TWI714378B - A large-angle optical raster scanning system for high speed deep tissue imaging - Google Patents

Abstract

The field of view (FOV) of a nonlinear optical microscope (NLOM) is expected to be large enough for employing high-speed raster scanning on a mesoscale volumetric biological sample. Concurrently, three-dimensional (3D) visualization of fine sub-micron biological structures requires high enough lateral and axial resolutions, enforcing a high numerical aperture (NA) objective lens to be employed, thereby limiting the FOV of an NLOM. The invention is directed to a laser scanning NLOM, or to a large-angle optical raster scanning system, for deep biological tissue imaging with a large FOV of more than one square millimeter, up to 1.6 × 1.6 mm², while simultaneously maintaining a sub-femtoliter effective 3D resolution by means of a high-NA and low magnification objective lens and further maintaining a high acquisition speed with synchronized sampling, limited by the repetition rate of a high repetition rate pulsed laser source, thereby exceeding Nyquist Criterion for resolving micro-optical resolution throughout a horizontal FOV of more than one millimeter.

Description

A wide-angle optical domain scanning system for high-speed deep tissue imaging

The invention relates to a large-angle optical scanning system for high-speed deep tissue imaging.

Compared with single-photon imaging systems based on charge-coupled elements (CCD), the deep penetration capability of nonlinear optical microscopes (NLOM) makes it suitable for three-dimensional (3D) imaging. In order to perform high-speed optical domain scanning of biological samples with a mesoscale volume, it is generally expected that the field of view (FOV) of the nonlinear optical microscope NLOM can be large enough. However, in order to expand the field of view (FOV) of a nonlinear optical microscope (NLOM), whether increasing the scanning angle or using a low magnification and low numerical aperture (NA) objective lens will reduce the imaging quality on the optical resolution. On the contrary, 3D visualization of fine submicron biological structures requires sufficiently high lateral and axial resolution, which requires the use of high numerical aperture (NA) objectives. In addition, the extended field of view FOV must meet the Nyquist criterion (Nyquist criterion). -Criterion) high-speed sampling. Therefore, without affecting the acquisition speed, it is a difficult task to extend the field of view FOV with sub-feeling effective 3D resolution to more than 1 square millimeter.

Several techniques for mesoscale imaging have been reported previously. Balu, Mihaela, and others, "An imaging platform based on a nonlinear optical microscope for rapid scanning of large areas of tissue." Patent Publication Number: WO 2018/075562 A1 discloses a non-linear optics (NLO) microscope design with an extended field of view of 0.8×0.8mm ² =0.64mm ² , which is less than one square millimeter, and a resolution of 1600×1600 Therefore, in the entire field of view (FOV) spanning 0.8×0.8 mm ² , for a submicron lateral resolution of 0.5±0.2 μm, the Nyquist criterion cannot be satisfied with an image size of 0.5 mm.

0.6)物鏡將視場(FOV)擴展到幾平方毫米，從而導致軸向分辨率差(進而導致3D分辨率差)，原因是軸向分辨率與物鏡的數值孔徑(NA)呈反平方關係。 Several researchers have published several non-linear optical microscopes, NLOM, which use low numerical aperture (NA) (

0.6) The objective lens expands the field of view (FOV) to several square millimeters, resulting in poor axial resolution (and thus poor 3D resolution), because the axial resolution and the numerical aperture (NA) of the objective are inversely squared.

Jonathan R. Bumstead et al., "Designing a two-photon microscope with a large field of view using optical invariant analysis", Neurophoton, 5(2), 025001 (2018), DOI: 10.1117/1.NPh.5.2.025001, discloses one Using the two-photon microscope for light invariance analysis, the FOV diameter of the field of view produced is 7mm, and the lateral and axial resolutions are 1.7μm and 28μm, respectively. It is achieved by using a 4x objective lens with a 0.22 numerical aperture NA. In addition, to solve the lateral resolution of 1.7μm on the FOV with a diameter of 7mm, that is, the square field of view FOV of 4.95×4.95mm ² , the Nyquist criterion Nyquist Criterion needs more than 5800×5800 pixels, but they Limited by the data collection speed of 1M samples per second.

Shin-Ichiro Terada et al., "Ultra-wide-field two-photon imaging with a micro-optic device moving in the rear objective lens space", Nature Communications (2018) 9: 3550, DOI: 10.1038/s41467-018-06058-8, A two-photon imaging system using a low-optic mechanical device in the rear eyepiece space is disclosed, resulting in a FOV of 1.2×3.5mm ² , which is stitched together by sequential imaging of multiple remote areas (>6mm). X axis The lateral resolution in the direction is 1.26±0.03 μm, the lateral resolution in the Y-axis direction is 0.88±0.07 μm, and the axial resolution of the 10X, 0.6 numerical aperture (NA) objective lens is 9.96±0.12 μm, which is further affected by the data acquisition speed limits.

Therefore, the existing technology is limited by less than one square millimeter field of view (FOV), or the axial resolution is poor, and/or the slower data acquisition speed meets the Nyquist criterion, so it is not suitable for high-resolution 3D imaging. That is, it cannot have an effective 3D resolution that meets the sub-feeling Nyquist in a field of view (FOV) of more than 1 square millimeter.

It is desired in the art to provide a nonlinear optical microscope NLOM to overcome the above-mentioned problems.

The present invention aims to provide a wide-angle optical domain scanning system for high-speed deep tissue imaging, which has an extended field of view (FOV) greater than one square millimeter, while maintaining a high-speed data acquisition system that exceeds the Nyquist criterion. Analytical and efficient 3D resolution. In doing so, in order not to affect the speed, each voxel acquisition is synchronized with each light pulse of a pulsed laser source with a high repetition rate, thereby pushing the acquisition speed to the maximum, even if it is limited The repetition rate of the pulsed laser source. The present invention uses a high numerical aperture (NA) and low magnification objective lens, and its resolution is not compromised. In order to extend the field of view (FOV) to more than one square millimeter, the present invention provides a special optical design that supports large scanning angles on both the fast X axis and the slow Y axis, and at the same time on the entire field of view (FOV) Maintain low optical aberrations.

While expanding the scanning angle (thus expanding the field of view (FOV)), the nonlinear speed of the resonant scanning mirror will cause image distortion along the fast X axis, which forces people to sample at unequal time points to maintain uniformity The pixel rate, which reduces the number of pixels, is not enough to meet the Nyquist criterion for a large field of view FOV with micro-optical resolution. The present invention relates to The point exceeds the Nyquist sampling in the entire scanning range, and then uses the graphics processing unit (GPU) accelerated interpolation algorithm to instantly repair the distortion caused by the resonance scanner. Compared with the center of the field of view (FOV), when more data points are collected near the edge (due to equidistant sampling in the entire nonlinear motion of the resonant scanning mirror), and due to the limited number of objective lens fields, It can further compensate for the decrease in signal intensity caused by vignetting near the edge of the FOV.

