Near-infrared two-zone line scanning optical tomography microscopic imaging system and method based on virtual digital slit

Abstract

The invention relates to the technical field of information optics, in particular to a near-infrared two-zone line scanning optical tomography microscopic imaging system and method based on a virtual digital slit, and the system comprises a near-infrared two-zone line scanning optical tomography microscopic imaging module. And the virtual digital slit image reconstruction module is used for reconstructing an image acquired by the near-infrared two-zone line scanning optical tomography microscopic imaging module. A digital micromirror device and a digital line array are introduced, exciting light is modulated by means of the high-speed signal modulation capacity of a DMD, the digital line array is used for replacing a physical slit in a traditional line scanning system, generation and rapid scanning of a parallel line array exciting light field are achieved, and the flexibility and imaging speed of the system are greatly improved; a virtual digital slit image reconstruction module is introduced to filter background noise, so that optical slicing is realized; a near-infrared two-area window is introduced into a line scanning optical tomography microscopic imaging system, so that the imaging depth, the resolution ratio and the signal-to-noise ratio of the system are further improved.

Description

Technical Field

[0001] The present invention relates to the field of information optics technology, and in particular to a near-infrared two-zone line scanning optical tomography microscopy imaging system and method based on a virtual digital slit. Background Art

[0002] Line-scanning optical sectioning microscopy (LSOSM) is a technique developed based on confocal microscopy. LSOSM uses a tiny slit instead of the pinhole aperture used in confocal microscopy to filter out out-of-focus background noise, forming light slices containing only in-focus information. By stacking the light slices at different axial positions, the three-dimensional structure of the sample can be restored. Compared with mainstream optical tomography microscopy, LSOSM's high-throughput characteristics effectively improve imaging speed, and it has the advantages of low phototoxicity and high resolution, making it more suitable for long-term dynamic observation of living biological tissues and rapid three-dimensional imaging. The imaging effect of LSOSM depends primarily on the width of the slit. The narrower the slit, the less fluorescence signal is collected by the detector, making it difficult to meet imaging requirements. However, if the slit is too wide, it cannot effectively filter out out-of-focus noise.

[0003] Traditional LSOSM systems typically use a physical slit, resulting in a relatively rigid operating system that makes it difficult to flexibly adjust the slit size. Furthermore, the entire scanning process is not automatically controlled by a microcomputer, which hinders further improvements in scanning speed and imaging accuracy. Furthermore, traditional LSOSM systems mostly operate in the visible light band (400-760nm), where light propagation is subject to interference from absorption and scattering. Furthermore, the autofluorescence generated by biological tissues when excited at specific wavelengths can also cause strong background noise, affecting the imaging depth and signal-to-noise ratio of the LSOSM system.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a near-infrared two-zone line-scanning optical tomography microscopy imaging system and method based on a virtual digital slit, aiming to solve the problem that the existing LSOSM system is difficult to flexibly control the size of the slit, and the imaging depth and signal-to-noise ratio are affected.

[0006] The technical solutions of the present invention are as follows:

[0007] A near-infrared two-zone line scanning optical tomography microscopy imaging system based on a virtual digital slit comprises a near-infrared two-zone line scanning optical tomography microscopy imaging module and a virtual digital slit image reconstruction module for reconstructing images acquired by the near-infrared two-zone line scanning optical tomography microscopy imaging module;

[0008] The near-infrared two-zone line scanning optical tomography microscopy module includes an excitation unit and a detection unit; the excitation unit includes a laser, a first 4f unit, a first reflector, a digital micromirror device, a second 4f unit, a first tube lens, a dichroic mirror, an objective lens, and an object stage, which are arranged in sequence along the excitation light path; the detection unit includes a second tube lens, a third 4f unit, a filter, and a detector, which are arranged in sequence along the detection light path; the second tube lens is arranged on the side of the dichroic mirror away from the objective lens.

[0009] The near-infrared two-zone line scanning optical tomography microscopy system based on virtual digital slit, wherein the laser is a continuous light laser with an output wavelength of 975nm; the light source power of the laser is 1W-2W, and the output spot diameter of the laser is 1mm-2mm.

[0010] The near-infrared two-zone line scanning optical tomography microscopy system based on virtual digital slit, wherein the first 4f unit, the second 4f unit and the third 4f unit are all composed of convex lenses; the operating bands of the convex lenses, the first tube lens, the objective lens and the second tube lens are all between 900nm and 1700nm.

