Patent Description:
Several techniques have been previously reported for mesoscale imaging. <CIT>, discloses a nonlinear optical (NLO) microscope design with extended FOV of <NUM> × <NUM><NUM> = <NUM><NUM>, i.e., less than one square millimeter, resolved by a pixel number of <NUM> × <NUM>, thereby being unable to fulfil Nyquist Criterion for a sub-micron lateral resolution of <NUM> ± <NUM> across the complete FOV of <NUM> × <NUM><NUM>, with a pixel size of <NUM>.

Quite a few researchers have disclosed several NLOMs extending the FOV up-to several square millimeters by means of employing low-NA (≤<NUM>) objectives, thus leading to poor axial resolution (and hence, poor 3D resolution), due to the fact that axial resolution has inverse square dependence on numerical aperture (NA) of the objective lens.

<NPL>, discloses a two-photon microscope using optical invariant analysis, yielding a FOV of <NUM> in diameter with lateral and axial resolutions of ~<NUM> and -<NUM>, respectively, achieved by a 4X, <NUM> NA objective. Besides, to resolve a lateral resolution of ~<NUM> across a FOV of <NUM> in diameter, i.e., a square-FOV of <NUM> × <NUM><NUM>, a pixel number of more than <NUM> × <NUM> is required by Nyquist Criterion, however, they are limited by data acquisition speed of <NUM> samples per second.

<NPL>, discloses a two-photon imaging system using micro-opto-mechanical device within post-objective space, leading to a FOV of <NUM> × <NUM><NUM>, being stitched together using sequential imaging of multiple distant areas (><NUM>), with lateral resolutions of <NUM> ± <NUM> for the X-axis direction and <NUM> ± <NUM> for the Y-axis direction, and axial resolution of <NUM> ± <NUM>, achieved by a 10X, <NUM> NA objective lens, further limited by data acquisition speed.

Consequently, the prior arts are limited by either less than one square millimeter FOV, or poor axial resolution, and/ or slower data acquisition speed to fulfil Nyquist Criterion, and hence, are not suitable candidate for high-resolution 3D imaging with sub-femtoliter Nyquist-fulfilled effective 3D resolution across a FOV of more than one square millimeter. <NPL> is hereby cited as further prior art.

<CIT> discloses an imaging platform based on nonlinear optical microscopy for rapid scanning large areas of tissue.

<CIT> discloses systems and methods for high-resolution imaging.

It is desired in the art to provide an NLOM to overcome the problems as stated above.

The invention is intended to provide a large-angle optical raster scanning system for deep tissue imaging with an extended FOV of more than one square millimeter, while simultaneously maintaining a high effective 3D resolution resolved by a high-speed data acquisition system exceeding Nyquist Criterion for the complete FOV. While doing so, to not compromise with the speed, each voxel acquisition is synchronized to each optical pulse from a pulsed laser source with a high repetition rate, thereby pushing the acquisition speed to the maximum, i.e., limited by the repetition rate of the pulsed laser source. The invention employs a high-NA and low magnification objective lens with resolution being uncompromised. In order to extend the FOV beyond one square millimeter, a specific optical design is invented supporting large scanning angle in both fast-X and slow-Y axes, while maintaining low optical aberrations across the FOV.

While extending the scanning angle (and thereby the FOV), the nonlinear speed of a resonant scanning mirror leads to image distortion along the fast X-axis, enforcing one to sample at non-equidistant time points maintaining uniform pixel rate and thereby resulting in lower pixel number, insufficient to fulfil Nyquist Criterion for large FOV with micro-optical resolution. The invention implicates Nyquist-exceeded sampling throughout the scanning range at equidistant time points and subsequently fixes resonant scanner induced distortions in real time by means of a graphics processing unit (GPU)-accelerated interpolation algorithm; while collecting a larger number of data points near the edges compared to the center of the FOV (due to equidistant sampling throughout the nonlinear motion of the resonant scanning mirror), further compensate for vignetting-induced reduced signal strength near the FOV edges due to limited field number of the objective lens.

According to the invention, to solve the above-mentioned problems encountered in the existing art, a large-angle optical raster scanning system for high-speed deep tissue imaging, being provided with field of view (FOV) of at least one square millimeter with sub-femtoliter effective 3D resolution resolved by Nyquist-exceeded synchronized sampling is disclosed, comprising:.