A_n×N_n，其中A_n是奈奎斯特(Nyquist)限制的採樣率，其係以4倍的水平FOV乘以共振掃描器頻率再除以理論上受目鏡限制的橫向分辨率，用以在超越>1mm的水平視場(FOV)上解析微光學分辨率，並且N_n是

1的整數來表示每個三維像素(voxel)的雷射脈衝數；一諧振掃描鏡，其光學耦合到該一或多個脈衝雷射源；一檢流計掃描鏡(galvanometer scanning mirror)，光學耦合到該諧振掃描鏡；一掃描透鏡，光學耦合到該檢流計掃描鏡；一專用管透鏡，包括三個平凸透鏡，每個平凸透鏡的有效焦距為500mm，該等透鏡組合在一起並光學耦合到該掃描透鏡；一與該專用管透鏡光學耦合的高數值孔徑(NA)和低倍物鏡，用以光域掃描一體積組織樣本，並用以收集樣本產生的一熒光信號，該熒光信號被導引 至一光電倍增管(PMT)以產生一電信號；以及一資料採集系統，耦合以從該光電倍增管(PMT)接收該電信號，其中每個採樣事件分別與來自一脈衝雷射源或最高重複率脈衝雷射源(如果有一或多個脈衝雷射源)的每個光脈衝同步，其中，該掃描透鏡和該專用管透鏡構成了一低倍率的擴束器，從而最大化了該視場(FOV)，但同時在高數值孔徑(NA)和低倍率物鏡的一後孔徑上提供了擴大的光束大小，以保持高激發數值孔徑(NA)，因而具有高分辨率。 According to the present invention, in order to solve the above-mentioned problems encountered in the prior art, a wide-angle optical domain scanning system for high-speed deep tissue imaging is disclosed, which has a field of view (FOV) of at least one square millimeter and has an ability to exceed Nyquist The sub-femtoliter effective 3D resolution of Nyquist's synchronous sampling analysis, including: one or more (1st to nth) pulsed laser sources for emitting one or more center wavelengths Is a laser beam of λ _n , and the trans-Nyquist repetition rate of the n-th pulsed laser source is R _n

A _n ×N _n , where A _n is the sampling rate limited by Nyquist, which is 4 times the horizontal FOV multiplied by the resonant scanner frequency and divided by the theoretical lateral resolution limited by the eyepiece. To resolve the micro-optical resolution in a horizontal field of view (FOV) beyond >1mm, and N _n is

An integer of 1 represents the number of laser pulses per voxel; a resonant scanning mirror, which is optically coupled to the one or more pulsed laser sources; a galvanometer scanning mirror, optical Coupled to the resonant scanning mirror; a scanning lens, optically coupled to the galvanometer scanning mirror; a special tube lens, including three plano-convex lenses, each plano-convex lens has an effective focal length of 500mm, and these lenses are combined and optical Coupled to the scanning lens; a high numerical aperture (NA) and low power objective lens optically coupled to the dedicated tube lens for scanning a volume of tissue sample in the optical domain, and for collecting a fluorescence signal generated by the sample, the fluorescence signal is Guided to a photomultiplier tube (PMT) to generate an electrical signal; and a data acquisition system coupled to receive the electrical signal from the photomultiplier tube (PMT), wherein each sampling event is associated with a pulsed laser source Or the highest repetition rate pulsed laser source (if there are one or more pulsed laser sources) each light pulse is synchronized, wherein the scanning lens and the special tube lens constitute a low-magnification beam expander, thereby maximizing The field of view (FOV), but at the same time provides an enlarged beam size on a rear aperture of a high numerical aperture (NA) and a low magnification objective lens to maintain a high excitation numerical aperture (NA), thus having a high resolution.

100: Large-angle optical domain scanning system

101: Input laser beam

102: Resonance Scanning Mirror

103: Galvanometer Scanning Mirror

104: Scanning lens

105, 106, 108: steering mirror

107: dedicated tube lens

109: High NA (numerical aperture) and low magnification objectives

110: focal length plane

111: Dichroic beam splitter

112: Focusing lens

113, 114: steering mirror

115: focusing lens

116: PMT photosensitive area

117: Transimpedance amplifier

119: Resonant Scanning Mirror Controller

200: Data Acquisition System

Figures 1(a) and 1(b) respectively show a wide-angle optical scanning and fluorescence detection optical device according to the present invention. Figure 1(c) shows a block diagram of the control and data acquisition system, which contains the control electronics.

Figures 2(a), 2(b) and 2(c) respectively show when the off-axis in the X direction is ±7.7°, when the off-axis in the X and Y directions is 0°, and in the Y direction The relationship between the modulus of the optical transfer function (OTF) and the spatial frequency (period/mm) when the off-axis of is ±7.7°.

Figures 3(a) and 3(b) show the transverse and axial cross-sectional views, respectively, with an average cross-section of 25 nano-beads (with a diameter of 220 nm), and the error bar represents the standard deviation. Gaussian fitting (Gaussian-fitting) produces effective two-photon lateral and axial resolutions of 0.483μm and 1.997μm respectively (ie full width and half maximum (FWHM)), that is, effective 3D resolution <0.5 soaring, and lateral resolution The average values of the standard deviation and standard error of the rate are 0.0342μm and 0.0068μm, and the average values of the axial resolution are 0.3027μm and 0.0605μm, respectively.

Figures 4(a) and 4(b) respectively show the 3D rendering volume of the volume tissue sample of the tdTomato positive isolated mouse medulla scanned under the large-angle optical domain scanning system of the present invention in oblique and top views. Figure 4 (c) shows the 3D zoom area cropped from the original volume shown in Figure 4(b). Figure 4(d) shows an image formed by the overlap of 10 frames in the depth range of 170μm to 173μm, which is extracted from the same volume as described in Figure 4(a) and (b) Taken. Figure 4(e) depicts the zoomed area cropped from the original image shown in Figure 4(d).

Table 1 shows the performance of the data acquisition system with full-field resolution beyond Nyquist according to the present invention.

Table 2 shows the comparison between the data acquisition system and the current system (Leica SP8 Confocal).

Table 3 shows the software dependencies and preparatory hardware for controlling and acquiring software for C++-based GPU acceleration according to the present invention.