[0011] The near-infrared two-zone line scanning optical tomography microscopy system based on virtual digital slit, wherein, along the direction of the excitation light path, the first 4f unit includes a first lens and a second lens; the second 4f unit includes a third lens and a fourth lens; and the third 4f unit includes a fifth lens and a sixth lens;

[0012] The focal length of the first lens is 10mm-30mm; the focal length of the second lens is 150mm-450mm; the focal length of the third lens is 20mm-150mm; the focal length of the fourth lens is 20mm-150mm; the focal length of the fifth lens is 20mm-200mm; the focal length of the sixth lens is 40mm-400mm; the focal length of the first tube lens is 150mm-250mm; the focal length of the objective lens is 6mm-10mm; and the focal length of the second tube lens is 150mm-250mm.

[0013] The near-infrared two-zone line scanning optical tomography microscopy system based on a virtual digital slit, wherein the second 4f unit further includes a second reflector and an aperture stop arranged between the third lens and the fourth lens; the second reflector is arranged close to the third lens, and the aperture stop is arranged close to the fourth lens.

[0014] The near-infrared two-zone line scanning optical tomography microscopy system based on virtual digital slits, wherein the dichroic mirror is a 1050nm long-pass short-reflection dichroic mirror; the detector is an InGaAs detector with a detection band of 900nm-1700nm.

[0015] A near-infrared two-zone line scanning optical tomography microscopy imaging method based on a virtual digital slit comprises the following steps:

[0016] Provide a near-infrared two-zone line scanning optical tomography microscopy system based on a virtual digital slit;

[0017] Loading a digital line array into a digital micromirror device, and using a detector to collect a fluorescence image corresponding to the object to be measured on the stage and the digital line array;

[0018] Obtain the line size of the digital line array generated by the digital micromirror device that excites the object to be measured and reaches the detection surface of the detector to obtain the slit width;

[0019] Based on the slit width, the fluorescence images are filtered using a virtual digital slit image reconstruction module, and the processed images are superimposed and summed in sequence to achieve near-infrared two-zone line scanning optical tomography microscopy imaging based on the virtual digital slit.

[0020] The method for near-infrared two-zone line scanning optical tomography microscopy based on a virtual digital slit, wherein the acquisition of the line size of the line array generated by the digital micromirror device that reaches the detection surface of the detector after exciting the object to be measured also includes determining the point spread function of the near-infrared two-zone line scanning optical tomography microscopy system based on the virtual digital slit; according to the Rayleigh criterion, the expression of the point spread function is

[0021] Where λ is the excitation wavelength and NA is the numerical aperture of the objective lens.

[0022] The near-infrared two-zone line scanning optical tomography microscopy imaging method based on virtual digital slit, wherein the slit width is expressed by the standard deviation corresponding to the point spread function, and the expression is

[0023] Where S is the width of a single line on the imaging surface.

[0024] The near-infrared two-zone line scanning optical tomography microscopy imaging method based on virtual digital slit, wherein the expression for the sequential superposition and summation of the processed images is:

[0025] Among them, I final is the single-layer sample image after virtual slit processing, Mask is the virtual digital slit, I iis the image of a certain position of the object under test collected by the detector, and N is the number of images obtained by the detector after scanning a layer of samples.

[0026] Beneficial effects: The present invention provides a near-infrared two-zone line-scanning optical tomography microscopy imaging system and method based on a virtual digital slit, the system comprising a near-infrared two-zone line-scanning optical tomography microscopy imaging module, and a virtual digital slit image reconstruction module for reconstructing images collected by the near-infrared two-zone line-scanning optical tomography microscopy imaging module; the near-infrared two-zone line-scanning optical tomography microscopy imaging module comprises an excitation unit and a detection unit; the excitation unit comprises a laser, a first 4f unit, a first reflector, a digital micromirror device, a second 4f unit, a first tube lens, a dichroic mirror, an objective lens, and an objective stage, which are sequentially arranged along an excitation light path; the detection unit comprises a second tube lens, a third 4f unit, a filter, and a detector, which are sequentially arranged along the detection light path; the second tube lens is arranged on the side of the dichroic mirror away from the objective lens. The present invention introduces a digital micromirror device (DMD) and a digital linear array, leverages the DMD's high-speed signal modulation capability to modulate the excitation light, and uses the digital linear array to replace the physical slit in the traditional line scanning system to achieve the generation and rapid scanning of parallel linear array excitation light fields, which can greatly improve the flexibility and imaging speed of the system. In addition, a virtual digital slit image reconstruction module is introduced to filter out background noise and achieve optical sectioning. At the same time, the near-infrared second zone window (NIR-II) is introduced into the line scanning optical tomography microscopy imaging system, further improving the imaging depth, resolution, and signal-to-noise ratio of the system. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic structural diagram of a near-infrared two-zone line scanning optical tomography microscopy imaging system based on a virtual digital slit according to the present invention;

[0028] Figure 2 Schematic diagram of the principle of NIR-IIDMD-LSOSM;