According to the invention, a large-angle optical raster scanning-system (as shown in <FIG>, <FIG>) is optimized using ZEMAX software (Radiant Zemax, LLC), with large scanning angle of up to ~±<NUM><NUM> on the back aperture of the high-NA and low magnification objective lens (Olympus-XLUMPlanFl, 20X, <NUM>. 95W, effective focal length (EFL) = <NUM>), producing square-FOV of up to <NUM> × <NUM><NUM>. To implement large scanning angle, the invention provides and optimizes a dedicated tube lens combining three plano-convex lenses (Edmund Optics: <NUM>-<NUM>), each with clear aperture and EFL of <NUM> and <NUM>, respectively, resulting in combined EFL of <NUM>, and producing beam magnification of <NUM> times with combination of a general scan lens (Thorlabs-LSM05-BB, EFL = <NUM>); hence, requiring scanning angle of up to ~±<NUM><NUM> over the scan lens to achieve a square-FOV of <NUM> × <NUM><NUM>.

Using an input beam at λ=<NUM> with a diameter of <NUM> and considering the high-NA and low magnification objective lens as a paraxial lens, the root mean square (RMS) wavefront errors (without defocus) and Strehl Ratios are found to be <<NUM>. 07λ and ><NUM>%, respectively, for <NUM><NUM> and ±<NUM><NUM> off-axis configurations (over the scan lens) in X and Y directions, confirming diffraction-limited performance at the edge-centers of the FOV of <NUM> × <NUM><NUM>, indicating ><NUM>% of the FOV (i.e., π × <NUM><NUM> mm<NUM> = <NUM><NUM> circular-FOV out of <NUM> × <NUM><NUM> = <NUM><NUM> square-FOV) being diffraction-limited (Maréchal Criterion). <FIG>, <FIG> and <FIG> plot modulus of the optical transfer function (OTF) vs spatial frequency (cycles/ mm) for angles (over the scan lens) of ±<NUM><NUM> off-axis in the X direction, <NUM><NUM> off-axis in X and Y directions and ±<NUM><NUM> off-axis in the Y direction, respectively. Besides, RMS wavefront errors (without defocus) at a fixed image plane simultaneously for all configurations of <NUM><NUM> and ±<NUM><NUM> off-axis over the scan lens in both X and Y directions are under <NUM>. 1λ, indicating a low field curvature of the system. For efficient collection of fluorescence signal, a relay system with demagnification factor of <NUM> is employed (<FIG>), resulting in ~<NUM> focused spot diameter throughout the scanning range, small enough to be inside a PMT photosensitive area. Acquiring a minimum pixel number demanded by Nyquist Criterion is crucial for retrieving the best optical resolution. FOV of <NUM> × <NUM><NUM> requires a pixel number of <NUM> × <NUM> for resolving the theoretical two-photon lateral resolution of ~<NUM> (λ=<NUM>, NA=<NUM>), with a pixel size of <NUM>. The invention introduces a Nyquist-exceeding data acquisition system capable of simultaneously sampling <NUM> channels at up to <NUM> samples per second (MSps) sampling-rate, reaching a single-frame pixel number of <NUM> × <NUM> for <NUM> channels, leading to ~<NUM> Gigapixels per frame, while maintaining ~<NUM> fps (frames per second). In the invention, an acquisition speed of <NUM> samples per second is implemented with synchronized sampling of <NUM> voxel per optical pulse from a femtosecond laser source (Coherent Fidelity-<NUM> Fiber Laser) with a <NUM> repetition rate, with the ability of scanning a <NUM> × <NUM> × <NUM><NUM> volume, with <NUM> × <NUM> × <NUM> (× <NUM> channels), i.e., <NUM> Giga-voxels, capturing ~<NUM> Terabyte of <NUM>-bit raw data with <NUM>-bit resolution in <<NUM> minutes at <NUM> Z-steps, and maintaining a Nyquist-exceeded voxel-size, a Nyquist-exceeded volume-scanning speed and a Nyquist-exceeded line-scanning speed of <<NUM> attoliter, ><NUM><NUM>/ ms and ><NUM>/ ms, respectively, while maintaining an effective pixel dwell time of <<NUM> ns, at up to an effective lateral resolution of <<NUM>. In the invention, an acquisition speed of <NUM> samples per second is further implemented with synchronized sampling of <NUM> voxel per optical pulse from a femtosecond laser source (Chromium-Forsterite Laser) with a <NUM> repetition rate, with the ability of scanning a <NUM> × <NUM> × <NUM><NUM> volume, with <NUM> × <NUM> × <NUM> (× <NUM> channels), i.e., <NUM> Tera-voxels, capturing -<NUM> Terabyte of <NUM>-bit raw data with <NUM>-bit resolution in <<NUM> minutes at <NUM> Z-steps, and maintaining a Nyquist-exceeded voxel-size, a Nyquist-exceeded volume-scanning speed and a Nyquist-exceeded line-scanning speed of <<NUM> attoliter, ><NUM><NUM>/ ms and ><NUM>/ ms, respectively, while maintaining an effective pixel dwell time of <<NUM> ns, at up to an effective lateral resolution of <<NUM>.