According to the present invention, the wide-angle optical scanning system (shown in Figure 1(a), 1(b)) is optimized using ZEMAX, which is effective in high NA and low magnification objectives (Olympus-XLUMPlanFl, 20X, 0.95W) The focal length (EFL)=9mm), the scanning angle on the aperture is as high as ±7.16°, which can produce a square field (FOV) up to 1.6×1.6mm ² . In order to achieve a large scanning angle, the present invention provides and optimizes a special tube lens, which combines three plano-convex lenses (Edmund Optics: 86-925), and the clear aperture and EFL of each lens are 73.5mm. And 500mm, resulting in a combined EFL of 166.7mm, combined with ordinary scanning lens (Thorlabs-LSM05-BB, EFL=110mm) can produce 1.515 times the beam magnification; therefore, the maximum scanning angle required on the scanning lens is about ±10.8° to achieve a square FOV of 1.6×1.6mm ² .

Using an input beam with a diameter of 9.25mm and a wavelength of λ=1070nm and treating a high NA and low magnification objective as a paraxial lens, it is found that the root mean square (RMS) wavefront error (no defocus) and Strehl are compared to the X The 0° and ±7.7° off-axis configurations (above the scanning lens) in the Y direction are <0.07λ and >80%, respectively, confirming that the diffraction limit performance at the center of the FOV edge is 1.6×1.6 mm², which means >78% of FOV (that is, π×0.8 in a square FOV of 1.6×1.6 square millimeters = 2.56 square millimeters, ² square millimeters = 2.01 square millimeters circular FOV) is limited by diffraction (Maréchal Criterion). Figure 2(a), 2(b) and 2(c) in the case of off-axis ±7.7° on the X axis, the relationship between the modulus of the optical transfer function (OTF) and the spatial frequency (period/mm) X axis The and Y axis are 0° axis and the Y direction is ±7.7° axis, respectively. In addition, in the X and Y directions, all configurations of 0° and ±7.7° off-axis on the scanning lens simultaneously have an RMS wavefront error (no defocus) below 0.1λ on the fixed image plane, which indicates a low-field curvature system . In order to effectively collect fluorescent signals, a relay system with a reduction magnification of 3.75 is used (Figure 1(b)), thereby forming a focused spot diameter of about 4mm in the entire scanning range, which is small enough to be located in the PMT photosensitive area Inside.

Obtaining the minimum number of pixels required by the Nyquist criterion is essential for obtaining the best optical resolution. The FOV of 1.6×1.6mm ² requires 7459×7459 pixels to resolve the theoretical two-photon lateral resolution of 429nm (λ=1070nm, NA=0.95), and the pixel size is 214.5nm. The present invention introduces a data acquisition system beyond Nyquist, which can sample 4 channels simultaneously at a sampling rate of up to 125M samples per second (MSps), so that the number of pixels in a single frame of 4 channels Reached 15720×16000, resulting in 1 gigapixel per frame, while maintaining 0.5fps (frames per second). In the present invention, by synchronously sampling 1 voxel from each light pulse from a femtosecond laser source (coherent fidelity 2 fiber laser) at a repetition rate of 70MHz, the realization of The acquisition speed of 70M samples, which can scan a volume of 1.6×1.6×1.6mm ³ , has 8800×8800×2000 (×4 channels), that is, 619.52Giga-voxels, which can be performed in 0.8μmZ steps in <39 minutes Capture 1.13TB of 16-bit raw data with 14-bit resolution, and maintain the size of 3D pixels beyond Nyquist, Nyquist volume scan speed and Nyquist line scan speed <27atliter, >1750μm ³ /ms and >12mm/ms, At the same time, the effective pixel dwell time is less than 40ns, and the effective resolution is less than 500nm. In the present invention, by synchronously sampling from a femtosecond laser source (chrome forsterite laser) with 1 voxel per light pulse at a repetition rate of 95MHz, the acquisition speed of 95M samples per second is further realized. 1.6×1.6×1.6mm ³ with a volume of 12000×12000×2000 (×4 channels), which is 1.152 Tera-voxels, which can capture about 2.1TB of 16-bit original in <53 minutes with 0.8μmZ-steps Data, with 14-bit resolution, and keeping Nyquist exceeding the 3D pixel size, Nyquist exceeding volume scanning speed and Nyquist exceeding line scanning speed <15atliter, >1288um ³ /ms and >12mm/ms, while maintaining an effective pixel dwell time less than 35ns , The effective lateral resolution is less than 420nm.

The present invention further uses a multi-programming control algorithm to synchronize the slow Y axis with the fast X axis, and does not send an external electrical frame trigger signal after each frame is completed, so as to achieve a frame rate of 983fps with a single frame of pixels, which has 15720 ×8 (×4 channels) pixels, that is, 125,760 (×4 channels) three-dimensional pixels with a sampling rate of 125M per second, including the real-time storage of 16-bit format acquisition data with 14-bit resolution, which can achieve the resonance scanner The limited frame rate confirms the stability of slow Y-axis synchronization. Table 1 and Table 2 respectively describe the collection capabilities of the data collection system of the present invention and its performance comparison with existing systems. The conclusion is that the system of the present invention provides FOV greater than 4 times and has a higher frame rate of 6 times. And compared with the current system (Leica SP8 Confocal), while maintaining a higher number of pixels> 4.5 times.

The resolution analysis of the system of the present invention uses Fluoresbrite® Multifluorescent microspheres (Polysciences, Inc.) with a diameter of 220 nm. The nanospheres were fixed by immersing the nanospheres in a 0.7% agarose solution, and the scanning FOV was 1.6×1.6mm ² , the number of pixels in a single frame was 8800×8800, and the pixel and voxel sizes were kept at 181.82nm and respectively. 9.92attoliter (the Z-step step length is 300nm). Figure 3 (a) and (b) depict the transverse and axial cross-sections of an average of 25 nanospheres, respectively, and the error bars represent the standard deviation. Applying Gaussian fitting, it is found that the effective two-photon lateral and axial resolution (ie, full width half maximum (FWHM)) are 0.483μm and 1.997μm, respectively, resulting in an effective 3D resolution of less than 0.5 soaring. The standard deviation and standard error of the average value of the lateral resolution are 0.0342μm and 0.0068μm, while the average value of the axial resolution is 0.3027μm and 0.0605μm.