[0029] Figure 3 :(a) Scanning pattern diagram when DMD is loaded with line array, (b) Line array diagram loaded into DMD, (c) Fluorescence image of uniform nanoparticle sample under line array excitation;

[0030] Figure 4 Schematic diagram for achieving synchronous triggering of DMD and InGaAs detector;

[0031] Figure 5 This is the result of reconstructing the electrospinning image collected by the NIR-IIDMD-LSOSM system using the DVS algorithm;

[0032] Explanation of the reference numerals: laser 10, first 4f unit 20, first lens 21, second lens 22, first reflector 30, digital micromirror device 40, second 4f unit 50, third lens 51, fourth lens 52, second reflector 53, aperture stop 54, first tube lens 60, dichroic mirror 70, objective lens 80, stage 90, second tube lens 100, third 4f unit 200, fifth lens 201, sixth lens 202, filter 300, detector 400. DETAILED DESCRIPTION

[0033] The present invention provides a near-infrared two-zone line-scanning optical tomography microscopy system and method based on a virtual digital slit. To clarify the objectives, technical solutions, and effects of the present invention, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention.

[0034] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art in the art to which the present invention belongs. It should also be understood that terms such as those defined in common dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0035] Compared with the visible light band, the second nearinfrared window (NIR-II) at 1000-1880nm has lower photon scattering, and the autofluorescence of biological tissues in this band almost completely disappears, which is conducive to further improving the penetration depth, resolution and signal-to-noise ratio of imaging.

[0036] Based on this, Figure 1 As shown, the present invention provides a near-infrared two-zone line scanning optical tomography microscopy imaging system based on a virtual digital slit, comprising a near-infrared two-zone line scanning optical tomography microscopy imaging module, and a virtual digital slit image reconstruction module for reconstructing images captured by the near-infrared two-zone line scanning optical tomography microscopy imaging module;

[0037] The near-infrared two-zone line scanning optical tomography microscopy module includes an excitation unit and a detection unit; the excitation unit includes a laser 10, a first 4f unit 20, a first reflector 30, a digital micromirror device 40, a second 4f unit 50, a first tube lens 60, a dichroic mirror 70, an objective lens 80, and an object stage 90, which are arranged in sequence along the excitation light path; the detection unit includes a second tube lens 100, a third 4f unit 200, a filter 300, and a detector 400, which are arranged in sequence along the detection light path; the second tube lens 100 is arranged on the side of the dichroic mirror 70 away from the objective lens 80.

[0038] In this embodiment, the provided near-infrared second-zone line scanning optical sectioning microscopy system (NIR-IIDMD-based line scanning optical sectioning microscopy, NIR-IIDMD-LSOSM) introduces a digital micromirror device (DMD) and a digital linear array, modulates the excitation light with the help of the high-speed signal modulation capability of the DMD, and uses the digital linear array to replace the physical slit in the traditional line scanning system to realize the generation and rapid scanning of the parallel linear array excitation light field, which can greatly improve the flexibility and imaging speed of the system; and by introducing a virtual digital slit image reconstruction module to filter out background noise, optical sectioning is achieved; at the same time, the near-infrared second zone window (NIR-II) is introduced into the line scanning optical sectioning microscopy system, further improving the imaging depth, resolution and signal-to-noise ratio of the system.

[0039] Specifically, the present invention utilizes the DMD's high-speed modulation capability for digital signals to replace the physical slit and scanning mirror combination in traditional LSOSM systems, greatly improving the system's flexibility and scanning speed. Using the DMD loaded with a digital linear array to modulate the excitation light and scan the object under test effectively increases imaging speed and reduces the complexity of system construction. Simultaneously, a virtual digital slit image reconstruction module is introduced to reconstruct the captured image and obtain three-dimensional structural information of the object under test. Furthermore, to further enhance the penetration depth of three-dimensional imaging, a near-infrared zone II window is introduced into the imaging system, overcoming the limitation of traditional linear scanning optical tomography systems that only operate in the visible light band. This window's extremely low photon absorption, scattering, and autofluorescence effectively enhances the imaging penetration depth and signal-to-noise ratio.

[0040] In the present invention, the NIR-IIDMD-LSOSM system modulates the excitation light with the help of the high-speed digital signal modulation capability of the DMD, and uses a digital linear array to replace the physical slit in the traditional line scanning system to achieve the generation and rapid scanning of the parallel linear array excitation light field, which greatly improves the flexibility and imaging speed of the system. Figure 2As shown, during the imaging process, the laser light source will generate a linear array beam at the objective lens after being modulated by the DMD. These beams are limited by the diffraction limit of the optical system and will form a Gaussian intensity distribution with a certain width at a position perpendicular to the line in the focal plane of the objective lens, and the illumination intensity is different at different positions. The object to be tested is scanned successively using linear array modulated excitation light, and a detector is used to collect line scan images of the same sample at different times. The line scan images at different times are superimposed to obtain the structural distribution information of a layer of the object to be tested. Then, different layers of the object to be tested are scanned, and the three-dimensional imaging of the object to be tested is completed by superimposing them.