The invention further utilizes a multithreaded control algorithm for synchronization of slow Y-axis with fast X-axis, without sending external electrical frame-trigger signals after completion of each frame, thereby achieving a frame rate of ~<NUM> fps with single-frame pixel number of <NUM> × <NUM> (× <NUM> channels), i.e., <NUM>,<NUM> (× <NUM> channels) voxels at <NUM> samples per second sampling-rate, including real-time storage of acquired data in <NUM>-bit format with <NUM>-bit resolution, reaching resonant scanner limited frame rate, confirming robustness of slow Y-axis synchronization. Table <NUM> and Table <NUM> depict acquisition capability of the invented data acquisition system and its performance comparison with a state-of-the-art system, respectively, concluding that the system of the invention provides ><NUM> times larger FOV with ~<NUM> times higher frame rate while maintaining ><NUM> times higher pixel number in comparison to a state-of-the-art system (Leica SP8 Confocal).

The resolution analysis of the invented system utilizes Fluoresbrite® Multifluorescent Microspheres (Polysciences, Inc. ), with diameter of <NUM>. Beads are immobilized by immersing them in <NUM>% agarose solution and are scanned for FOV of <NUM> × <NUM><NUM>, with a single-frame pixel number of <NUM> × <NUM>, maintaining a pixel and voxel size of ~<NUM> and ~<NUM> attoliter (with Z-steps of <NUM>), respectively. <FIG> and <FIG> depict lateral and axial cross sections, respectively, averaging <NUM> beads, error bars indicating the standard deviations. Applying gaussian fitting, effective two-photon lateral and axial resolutions (i.e., full width half maximum (FWHM)) are found to be <NUM> and <NUM>, respectively, resulting in effective 3D resolution of <<NUM> femtoliter. The standard deviation and standard error of the mean for lateral resolution are <NUM> and <NUM>, respectively and for axial resolution are <NUM> and <NUM>, respectively.