Refer to the present invention. Figures 1(a) and 1(b) show the large-angle optical domain scanning system and its fluorescence detection optics, where 101: input laser beam, 102, 103: resonance and galvanometer scanning mirrors, 104, 107: Scanning lens and dedicated tube lens respectively, 109: High NA (numerical aperture) and low power objective lens, 110: Focal length plane, 111: Dichroic beam splitter, 112, 115: Focus lens, 116: PMT photosensitive area , 105, 106, 108, 113, 114: turning mirror. Figures 2(a), 2(b), and 2(c) respectively show the off-axis ±7.7° on the X-axis, 0° off-axis on the X-axis and Y-axis, and ±7.7° on the Y-axis. The relationship between the modulus of the off-axis optical transfer function (OTF) and the spatial frequency (period/mm). Figures 3(a) and 3(b) show the transverse and axial cross-sections, respectively, with an average of 25 nanospheres (beads, 220nm in diameter), and the error bar represents the standard deviation. Gaussian fitting produces effective two-photon lateral and axial resolutions of 0.483μm and 1.997μm (that is, full-width half-maximum (FWHM)), that is, the effective 3D resolution is less than 0.5 soaring, and the standard deviation and lateral resolution are The average values are 0.0342μm and 0.0068μm, while the average values of the axial resolution are 0.3027μm and 0.0605μm. Figures 4(a) and 4(b) depict the 3D rendered volume in an oblique view and a top view, respectively. The volume is 1.6×1.6×0.5mm ³ , which is based on the invented wide-angle optical domain scanning system 8800×8800×1711 voxels (three-dimensional pixels), that is, the total number is 132.5Giga-voxels, keeping the Z-step (Z-step) at 300nm, in which the tdTomato positive organism in vitro mouse medulla is used as a volume tissue sample for two-photon imaging, The laser light source (Coherent Fidelity-2 fiber laser) with a femtosecond excitation center frequency of 1070nm has a repetition frequency of 70MHz, and 3D rendering is performed using Amira 5.3.2 (Visage Imaging Inc., San Diego, California) software. Use stitching and/or inlay. Fig. 4(c) depicts the 3D zoom area cropped from the original volume shown in Fig. 4(b), that is, the area is marked by the white dashed frame in Fig. 4(b). Figure 4(d) depicts an image formed by the overlap of 10 frames in the depth range of 170μm to 173μm, which is the same as described in Figures 4(a) and 4(b) Extracted from the volume, the two-dimensional (2D) FOV is 1.6×1.6mm ² , and the size of the Nyquist super pixel is about 181.82 nm. Figure 4(e) shows the zoomed area cropped from the original image shown in Figure 4(d), that is, the area is marked by the white dashed frame in Figure 4(d) with a pixel size beyond Nyquist It is 181.82nm.

Opto-mechanical settings for wide-angle optical scanning: Maximization of FOV requires low magnification of the scanning lens and tube lens pair. At the same time, the maximization of the excitation NA of the objective requires the incident beam to meet its rear aperture, which requires the largest possible beam diameter to hit the scanning lens to achieve the best conditions. Therefore, a 4kHz resonance scanner from Cambridge Technology (Figure 1(a)-102) was selected for fast X-axis scanning, with a large clear aperture of 12mm×9.25mm. For the slow Y axis, Cambridge Technology's galvanometer scanner (Figure 1(a)-103) was selected, with an aperture of 14mm. A pulsed laser source (Coherent Fidelity-2 fiber laser) operating at 1070nm with a repetition rate of 70MHz or/and a pulsed laser source (Chrome forsterite laser) operating at 1260nm with a repetition rate of 95MHz It is used as a light source for nonlinear stimulation of volume tissue samples (Figure 1(a)-101). Use a beam expander with a magnification ratio of 1:5 to fully expand the beam so that the resonant scanning mirror is overfilled. Use ThorLabs-LSM05-BB as a scanning lens with 110mm EFL (Figure 1(a)-104) and a special tube lens or a custom-designed tube lens, that is, three flat convex lenses A combination of mirrors (Figure 1b-104). 1((a)-107)(Edmund Optics: 86-925, EFL=500mm), the combined EFL is 166.7mm, and the beam magnification is 1.515, resulting in a beam size> 14mm (maximum 18mm) behind the high NA and low magnification objectives Aperture (mm) (Figure 1(a)-109) (Olympus XLUMPlanFl, 20X, 0.95W, EFL=9mm, pupil diameter 17mm).

Signal collection photoelectric system: Figure 1(b) shows an oblique view of the signal collection optical design, which is part of the large-angle optical domain scanning system. High magnification and low magnification objectives (Figure 1(b)-109) collect the fluorescence signal emitted from the volumetric tissue sample at the focal plane (Figure 1(b)-110), and pass the dichroic beam splitter (FF801-Di02) , Semrock) (Figure 1(b)-111) reflects towards the detection unit. The detection unit includes a relay system with two EFLs with 150mm (Edmund Optics: 32-982, double convex) (Figure 1(b)-112) and 40mm (Edmund Optics: 48-654, plano-convex) (Figure 1(b)-115), with 49mm and 39mm through holes, respectively; the outgoing fluorescent beam collected by the high NA and low magnification objective lens is reduced by 3.75 times, thereby providing a focused light spot of about 4mm in the entire scanning range The diameter is small enough to be located in the photosensitive area of the PMT (Figure 1(b)-116) (R10699, Hamamatsu, photosensitive area=24×8mm2 ⁾ . To ensure that the two-photon fluorescence signal of tdTomato is detected, a band-pass filter (FF01-580/60-25-D, Semrock) is placed before the PMT photocathode. For the current to voltage conversion, the signal from the PMT passes through the transimpedance amplifier (C6438-01, Hamamatsu), and its output is digitized using an AlazarTech ATS9440 digitizer with 14-bit resolution.

Use ZEMAX for performance analysis: As shown in Figure 1(a), the resonance scanning and galvanometer scanning mirrors (labeled as Figure 1(a)-102 and Figure 1(a)-103, respectively) are separated by a distance of 12mm, so that the two The mirror cannot be equidistant from the scanning lens, Therefore, the performance in the X and Y directions is different (Figure 1(a)-104). In order to optimize the design, use ZEMAX to perform a complete 3D simulation of the optical domain scanning system, and simultaneously configure the resonance and galvanometer scanning mirrors in the X and Y directions (ie, off-axis 0° and ±7.7°). The configuration of the optical axis in the X and Y directions (on the scanning lens). Considering the high NA and low magnification objectives (Figure 1(a)-109, Olympus-XLUMPlanFl, 20X, 0.95W) as a paraxial lens with 9mm EFL, the system is optimized at 1070nm. The performance of the optical system further depends on the size of the input laser beam. The fact is that for filling the rear aperture of the objective lens to maximize the larger input beam diameter required to excite NA, the scanning lens (Figure 1(a)-107) and the dedicated The total optical aberration caused by the tube lens (Figure 1(a)-104) is particularly important, especially for larger scanning angles, the overall performance will decrease. Therefore, in order to evaluate the real performance of the scanning system, the input beam diameter of 9.25mm (that is, the minimum size of the 4kHz resonant scanning mirror) was used when performing the simulation.