[0041] In some embodiments, the NIR-IIDMD-LSOSM imaging technique can be considered as a wide-field fluorescence microscopy technique using structured light field illumination, and its imaging process can be expressed as: Where e(x) is the line array excitation mode, and the line array image detected by the system is the product of the structure of the object to be measured and the system detection point spread function.

[0042] In some embodiments, the stage is disposed on a motorized nano-displacement stage, and the motorized nano-displacement stage can be used to adjust the Z-axis position of the stage to scan different layers of the object to be measured.

[0043] In some embodiments, the virtual digital slit-based near-infrared second-zone line scanning optical tomography microscopy system further includes a NIR-II fluorescent probe; preferably, the NIR-II fluorescent probe is a rare earth-doped nanoparticle with an emission peak at 1550 nm.

[0044] In some embodiments, the laser is a continuous light laser with an output wavelength of 975 nm; the light source power of the laser is 1 W-2 W, and the output spot diameter of the laser is 1 mm-2 mm.

[0045] Specifically, in order to match the NIR-II fluorescent probe, the system uses a continuous light laser with an output wavelength of 975 nm as the excitation light source, the light source power is 1 W, and the output spot diameter is 1 mm.

[0046] In some embodiments, the first 4f unit, the second 4f unit, and the third 4f unit are all composed of convex lenses; the operating bands of the convex lenses, the first tube lens, the objective lens, and the second tube lens are all between 900nm and 1700nm.

[0047] In some embodiments, along the direction of the excitation light path, the first 4f unit 20 includes a first lens 21 and a second lens 22; the second 4f unit 50 includes a third lens 51 and a fourth lens 52; the third 4f unit 200 includes a fifth lens 201 and a sixth lens 202;

[0048] The focal length of the first lens is 10mm-30mm; the focal length of the second lens is 150mm-450mm; the focal length of the third lens is 20mm-150mm; the focal length of the fourth lens is 20mm-150mm; the focal length of the fifth lens is 20mm-200mm; the focal length of the sixth lens is 40mm-400mm; the focal length of the first tube lens is 150mm-250mm; the focal length of the objective lens is 6mm-10mm; and the focal length of the second tube lens is 150mm-250mm.

[0049] In some embodiments, the second 4f unit 50 further includes a second reflector 53 and an aperture diaphragm (Iris) 54 disposed between the third lens 51 and the fourth lens 52; the second reflector 53 is disposed close to the third lens 51, and the aperture diaphragm 54 is disposed close to the fourth lens 52.

[0050] Specifically, after the excitation light is emitted, it first passes through the first 4f unit for expansion and collimation. The expanded excitation light is reflected at a specific angle by the first reflector onto the digital micromirror device (DMD) panel, and then enters the second 4f unit through DMD reflection. The aperture stop is placed on the Fourier surface of the second 4f unit to block the reflected light of the excess diffraction pole. The light then converges at the back focal plane of the second 4f unit to form a uniformly distributed line stripe array. After being reduced by the first tube lens and objective lens, it is irradiated onto the object to be tested on the stage, forming a multi-line parallel excitation mode. After being excited by the light source, the fluorescence generated by the object to be tested enters the detection light path through the dichroic mirror, is magnified to a suitable size by the second tube lens and the third 4f unit, and then the stray light is filtered out by the filter. The final object to be tested signal is collected by the detector.

[0051] In some embodiments, the incident light received by the digital micromirror device is at a 24° angle to the normal of the panel of the digital micromirror device to avoid distortion of the excitation mode; the digital micromirror device is mainly used to generate regular sparsely distributed stripes to modulate the excitation light. When the incident light is at a 24° angle to the DMD normal, the micromirror in the "on" state will reflect the incident light in a direction perpendicular to the DMD surface into the subsequent second 4f unit and focus it on the surface of the object to be measured; while the micromirror in the "off" state will cause the incident light to deviate from the optical axis, thereby reducing the impact of stray light on imaging. Therefore, during the optical path construction process, it is necessary to ensure that the angle between the incident light and the DMD surface is appropriate.

[0052] In some embodiments, the dichroic mirror is a 1050 nm long pass short reflection dichroic mirror; and the detector is an InGaAs detector with a detection band of 900 nm-1700 nm.

