of the experimental results of optical slice imaging of a cross-section sample of a lily stigma in wide-field mode and in the OS-SIM mode of an embodiment of the present invention, respectively. Figure 7 (a) is a wide-field illumination image, Figure 7 (b) is the OS-SIM imaging result. Figure 7 The right panels (a) and (b) show widefield and OS-SIM images of the boxed area in the left image at different depths (4μm, 8μm, 12μm, and 16μm), respectively. Comparing widefield and OS-SIM images at different axial positions reveals that OS-SIM can suppress out-of-focus background at different depths of the sample, demonstrating its excellent optical sectioning capabilities.

[0111] In the several embodiments provided herein, it should be understood that the apparatus and method disclosed herein can be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative. For example, the module division is merely a logical functional division. In actual implementation, other division methods may be used. For example, multiple modules or components may be combined or integrated into another system, or some features may be ignored or not implemented.

[0112] In addition, the functional modules in various embodiments of the present invention may be integrated into a single processing module, each module may exist physically separately, or two or more modules may be integrated into a single module. The aforementioned integrated modules may be implemented in the form of hardware or hardware plus software functional modules.

[0113] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

Claims

1. A light section microscopy imaging device based on monochromatic broad structured light illumination, characterized in that: It includes a monochromatic extended illumination generation module, a beam modulation module, an imaging module and an image processing module, wherein: The monochromatic extended illumination generation module is used to generate partially coherent monochromatic extended illumination light with a certain angular distribution using a single-frequency laser; The beam modulation module is used to modulate the monochromatic extended illumination light to generate stripe structured light with different phase shift amounts; The imaging module is used to use the stripe structured light with different phase shift amounts to perform layer-by-layer imaging on different axial positions in the thickness direction of the sample, and obtain intensity images of the sample at each axial position under the illumination of different stripe structured light; The image processing module is used to obtain a light slice image at the current axial position based on the multiple intensity images at each axial position, and to reconstruct a three-dimensional image of the sample based on the light slice images at different axial positions; The monochromatic extended illumination generation module comprises a laser (1), and a first microscope objective lens (2), a ground glass sheet (3), and a first lens (4) arranged in sequence along the optical axis direction of the laser (1), wherein: The laser (1) is used to emit single-frequency laser light; The first microscope objective lens (2) and the first lens (4) are used to expand the single-frequency laser light generated by the laser (1) to cover the digital projection device (7) in the beam modulation module; The frosted glass sheet (3) is arranged perpendicular to the optical axis of the single-frequency laser, and the axis of the frosted glass sheet (3) deviates from the optical axis. The frosted glass sheet (3) can rotate around the optical axis to generate dynamically scattered partially coherent light. The distance between the frosted glass sheet (3) and the focus of the first lens (4) is 7.5 to 15 mm, so as to generate extended illumination light with a certain illumination angle distribution. The beam modulation module comprises a digital projection device (7), a second lens (8), a spectrum filter (9) and a third lens (10) which are sequentially arranged along the optical axis direction, wherein: The digital projection device (7) is used to perform light modulation on the monochromatic extended illumination light from the monochromatic extended illumination generation module to generate stripe structured light with different phase shift amounts; The second lens (8) is used to focus the stripe structured light on the spectrum filter (9); The spectrum filter (9) is used to perform spectrum selection on the stripe structured light modulated by the digital projection device (7), and only allows the spectrum of ±1 diffracted light to pass through; The third lens (10) is used to expand and collimate the light beam spectrally selected by the spectrum filter into parallel illumination light in different directions.

2. The light sectioning microscopy imaging device based on monochromatic broad structured light illumination according to claim 1, characterized in that: The digital projection device (7) is a digital micromirror array or a spatial light modulator, which is used to load binary structured light stripes and perform 0, 2π / 3, and 4π / 3 phase shift operations in sequence.

3. The light sectioning microscopy imaging device based on monochromatic broad structured light illumination according to claim 1, characterized in that: The light beam modulation module further comprises a first reflector (5) and a second reflector (6) arranged obliquely between the first lens (4) and the digital projection device (7), and used for reflecting the monochromatic expanded illumination light from the first lens (4) to the surface of the digital projection device (7).

4. The light sectioning microscopy imaging device based on monochromatic broad structured light illumination according to claim 1, characterized in that: The imaging module comprises a third reflector (11), a dichroic mirror (12), a fourth lens (13), a fourth reflector (14), a piezoelectric shift stage (15), a second objective lens (16), a filter (18) and a camera (19), wherein: The third reflector (11), the dichroic mirror (12), the fourth lens (13), the fourth reflector (14), the piezoelectric shift stage (15) and the second objective lens (16) are arranged in sequence along the optical axis direction, the sample (17) is arranged on a side of the second objective lens (16) away from the piezoelectric shift stage (15), and the piezoelectric shift stage (15) is capable of adjusting the axial distance between the sample (17) and the second objective lens (16) so that the stripe structured light from the second objective lens (16) is focused on different layers in the thickness direction of the sample (17); The filter (18) and the camera (19) are sequentially arranged on a side of the dichroic mirror (12) away from the fourth lens (13) for receiving the light beam reflected from the sample (17) and obtaining a single-layer light slice image.

5. The light sectioning microscopic imaging device based on monochromatic broad structured light illumination according to claim 4, characterized in that: The image processing module includes a data acquisition card (20) and a computer (21), wherein: The data acquisition card (20) is electrically connected to the camera (19) and is used to transmit the intensity images recorded by the camera (19) under different stripe structured light illuminations and send them to the computer (21); The computer (21) is used to obtain a light slice image at a current thickness position based on a plurality of intensity images at corresponding thickness positions of the sample, and to reconstruct a three-dimensional image of the sample based on the light slice images at different thickness positions.

6. A light section microscopy imaging method based on monochromatic broad structured light illumination, characterized in that: include: S1: using the light sectioning microscopy imaging device based on monochromatic broad structured light illumination according to any one of claims 1 to 5 to obtain multiple intensity images of the sample under structured light illumination; S2: obtaining a light slice image of the sample using the original intensity images under the multiple structured light illuminations; S3: By moving the sample or objective lens along the axial direction and repeatedly obtaining multiple single-layer optical slice images of the sample at different thickness positions, plane images at different thickness positions are obtained to form a three-dimensional image of the sample.

7. The light section microscopy imaging method based on monochromatic broad structured light illumination according to claim 6, characterized in that: The S2 includes: Get three single-layer optical slice images of the sample at the current thickness position: D m (x,y)=D in (x,y)·{1+cos[2πkx+2(m-1)π / 3]}+D out (x,y), Among them, D in (x,y) represents the in-focus component of a single-layer light slice image, D out (x,y) represents the defocused component of a single-layer light slice, m represents the number of phase shifts, and k is the spatial frequency of the fringes; Let m = 1, 2, 3 form a set of equations, and solve the coke component D according to the set of equations. in (x,y): In-focus component D in (x, y) performs deconvolution operation to obtain the plane image of the sample at the current thickness position: in, Indicates the coke component D in The Fourier spectrum of (x,y), represents the optical transfer function, represents the two-dimensional inverse Fourier transform, is the apodization function in the frequency domain, and w is the Wiener coefficient.

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