Data acquisition system with full-field resolution beyond Nyquist: Fig. 1(c) shows a data acquisition system 200 with control electronics. In the data acquisition system 200, the transimpedance amplifier (Figure 1(c)-117) is used to convert the output signal from the PMT (Figure 1(c)-116) from current to voltage. The output of the amplifier is digitized using AlazarTech's digitizer ATS9440 (Figure 1(c)-118a). National Instrument's control card PCIe-6341 (Figure 1(c)-118b) is used to synchronize the slow Y axis with the fast X axis. The resonant scanning mirror controller (Figure 1(c)-119) and the galvo scanning mirror controller (Figure 1(c)-121) (electronic drive board) are used to control the resonant scanning mirror (Figure 1(c))- 102) and galvanometer scanning mirror (Figure 1(c)-103). The two elements of Figure 1(c)-118a and Figure 1(c)-118b both receive the synchronization signal (4kHz digital signal) from the controller (Figure 1(c)-119) from the resonant scanning mirror, and each edge represents the resonance scanning The change of the direction of movement of the mirror) controller. The components of Figure 1(c)-118b pass through its controller unit (Figure 1(c)-119) The amplitude of the resonant scanning mirror (Figure 1(c)-102) can be controlled. Use a 16-bit digital-to-analog converter (DAC) (Figure 1(c)-120) to convert the 16-bit digital data word generated in Figure 1(c)-118b (by the control and acquisition software (Figure 1(c)) -122) to calculate and issue instructions) and convert it into a voltage, and provide the specific voltage to the galvanometer scanning mirror controller (Figure 1(c)-121) to generate the specific direction/angle of the slow Y-axis mirror (Figure 1(c)-103). Components Figure 1(c)-122 is a custom-developed C++-based GPU acceleration control and adoption software that can control the following components: Figure 1(c)-118a, 118b, 119, 120, 121, 102, and 103.

The control and acquisition software developed (Figure 1(c)-122) is a multi-programming application written in C++ and C# (using Visual Studio 2017), combined with C/C++ supported by AlazarApi and NIDAQmx, and can provide separate Control of ATS9440 (from AlazarTech) (Figure 1(c)-118a) and PCIe-6341 (from National Instruments) (Figure 1(c)-118b). The digitizer ATS9440 (Figure 1(c)-118a) can sample 4 channels simultaneously with a 14-bit resolution and a sampling rate of up to 125M samples per second, and further provides dual-port memory support, so that data can be stored in the data Transfer to the host memory at the same time during acquisition. In order to process the collected data at high speed, the custom-developed control and acquisition software uses NVIDIA CUDA (Computer Unified Device Architecture) (version: 10.1) and accelerated OpenCV (image processing library) (version: 4.1.1). Use a computer equipped with Intel® Core ^TM i7-9800X processor and Nvidia Quadro RTX 8000 graphics card to run the control and capture software.

The custom-developed control and acquisition software also involves a multi-programming control algorithm for synchronizing the slow Y axis with the fast X axis without sending an external electronic frame trigger signal after each frame is completed. A 16-bit DAC module (Cambridge Technology's 6757 type) (Figure 1(c)-120) is connected to the MicroMax ^TM 671 series (Cambridge Technology's galvanometer scanning mirror driver module) to form an interface (Figure 1(c)- 121)) to achieve precise movement of the slow Y axis. In order to send 16-bit data words to the 6757 DAC module (Figure 1(c)-120), a control card PCIe-6341 (National Instruments, Figure 1(c)-118b) (with 24 digital I/Os) is required ) Tip) is directly connected to the computer motherboard, so that the slow Y-axis motion can be directly controlled through the control and acquisition software. Background programming continuously monitors the line trigger signal (synchronization signal) from the resonant scanner, and uses 16-bit resolution to generate 16-bit data words, which is the angle positioning step to the DAC module. Using 15720×8 (×4 channels) single frame pixels (ie 125,760 (×4 channels) voxels), at a sampling rate of 125M samples per second to achieve a frame rate of about 983fps, including real-time storage and 14-bit resolution Rate acquisition of 16-bit format data, reaching the maximum frame rate limited by the resonant scanner frequency, confirming the stability of slow Y-axis synchronization.

An×Nn，其中An是奈奎斯特(Nyquist)限制的採樣率，係以4倍的水平FOV乘以共振掃描儀頻率除以理論上受目鏡限制的橫向分辨率，用以在大於1mm的水平FOV上解析微光學分辨率，並且Nn是

1的整數來表示每個voxel(三維像素)的雷射脈衝數；一諧振掃描鏡102，光學耦合到該一或多個脈衝雷射源；一檢流計掃描鏡103(galvometer scanning mirror)，光學耦合到該諧振掃描鏡； 一光學耦合到該檢流計掃描鏡103的掃描透鏡104；一專用管透鏡107，包括三個平凸透鏡，每個平凸透鏡的有效焦距為500mm，該等平凸透鏡組合在一起並光學耦合至該掃描透鏡104；一光學耦合到該專用管透鏡107的高數值孔徑(NA)和低倍率物鏡109，用以光域掃描放置在一焦距平面110上的體積組織樣本，並用以收集樣本產生的熒光信號，該螢光信號被導向一光電倍增管(PMT)116產生一電信號；以及一資料採集系統200，耦合以從PMT 116接收電信號，其中每個採樣事件分別與一個脈衝雷射源或最高重複率脈衝雷射源(如果是一個或多個脈衝雷射源)的每個光脈衝同步，該資料採集系統包含一跨阻抗放大器117，一AlazarTech數位器118a，一National Instrument卡118b，一諧振掃描鏡控制器119、一16位元數位至類比轉換器(DAC)120，一檢流器掃描鏡控制器121以及控制和採取軟體122，其中，該掃描透鏡和該專用管透鏡構成了一低倍率的擴束器，從而最大化了視場(FOV)，但同時在高NA和低倍率物鏡的後孔徑上提供了擴大的光束大小，以保持高激發NA，因而具有高分辨率。 Refer to Figures 1 to 4. 1(a), 1(b) and 1(c), the essence of the preferred embodiment of the present invention is summarized as follows. The present invention provides a large-angle optical domain scanning system 100 for high-speed deep tissue imaging, which has a field of view (FOV) of at least one square millimeter, and has a sub-feeling higher than Nyquist-exceeded synchronous sampling analysis Effective 3D resolution, including: one or more pulsed laser sources, that is, the first to nth pulsed laser sources 101, one or more center wavelengths used to emit the nth pulsed laser source are λn and exceed Nyquist repetition rate Rn

An×Nn, where An is the sampling rate limited by Nyquist, which is 4 times the horizontal FOV multiplied by the resonant scanner frequency divided by the theoretically limited lateral resolution by the eyepiece, which is used in Resolve micro-optical resolution on horizontal FOV, and Nn is