[0053] In a specific embodiment, in order to more clearly describe the working principle of the system and the connection process between the various components, the entire near-infrared second-zone line scanning optical tomography microscopy module is divided into an excitation unit and a detection unit; specifically as follows:

[0054] In the construction of the excitation unit, it is first necessary to select a suitable beam expansion factor. The spot size of the laser beam after beam expansion is just slightly larger than the reflective panel of the DMD, so as to maximize the utilization efficiency of the light source. The spot diameter of the system light source is 1mm, the single pixel size of the DMD is 10.8μm×10.8μm, the pixel number is 1024×768, and the effective modulation reflection surface is 1.1cm×0.83cm. In order to make the light spot cover the modulation surface of the DMD, a lens composed of the first lens (f1=10mm) and the second lens (f2 =10mm), the first 4f unit, consisting of a third lens (f3 = 75mm) and a fourth lens (f4 = 75mm), expands the light source's spot diameter by 15 times. Secondly, during the optical path construction process, the incident angle of the light beam needs to be 24° with the normal to the DMD surface to avoid distortion of the excitation pattern. The DMD reflects the incident light perpendicular to the DMD surface into the second 4f unit, consisting of a third lens (f3 = 75mm) and a fourth lens (f4 = 75mm). The light is then reduced by 25 times by the first tube lens (f = 200mm) and the objective lens (f = 8mm) before reaching the surface of the object to be measured, achieving parallel linear array excitation.

[0055] In the detection unit, a near-infrared InGaAs detector is used to collect the fluorescence signal of the object under test. The detector has an operating band of 900-1700nm and a single pixel size of 15μm. After the object under test is excited, the generated fluorescence signal passes through the objective lens and then enters the detection optical path through a dichroic mirror. Because the excitation light wavelength is 975nm, and the fluorescence emission of the probe is between 1500-1700nm, a 1050nm long-pass short-reflection dichroic mirror is selected. After entering the detection optical path, the fluorescence is magnified 25 times by the objective lens and the tube lens (f=200mm) of the detection optical path. It is then magnified 2 times by the third 4f unit composed of a fifth lens (f5=40mm) and a sixth lens (f6=80mm). It is then filtered through a 1300nm long-pass filter to remove stray light before being collected by the detector. In addition, during the system construction process, care should be taken to maintain the conjugate relationship between the DMD surface and the object under test plane to prevent spot deformation from affecting the imaging effect.

[0056] Specifically, the workflow of the NIR-IIDMD-LSOSM system includes scanning of the object to be tested and collection of fluorescence data. During the imaging process of the system, the DMD can be used to simultaneously realize the generation of the line excitation array and the scanning of the object to be tested; DMD is a micro-mechanical optical device, and its display panel is composed of several miniature square mirrors. These mirrors are quickly switched between ±12° through the hinge below, corresponding to the "on" and "off" in the display mode. In the NIR-IIDMD-LSOSM system, DMD is mainly used to generate a regular focusing line array, and then focus on the sample surface after the subsequent optical path is narrowed to stimulate the fluorescence of the structure of the object to be tested. When the incident light is 24° to the normal of the DMD surface, the "on" state micro-mirror will turn the incident light to the sample surface. The light is reflected into the subsequent optical path at an angle perpendicular to the DMD surface, and forms a diffraction-limited focused spot at the corresponding position on the surface of the object to be measured. The micro-mirror in the "off" state will deviate the incident light from the optical axis at a large angle; therefore, as long as the micro-mirror in a specific position remains in the "on" state, a regular array of line stripes can be generated. The DMD needs to be used in conjunction with the control system to achieve precise control of the micro-mirror, ensure that the control software can work well with the DMD hardware, can quickly respond and accurately adjust the switching state of the micro-mirror, and when debugging the system, attention should also be paid to communication delays and data transmission stability to ensure the real-time performance of the scanning process.

[0057] In actual operation, the DMD's reflective panel can be regarded as a 1024×768 black and white screen. Each pixel of the screen corresponds to a micro-mirror, where white pixels correspond to micro-mirrors in the "on" state and black pixels correspond to micro-mirrors in the "off" state. According to the duty cycle of the "on / off" state of each micro-mirror, a corresponding line stripe binary image is created. The state of the micro-mirrors is controlled by controlling the binary image loaded into the DMD. When using the NIR-II DMD-LSOSM system to scan the object to be tested, a series of binary stripe patterns need to be loaded into the DMD in advance. When the DMD switches the display mode according to the order of the loaded patterns, the position of the white pixels will change with the Figure 3 Move in the direction indicated by the arrow, one pixel at a time, until the scanning of one layer of samples is completed. The principle is as follows Figure 3 As shown in (a) in .