An integer of 1 represents the number of laser pulses per voxel (three-dimensional pixel); a resonant scanning mirror 102 is optically coupled to the one or more pulsed laser sources; a galvometer scanning mirror 103 (galvometer scanning mirror), Optically coupled to the resonant scanning mirror; a scanning lens 104 optically coupled to the galvanometer scanning mirror 103; a special tube lens 107, including three plano-convex lenses, each plano-convex lens has an effective focal length of 500mm, the plano-convex lenses Assembled together and optically coupled to the scanning lens 104; a high numerical aperture (NA) and low magnification objective lens 109 optically coupled to the dedicated tube lens 107 for optical scanning of a volumetric tissue sample placed on a focal plane 110 , And used to collect the fluorescent signal generated by the sample, the fluorescent signal is directed to a photomultiplier tube (PMT) 116 to generate an electrical signal; and a data acquisition system 200 coupled to receive the electrical signal from the PMT 116, wherein each sampling event Synchronize with each light pulse of a pulsed laser source or a pulsed laser source with the highest repetition rate (if one or more pulsed laser sources). The data acquisition system includes a transimpedance amplifier 117 and an AlazarTech digitizer 118a , A National Instrument card 118b, a resonant scanning mirror controller 119, a 16-bit digital-to-analog converter (DAC) 120, a galvanometer scanning mirror controller 121 and control and acquisition software 122, where the scanning lens This special tube lens constitutes a low-magnification beam expander, thereby maximizing the field of view (FOV), but at the same time provides an enlarged beam size on the rear aperture of the high NA and low-magnification objective lens to maintain high excitation NA , Which has high resolution.

An×Nn進行同步採樣，或者在一個或多個脈衝雷射源的情況下，以相等於最高重複率脈衝雷射源的採樣率進行同步採樣。在一個或多個脈衝雷射源的情況下，而其每個採樣事件與每個光脈衝同步，從而獲得超越使Nyquist(超越Nyquist準則)的像素數目，俾在不縮小FOV大小的情況下，解決了水平FOV大於1mm的微光學分辨率。 According to the large-angle optical domain scanning system 100 of the present invention, the data acquisition system 200 can achieve a repetition rate Rn equal to the nth pulsed laser (that is, a pulsed laser source)

An×Nn performs synchronous sampling, or in the case of one or more pulsed laser sources, performs synchronous sampling at a sampling rate equal to the highest repetition rate pulsed laser source. In the case of one or more pulsed laser sources, and each sampling event is synchronized with each light pulse, so as to obtain the number of pixels exceeding Nyquist (exceeding the Nyquist criterion), so that without reducing the FOV size, The micro-optical resolution with horizontal FOV greater than 1mm is solved.

20X。 In the large-angle optical domain scanning system of the present invention, the numerical aperture of the high NA and low magnification objective lens 109 is greater than 0.9, and the effective magnification

20X.

According to the present invention, the frequency of the resonant scanning mirror 102 is at least 4kHz, and the resonant scanning mirror 102 provides a 12mm×9.25mm clear aperture, which is overfilled by one or more input laser beams to maximize the size of the scanning beam .

In the present invention, the first pulsed laser source operates at a repetition frequency of 70 MHz centered at 1070 nm, and the second pulsed laser source operates at a repetition frequency of 95 MHz centered at 1260 nm.

According to the present invention, the dedicated tube lens 107 includes three plano-convex lenses combined together, and each plano-convex lens has an effective focal length of 500mm, resulting in a combined effective focal length of 166.7mm and a large net focal length greater than >60mm in diameter. The resonant scanning mirror and galvanometer scanning mirror support a larger scanning angle.

In an embodiment of the large-angle optical domain scanning system 100 of the present invention, the scanning lens 104 and the dedicated tube lens 107 respectively having an effective focal length of 110 mm and 166.7 mm constitute a low magnification relay system with a magnification of 1.515, This provides a scan angle of up to ±7.16° on the rear aperture of high NA and low magnification objectives, and a scan angle of up to ±10.8° on the entire scan lens, thus providing square and circular fields of view (FOV) respectively Up to 1.6×1.6mm ² and a diameter of 2.26mm, but at the same time, it provides an enlarged beam size greater than 14mm (maximum 18mm) on the rear aperture of high NA and low magnification objectives (NA>0.9), thereby providing high resolution.

In another embodiment of the large-angle optical domain scanning system 100 of the present invention, an input beam with a diameter of λ=1070 nm with a diameter of 9.25 mm and a high NA and low-magnification paraxial lens simulated as a paraxial lens, To produce 0° and ±7.7° off-axis configurations (on the scan lens) for the X and Y directions, the root mean square (RMS) wavefront error (without defocus) and the Strehl ratio are <0.07λ and >80%, respectively , To ensure that the diffraction limit performance at the edge center of the FOV is 1.6×1.6mm ² , indicating that the FOV is >78%, that is, π×0.8 ² mm ² =2.01mm ² Circular FOV (at 1.6×1.6mm ² =2.56mm ² square FOV) is diffraction limited (Maréchal standard).

In another embodiment of the present invention, an input beam with a diameter of λ=1070nm with a diameter of 9.25mm and a high NA and low magnification objective lens modeled as a paraxial lens produce RMS wavefront error (without dispersion) on the fixed image plane. Focus), and all configurations of the off-axis 0° and ±7.7° of the scanning lens in the X and Y directions are simultaneously below 0.1λ, which means that the field curvature of the system is low.

In addition, a relay system with a reduction magnification of 3.75 can effectively collect fluorescent signals, so that the 4mm focal spot diameter is located in the photosensitive area of the PMT.

According to the present invention, a first turning mirror 105 and a second turning mirror 106 are optically coupled to the scanning lens 104, the dedicated tube lens 107 is optically coupled to the second turning mirror 106, and a third turning mirror 108 is optically coupled to the second lens. 106 is optically coupled, and the dedicated tube lens 107 and the high NA low power objective lens 109 are optically coupled to the third turning mirror to achieve a portable form factor.

In addition, a dichroic beam splitter 111, a lenticular lens 112, a fourth turning mirror 113, and a fifth are sequentially provided between the rear aperture of the high NA and low magnification objective lens 109 and the power multiplier tube (PMT) 116. Turning mirror 114 and a plano-convex lens 115.

In the present invention, in order to exceed the requirements of the Nyquist criterion for a complete FOV with submicron lateral optical resolution, the data acquisition system provides the ability to sample 4 channels simultaneously at a sampling rate of up to 125M per second, with simultaneous The 4-channel 14-bit resolution performs data acquisition, transmission, processing, preview and storage of 16-bit original data, and achieves a single frame pixel number of 15720×16000 (×4 channels), and results in about 1 gigapixel per frame Frame acquisition while maintaining 0.5fps.