[0058] For a better understanding, this embodiment uses rare earth-doped nanoparticles with an emission wavelength of 1550 nm to verify the excitation and scanning performance of the NIR-IIDMD-LSOSM system on the object to be measured. First, the nanoparticles are diluted a certain multiple and then smeared on a glass slide. After they are completely dry, they are sealed with a mounting medium to produce a uniformly distributed sample. When creating a stripe binary image, the appropriate scanning step size (i.e., the width of the line) should be selected first to increase the imaging range of the system while avoiding artifacts during the reconstruction process. The line width selected in this embodiment is the size of a single pixel of the DMD. In addition, when scanning the sample, the density of the stripes loaded into the DMD will affect the signal-to-noise ratio of the imaging. Too dense stripes will increase the scattered light signal between the line arrays and generate strong background noise, while too sparse stripes will prolong the scanning time. By loading stripe arrays of different densities into the DMD and analyzing the signal-to-noise ratio of the scanned image, the number of lines in a binary stripe image is set to 7. Figure 3 (b) is the sparse line array image loaded into DMD. Figure 3 (c) in the figure shows a uniform fluorescent sample image generated using the linear array. To maximize the imaging speed of the system, the number of scan steps (i.e., the number of binary images required to scan a single layer of the sample) should be minimized while maintaining a constant scan step size.

[0059] During the fluorescence data acquisition process, in order to synchronously control the hardware in the NIR-IIDMD-LSOSM system, the present invention uses Labview program (National Instruments) to communicate between the Z-axis electric nano-displacement stage, DMD and InGaAs detector. All hardware are connected to the same workstation for data transmission and command sending, thereby realizing the switching of scanning modes, the synchronous acquisition of fluorescence signals by the detector and the change of the number of scanning layers; the Z-axis electric nano-displacement stage is used to control the longitudinal scanning step length. When the Z-axis electric nano-displacement stage moves to the set position, as a series of binary images are loaded into the DMD, the InGaAs detector acquires the original fluorescence image sequence; when acquiring data, each scanning position corresponds to an exposure of the detector. Whenever the line stripe array moves to the next position, the detector ends the previous exposure and immediately enters the next synchronous acquisition; during the image acquisition process, it is necessary to use a data acquisition card to synchronize the scanning process with the data acquisition process of the detector. The principle of synchronous triggering of the DMD and InGaAs detector is as follows Figure 4As shown in the figure, this is the timing diagram of the hardware control; the data acquisition card is used as the main clock source to generate two synchronously triggered square wave signals, one of which is the trigger signal for the DMD and the other is the trigger signal for the InGaAs detector. These two signals have the same initial phase and frequency and are used to control the switching of the DMD excitation mode and the data acquisition of the detector respectively; when the imaging system is running, the data acquisition card sends two synchronous signals. Whenever the DMD receives a new rising edge signal, it reads a new binary image from the memory to replace the current image, thereby adjusting the position of the focused light on the sample surface; at the same time, the detector will end the current data acquisition and quickly enter a new acquisition cycle under the action of the trigger signal. The exposure time of each time is the time interval between the two rising edges. Therefore, the frequency of the trigger signal can be adjusted according to the characteristics of the object to be measured to achieve precise control of the scanning speed and single-frame exposure time; after the DMD and detector have collected a layer of data, the Z-axis motorized nano-stage moves one step and cycles in sequence, thereby completing the three-dimensional data acquisition of the sample.

[0060] In addition, the present invention also provides a near-infrared two-zone line scanning optical tomography microscopy imaging method based on a virtual digital slit, comprising the steps of:

[0061] Step S10: providing a near-infrared two-zone line scanning optical tomography microscopy imaging system based on a virtual digital slit;

[0062] Step S20: loading a digital line array into the digital micromirror device, and using a detector to collect a fluorescence image corresponding to the object to be tested on the stage and the digital line array;

[0063] Step S30: obtaining the size of the line that reaches the detection surface of the detector after the digital line array generated by the digital micromirror device excites the object to be tested, and obtaining the slit width;

[0064] Step S40: Based on the slit width, the fluorescence images are filtered using a virtual digital slit image reconstruction module, and the processed images are sequentially superimposed and summed to achieve near-infrared two-zone line scanning optical tomography microscopy imaging based on the virtual digital slit.

[0065] In this embodiment, by introducing a digital micromirror device (DMD) and a digital linear array, the excitation light is modulated with the help of the high-speed signal modulation capability of the DMD, and the digital linear array is used to replace the physical slit in the traditional line scanning system to realize the generation and rapid scanning of the parallel linear array excitation light field, which can greatly improve the flexibility and imaging speed of the system; and by introducing a virtual digital slit image reconstruction module to filter out background noise, optical sectioning is achieved; at the same time, the near-infrared second zone window (NIR-II) is introduced into the line scanning optical tomography microscopy imaging system, further improving the imaging depth, resolution and signal-to-noise ratio of the system.