Or, through a pulse laser source pulsed at a repetition rate of 70MHz, the data acquisition system's acquisition speed can be maximized at a speed of 70M samples per second, and each light pulse synchronously samples 1 voxel, and can scan 1.6× 1.6×1.6mm ³ volume, with 8800×8800×2000 (×4 channels), that is, 619.52Giga-voxels, which can capture 1.13TB with 14-bit resolution in <39 minutes with a 0.8μm Z-step The original 16-bit data, and keep surpassing the size of Nyquistvoxel, surpassing Nyquist volume scanning speed and surpassing Nyquist line scanning speed <27attoliter, >1750um ³ /ms and >12mm/ms, while keeping the effective lateral resolution less than 500nm Effective pixel dwell time of 40us.

In yet another embodiment of the present invention, a pulsed laser source is used to pulse at a repetition frequency of 95MHz to maximize the acquisition speed of the data acquisition system at a speed of 95M samples per second, and each optical pulse is synchronously sampled 1 A voxel, capable of scanning a volume of 1.6×1.6×1.6mm ³ , with 12000×12000×2000 (×4 channels), that is 1.152 Tera-voxels, can capture about 2.1TB in less than 53 minutes when the Z step is 0.8μm The 16-bit original data, the resolution is 14 bits, and maintains the size of Nyquist voxel, the ultra-Nyquist volume scan speed and the ultra-Nyquist line scan speed <15attoliter, >1288um ³ /ms and >12mm/ms, at the same time at the highest < Under the effective lateral resolution of 420nm, an effective pixel dwell time of <35ns is maintained.

Furthermore, the data acquisition system 200 includes a multi-programming control algorithm, which is used to synchronize the slow Y-axis scanning of the galvanometer scanning mirror (with 16-bit precision movement) and the fast X-axis scanning of the resonant scanning mirror. No need to send external power. After each frame is completed, the frame trigger signal is triggered, so that the limited frame rate of the resonance scanner of 983fps is reached with 15720×8 (×4 channels) voxels per frame. In addition, the data acquisition system 200 enables GPU accelerated real-time calibration to correct the distortion along the fast X axis caused by the nonlinear velocity distribution of the resonant scanning mirror.

According to the large-angle optical domain scanning system 100 of the present invention, through the entire field (without reducing FOV) The effective two-photon lateral and axial resolutions beyond the analysis of Nyquist sampling are <0.5μm and <2μm, respectively, resulting in an effective 3D resolution of <0.5 soaring, the standard deviation of the lateral resolution and the standard of the average The errors are respectively <0.04μm and <0.007μm, and the axial resolution is <0.31μm and <0.061μm respectively

100: Large-angle optical domain scanning system

101: Input laser beam

102: Resonance Scanning Mirror

103: Galvanometer Scanning Mirror

104: Scanning lens

107: dedicated tube lens

109: High numerical aperture (NA) and low magnification objectives

110: focal plane

Claims (18)

A wide-angle optical domain scanning system for high-speed deep tissue imaging, with a field of view (FOV) of at least one square millimeter, and a sub-femtoliter (sub-femtoliter) that is resolved by synchronous sampling beyond Nyquist Effective 3D resolution, including; one or more (1st to nth) pulsed laser sources, used to emit one or more laser beams with a center wavelength of λ _n , and the nth pulsed laser source Beyond the Nyquist repetition rate R _n

A _n ×N _n , where A _n is the sampling rate limited by Nyquist, which is 4 times the horizontal field of view (FOV) multiplied by the resonant scanner frequency and divided by the theoretically limited horizontal field of the eyepiece Resolution, used to resolve the micro-optical resolution in the horizontal field of view (FOV) beyond >1mm, and N _n is

An integer of 1 represents the number of laser pulses per voxel; a resonant scanning mirror, which is optically coupled to the one or more pulsed laser sources; a galvanometer scanning mirror, optical Coupled to the resonant scanning mirror; a scanning lens, optically coupled to the galvanometer scanning mirror; a special tube lens, including three plano-convex lenses, each plano-convex lens has an effective focal length of 500mm, and these lenses are combined and optical Coupled to the scanning lens; a high numerical aperture (NA) and low power objective lens optically coupled to the dedicated tube lens for scanning a volume of tissue sample in the optical domain, and for collecting a fluorescence signal generated by the sample, the fluorescence signal is Guided to a photomultiplier tube (PMT) to generate an electrical signal; and a data acquisition system coupled to receive the electrical signal from the photomultiplier tube (PMT), wherein each sampling event is associated with a pulsed laser source Or the highest repetition rate pulsed laser source (if there are one or more pulsed laser sources) each light pulse is synchronized, wherein the scanning lens and the special tube lens constitute a low-magnification beam expander, thereby maximizing The field of view (FOV), but at the same time provides an enlarged beam size on the rear aperture of a high numerical aperture (NA) and a low magnification objective lens to maintain a high excitation numerical aperture (NA). The wide-angle optical domain scanning system for high-speed deep tissue imaging as described in item 1 of the scope of patent application, wherein the data acquisition system can achieve a repetition rate equal to the nth pulsed laser source R _n

The sampling rate of A _n ×N _n is synchronized sampling, that is, the laser source or the highest repetition rate pulsed laser source (if it is one or more pulsed laser sources), and each sampling event is related to each The two light pulses are synchronized to achieve a number of pixels exceeding Nyquist (exceeding the Nyquist criterion) to resolve the micro-optical resolution across the horizontal field of view (FOV)>1mm. The wide-angle optical scanning system for high-speed deep tissue imaging as described in the first item of the patent application, wherein the numerical aperture of the high numerical aperture (NA) and low magnification objective lens is greater than 0.9, and the effective magnification