[0066] Specifically, the NIR-IIDMD-LSOSM system uses one-dimensional line scanning excitation to replace the point scanning excitation in the traditional confocal system, which can greatly improve the imaging speed; and, no physical slits or masks are used during the system construction process, thereby effectively improving the flexibility of the imaging system, reducing the difficulty of system debugging and the errors caused by components. However, since no physical slit is introduced to filter the fluorescence generated by the sample, the three-dimensional fluorescence image obtained by the NIR-IIDMD-LSOSM system can only be approximated as a superposition of wide-field images, and cannot effectively avoid the interference caused by non-focal background noise on the imaging. In order to solve this problem, the present invention introduces a virtual digital slit (DVS) into the reconstruction process of the NIR-IIDMD-LSOSM image, and replaces the traditional physical slit with a simulated virtual digital slit to achieve the filtering of stray signals. The method of introducing DVS for image reconstruction can not only effectively simplify the imaging system, but also improve the system's utilization of excitation light energy.

[0067] In some embodiments, the acquisition of the line size of the line array generated by the digital micromirror device and reaching the detection surface of the detector after exciting the object to be measured also includes determining the point spread function of the near-infrared two-zone line scanning optical tomography microscopy imaging system based on the virtual digital slit; according to the Rayleigh criterion, the expression of the point spread function is

[0068] Where λ is the excitation wavelength and NA is the numerical aperture of the objective lens.

[0069] In some embodiments, the slit width is expressed using the standard deviation corresponding to the point spread function, expressed as

[0070] Where S is the width of a single line on the imaging surface.

[0071] In some embodiments, the expression for the sequential superposition and summation of the processed images is:

[0072] Among them, I final is the single-layer sample image after virtual slit processing, Mask is the virtual digital slit, I i is the image of a certain position of the object under test collected by the detector, and N is the number of images obtained by the detector after scanning a layer of samples.

[0073] In some embodiments, the filtering process includes: first using a spectral filtering algorithm to reduce the noise of the fluorescence image, converting the image to the spectral domain through Fourier transform, filtering out excess background noise through a high-pass filter, and setting the cutoff frequency according to the transfer function of the filter, and then using an inverse Fourier transform to obtain the filtered fluorescence image to complete the filtering process; finally, using a virtual digital slit to reconstruct the image after noise reduction using spectral filtering, thereby removing stray signals and achieving the purpose of optical slicing.

[0074] In this embodiment, as an example, when using DVS to filter the collected fluorescence image, it is first necessary to determine the size of the line stripes that reach the detection surface of the detector after the line array generated by the DMD excites the object to be tested, and calculate the appropriate slit width based on the line stripe size of the imaging surface. In the constructed NIR-IIDMD-LSOSM system, a line array template containing 7 parallel lines is used to scan the object to be tested, where the single line width is 10.8μm (the size of a single micromirror of the DMD) and the line length is 3628.8μm (the size of 336 DMD micromirrors). 48 line array stripe patterns are required to complete the scanning of one layer of sample. The focused line array is imaged onto the sample surface after being reduced by 25 times by the tube lens and the objective lens. The line width corresponding to the sample surface is 0.432μm. The probe excitation wavelength λ used in this specific example is 975nm, and the objective lens used by the system is a 25X water mirror (NA is 1.1). The results of reconstructing the electrospinning image collected by the NIR-II DMD-LSOSM system using the DVS algorithm are shown in the figure. Figure 5 shown.

[0075] In summary, the present invention provides a near-infrared two-zone line-scanning optical tomography microscopy system and method based on a virtual digital slit. The system includes a near-infrared two-zone line-scanning optical tomography microscopy module and a virtual digital slit image reconstruction module for reconstructing images captured by the near-infrared two-zone line-scanning optical tomography microscopy module; the near-infrared two-zone line-scanning optical tomography microscopy module includes an excitation unit and a detection unit; the excitation unit includes a laser, a first 4f unit, a first reflector, a digital micromirror device, a second 4f unit, a first tube lens, a dichroic mirror, an objective lens, and an object stage arranged in sequence along the excitation light path; the detection unit includes a second tube lens, a third 4f unit, a filter, and a detector arranged in sequence along the detection light path; the second tube lens is arranged on the side of the dichroic mirror away from the objective lens. The present invention introduces a digital micromirror device (DMD) and a digital linear array, leverages the DMD's high-speed signal modulation capability to modulate the excitation light, and uses the digital linear array to replace the physical slit in the traditional line scanning system to achieve the generation and rapid scanning of parallel linear array excitation light fields, which can greatly improve the flexibility and imaging speed of the system. In addition, a virtual digital slit image reconstruction module is introduced to filter out background noise and achieve optical sectioning. At the same time, the near-infrared second zone window (NIR-II) is introduced into the line scanning optical tomography microscopy imaging system, further improving the imaging depth, resolution, and signal-to-noise ratio of the system.

[0076] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A near-infrared two-zone line scanning optical tomography microscopy system based on a virtual digital slit, characterized in that: It includes a near-infrared two-zone line scanning optical tomography microscopy imaging module and a virtual digital slit image reconstruction module for reconstructing images collected by the near-infrared two-zone line scanning optical tomography microscopy imaging module; The near-infrared two-zone line scanning optical tomography microscopy module includes an excitation unit and a detection unit; the excitation unit includes a laser, a first 4f unit, a first reflector, a digital micromirror device, a second 4f unit, a first tube lens, a dichroic mirror, an objective lens, and an object stage, which are arranged in sequence along the excitation light path; the detection unit includes a second tube lens, a third 4f unit, a filter, and a detector, which are arranged in sequence along the detection light path; the second tube lens is arranged on the side of the dichroic mirror away from the objective lens.

2. The near-infrared two-zone line scanning optical tomography microscopy system based on virtual digital slit according to claim 1, characterized in that: The laser is a continuous light laser with an output wavelength of 975 nm; the light source power of the laser is 1 W to 2 W, and the output spot diameter of the laser is 1 mm to 2 mm.

3. The near-infrared two-zone line scanning optical tomography microscopy system based on virtual digital slit according to claim 1, characterized in that: The first 4f unit, the second 4f unit and the third 4f unit are all composed of convex lenses; the operating bands of the convex lenses, the first tube lens, the objective lens and the second tube lens are all between 900nm and 1700nm.

4. The near-infrared two-zone line scanning optical tomography microscopy system based on virtual digital slit according to claim 1, characterized in that: Along the direction of the excitation light path, the first 4f unit includes a first lens and a second lens; the second 4f unit includes a third lens and a fourth lens; and the third 4f unit includes a fifth lens and a sixth lens; The focal length of the first lens is 10mm-30mm; the focal length of the second lens is 150mm-450mm; the focal length of the third lens is 20mm-150mm; the focal length of the fourth lens is 20mm-150mm; the focal length of the fifth lens is 20mm-200mm; the focal length of the sixth lens is 40mm-400mm; the focal length of the first tube lens is 150mm-250mm; the focal length of the objective lens is 6mm-10mm; and the focal length of the second tube lens is 150mm-250mm.

5. The near-infrared two-zone line scanning optical tomography microscopy system based on virtual digital slit according to claim 4, characterized in that: The second 4f unit further includes a second reflecting mirror and an aperture stop disposed between the third lens and the fourth lens; the second reflecting mirror is disposed close to the third lens, and the aperture stop is disposed close to the fourth lens.

6. The near-infrared two-zone line scanning optical tomography microscopy system based on virtual digital slit according to claim 1, characterized in that: The dichroic mirror is a 1050nm long-pass short-reflection dichroic mirror; the detector is an InGaAs detector with a detection band of 900nm-1700nm.

7. A near-infrared two-zone line scanning optical tomography microscopy method based on a virtual digital slit, characterized in that: Including steps: Provided is a near-infrared two-zone line scanning optical tomography microscopy system based on a virtual digital slit according to any one of claims 1 to 6; Loading a digital line array into a digital micromirror device, and using a detector to collect a fluorescence image corresponding to the object to be measured on the stage and the digital line array; Obtain the line size of the digital line array generated by the digital micromirror device that reaches the detection surface of the detector after exciting the object to be measured, and obtain the slit width; Based on the slit width, the fluorescence images are filtered using a virtual digital slit image reconstruction module, and the processed images are superimposed and summed in sequence to achieve near-infrared two-zone line scanning optical tomography microscopy imaging based on the virtual digital slit.

8. The near-infrared two-zone line scanning optical tomography microscopy imaging method based on virtual digital slit according to claim 7, characterized in that: The acquisition of the line size of the line array generated by the digital micromirror device and reaching the detection surface of the detector after exciting the object to be tested also includes determining the point spread function of the near-infrared two-zone line scanning optical tomography microscopy imaging system based on the virtual digital slit; according to the Rayleigh criterion, the expression of the point spread function is Where λ is the excitation wavelength and NA is the numerical aperture of the objective lens.

9. The near-infrared two-zone line scanning optical tomography microscopy imaging method based on virtual digital slit according to claim 8, characterized in that: The slit width is expressed using the standard deviation corresponding to the point spread function, expressed as: Where S is the width of a single line on the imaging surface.

10. The near-infrared two-zone line scanning optical tomography microscopy imaging method based on virtual digital slit according to claim 7, characterized in that: The expression for the sequential superposition and summation of the processed images is: Among them, I final is the single-layer sample image after virtual slit processing, Mask is the virtual digital slit, I i is the image of a certain position of the object under test collected by the detector, and N is the number of images obtained by the detector after scanning a layer of samples.

Images