20X. The wide-angle optical domain scanning system for high-speed deep tissue imaging as described in item 1 of the scope of patent application, wherein the frequency of the resonant scanning mirror is at least 4 kHz. The wide-angle optical domain scanning system for high-speed deep tissue imaging as described in item 1 of the scope of patent application, wherein the resonant scanning mirror provides a clear aperture of 12mm×9.25mm, and the clear aperture is inputted by one or more The laser beam is overfilled to maximize the size of the scanning beam. The wide-angle optical domain scanning system for high-speed deep tissue imaging as described in the first item of the scope of patent application, wherein a first pulsed laser source is operated at 1070nm with a repetition frequency of 70MHz, and a second pulsed laser The radiation source is operated at 1260nm as the center and the repetition frequency is 95MHz. The wide-angle optical domain scanning system for high-speed deep tissue imaging as described in item 1 of the scope of patent application, wherein the dedicated tube lens includes the three plano-convex lenses combined together, and each plano-convex lens has a 500 mm Therefore, a combined effective focal length of 166.7 mm is generated, and a large clear aperture with a diameter greater than 60 mm is provided to support the large scanning angle of the resonance scanning mirror and the galvanometer scanning mirror. The wide-angle optical scanning system for high-speed deep tissue imaging as described in item 1 of the scope of patent application, wherein the scanning lens and the dedicated tube lens respectively having an effective focal length of 110mm and 166.7mm constitute a 1.515 A low-magnification relay system with high magnification to provide a scan angle of up to ±7.16° on the back aperture of a high numerical aperture and low-power objective lens, and a scan angle of up to ±10.8° on the entire scanning lens, thereby providing The maximum diameters of the square and circular fields of view (FOV) are 1.6×1.6mm ² and 2.26mm, respectively, but at the same time, it provides more than 14mm (maximum 18mm) on the back aperture of high NA and low magnification objectives (NA>0.9) Expand the beam size. The wide-angle optical scanning system for high-speed deep tissue imaging as described in the first item of the patent application, in which an input beam with a diameter of 9.25mm and λ=1070nm and a high numerical aperture simulated as a paraxial lens NA and low-magnification objectives, generating root mean square (RMS) in the X and Y directions 0° and ±7.7° off-axis configuration (on the scanning lens) wavefront error (no defocus) and Strehl ratio are < 0.07λ and >80%, confirming diffraction-limited performance at 1.6×1.6mm ² at the edge center of FOV. Under 1.6×1.6mm ² =2.56mm ² square FOV, it shows that the FOV of the field of view is >78%, which is π ×0.8 ² mm ² =2.01mm ² The diffraction limit of a circular FOV (Maréchal standard). The wide-angle optical scanning system for high-speed deep tissue imaging as described in the first item of the patent application, in which an input beam with a diameter of 9.25mm and λ=1070nm and a high numerical aperture simulated as a paraxial lens NA and low magnification objective lenses produce RMS wavefront error (no defocus) in a fixed image plane, and at the same time, the scanning lens in the X and Y directions at 0° and ±7.7° off-axis in all configurations are 0.1λ In the following, this means the low field curvature of the system. The wide-angle optical domain scanning system for high-speed deep tissue imaging as described in item 1 of the scope of patent application, wherein the effective collection of the fluorescent signal is realized by a relay system with a reduction magnification of 3.75, resulting in A focused spot diameter of 4 mm is located in a photosensitive area of the photomultiplier tube PMT. The wide-angle optical domain scanning system for high-speed deep tissue imaging as described in item 1 of the scope of patent application, wherein a first turning mirror and a second turning mirror are optically coupled with the scanning lens, and the dedicated tube lens and the The second turning mirror is optically coupled, a third turning mirror is optically coupled to the dedicated tube lens, and the high numerical aperture NA and the low magnification objective lens are optically coupled to the third turning mirror to achieve a portable size. The wide-angle optical domain scanning system for high-speed deep tissue imaging as described in item 1 of the scope of patent application, wherein, the rear aperture of the high numerical aperture NA and low power objective lens and the photomultiplier tube (PMT) It includes a dichroic beam splitter, a biconvex mirror, a fourth turning mirror, a fifth turning mirror and a plano-convex lens in sequence. The wide-angle optical domain scanning system for high-speed deep tissue imaging as described in item 1 of the scope of patent application, wherein, in order to exceed the requirements of the Nyquist criterion of a full field of view FOV with submicron lateral optical resolution, The data acquisition system provides the ability to sample 4 channels simultaneously, with a sampling rate of up to 125M samples per second, with data acquisition, transmission, processing, preview and The ability to store 4 channels of 14-bit resolution 16-bit original data at the same time, and the number of pixels in a single frame is 15720×16000 (×4 channels), and each frame obtains 1G pixels while maintaining 0.5fps. The large-angle optical domain scanning system for high-speed deep tissue imaging as described in the first item of the scope of patent application, wherein the acquisition speed of the data acquisition system is achieved by a pulsed laser source pulsed at a repetition rate of 70MHz at a rate per second The speed of 70M samples is maximized, and each light pulse samples 1 3D pixel, which can scan a volume of 1.6×1.6×1.6mm ³ with 8800×8800×2000 (×4 channels), that is, 619.52Giga-voxels, capture 1.13TB of 16-bit raw data is processed in 39 minutes with a 0.8μm Z step size and 14-bit resolution, and the Nyquist is kept beyond the 3D pixel size, and the Nyquist exceeds the volumetric scanning speed and Nyquist Frost line scanning velocity exceeds <27attoliter,> 1750μm ³ / ms and> 12mm / ms, while maintaining <40ns effective pixel dwell time, the most effective lateral resolution of <500nm. The wide-angle optical domain scanning system for high-speed deep tissue imaging as described in item 1 of the scope of patent application, in which a pulsed laser source pulsed at a repetition rate of 95MHz is used at a speed of 95M samples per second. The collection speed of the data collection system is maximized. Each light pulse samples 1 three-dimensional pixel, can scan a volume of 1.6×1.6×1.6mm ³ , and has 12000×12000×2000 (×4 channels), that is, 1.152 Tera- voxels, capturing 2.1TB of 16-bit raw data in <53 minutes with 0.8μm Z step size and 14-bit resolution for data processing, and keeping Nyquist beyond the voxel size, Nyquist beyond the volume scan speed and Nyquist beyond the line scan The speed is <15attoliter, >1288μm ³ ms and >12mm/ms, while maintaining the effective pixel dwell time of <35ns, and the highest effective lateral resolution is <420nm. The wide-angle optical scanning system for high-speed deep tissue imaging as described in item 1 of the scope of patent application, wherein the data acquisition system further includes a multi-programming control algorithm for passing the The galvanometer scanning mirror synchronizes the slow Y-axis scanning (to maintain 16-bit precision motion) and fast X-axis scanning through the resonance scanning mirror, without the need to send an external electronic frame trigger signal after each frame is completed, so as to achieve resonance scanning The limited frame rate of the camera is 983fps, with 15720×8 (×4 channels) voxels per frame. The wide-angle optical domain scanning system for high-speed deep tissue imaging as described in item 1 of the scope of patent application, in which the full field of view (without reducing the FOV of the field of view) and the effective two of the Nyquist sampling The photon lateral and axial resolutions are <0.5μm and <2μm, respectively, resulting in an effective 3D resolution of <0.5 soaring. The standard deviation of the lateral resolution and the standard error of the average are <0.04μm and <0.007μm, respectively. The direction resolution is <0.31μm and <0.061μm.

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