Referring to the invention, <FIG> and <FIG> show the large-angle raster scanning system with its fluorescence detection optics, respectively with <NUM>: input laser beam, <NUM>, <NUM>: resonant and galvanometer scanning mirror, respectively, <NUM>, <NUM>: scan lens and dedicated tube lens, respectively, <NUM>: high-NA and low magnification objective lens, <NUM>: focal plane, <NUM>: dichroic beam splitter, <NUM>, <NUM>: focusing lenses, <NUM>: PMT photosensitive area, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>: turning mirrors. <FIG>, <FIG> and <FIG> show modulus of the optical transfer function (OTF) vs spatial frequency (cycles/ mm) for angles (over the scan lens) of ±<NUM><NUM> off-axis in the X direction, <NUM><NUM> off-axis in the X and Y directions and ±<NUM><NUM> off-axis in the Y direction, respectively. <FIG> and <FIG> show lateral and axial cross sections, respectively, averaging <NUM> beads (with diameter of <NUM>) and error bars indicating the standard deviations. Gaussian-fitting results in effective two-photon lateral and axial resolutions (i.e., full width half maximum (FWHM)) of <NUM> and <NUM>, respectively, i.e., effective 3D resolution of <<NUM> femtoliter with standard deviation and standard error of the mean of <NUM> and <NUM>, respectively for lateral resolution and <NUM> and <NUM>, respectively for axial resolution. <FIG> and <FIG> depict a 3D rendered volume in inclined and top views, respectively, with a volume size of <NUM> × <NUM> × <NUM><NUM>, being scanned under the invented large-angle optical raster scanning system, with <NUM> × <NUM> × <NUM> voxels, i.e., a total number of <NUM> Giga-voxels maintaining Z-step size of <NUM>, with Nav1. <NUM>-tdTomato positive ex vivo mouse medulla being used as a volumetric tissue-sample for two-photon imaging, being excited with a femtosecond laser source (Coherent Fidelity-<NUM> Fiber Laser) with a <NUM> repetition rate at a <NUM> central wavelength, 3D rendered using Amira <NUM>. <NUM> (Visage Imaging Inc. , San Diego, CA) software, with no stitching and/ or mosaicking being applied. <FIG> depicts a 3D zoomed region cropped from the original volume shown in <FIG>, i.e., the region being marked by the white dotted box in <FIG>. <FIG> depicts an image formed by overlapping of <NUM> frames within a depth range of ~<NUM> to ~<NUM>, extracted from the same volume being described in <FIG> and <FIG>, with a two-dimensional (2D) FOV of <NUM> × <NUM><NUM>, with a Nyquist-exceeded pixel size of -<NUM>. <FIG> depicts a zoomed region cropped from the original image shown in <FIG>, i.e., the region being marked by the white dotted box in <FIG>, resolving micro-optical resolution with a Nyquist-exceeded pixel size of -<NUM>.

Maximization of FOV demands a low magnification ratio by the scan lens and tube lens pair. Concurrently, maximization of excitation-NA of the objective lens demands incident beam to fulfil its back aperture, necessitating largest possible beam diameter to hit the scan lens for the optimum condition. Therefore, a <NUM> resonant scanner (<FIG>-<NUM>) from Cambridge Technology, MA, USA is chosen for fast X-axis scanning, with large clear aperture of <NUM> × <NUM>. For slow Y-axis, a galvanometer scanner (<FIG>-<NUM>) from Cambridge Technology is chosen with a clear aperture of <NUM>. A pulsed laser source (Coherent Fidelity-<NUM> Fiber Laser) operating at a repetition rate of <NUM> centered at <NUM> or/ and a pulsed laser source (Chromium-Forsterite Laser) operating at a repetition rate of <NUM> centered at <NUM> is/ are used as source(s) (<FIG>-<NUM>) for nonlinear excitation of the volumetric tissue-sample. A beam expander with <NUM>:<NUM> magnification is used to expand the beam sufficiently, overfilling the resonant scanning mirror. Employing ThorLabs-LSM05-BB as scan lens (<FIG>-<NUM>) with EFL of <NUM> and a dedicated tube lens, or a custom designed tube lens, i.e., a combination of three plano-convex lenses (<FIG>-<NUM>) (Edmund Optics: <NUM>-<NUM>, EFL = <NUM>) with combined EFL of <NUM>, a beam magnification by a factor of <NUM> is achieved resulting in a beam size of ><NUM> (up to <NUM>) over the back aperture of the high-NA and low magnification objective lens (<FIG>-<NUM>) (Olympus XLUMPlanFl, 20X, <NUM>. 95W, EFL = <NUM>, pupil-diameter ~<NUM>).

<FIG> illustrates the inclined view of the signal collecting optical design, a part of the large-angle optical raster scanning system. The generated fluorescence signal emerging from the volumetric tissue-sample at the focal plane (<FIG>-<NUM>) is collected by the high-NA and low magnification objective lens (<FIG>-<NUM>) and is reflected towards the detection unit by a dichroic beam-splitter (FF801-Di02, Semrock) (<FIG>-<NUM>). The detection unit comprises a relay system with two lenses having EFL of <NUM> (Edmund Optics: <NUM>-<NUM>, bi-convex) (<FIG>-<NUM>) and <NUM> (Edmund Optics: <NUM>-<NUM>, plano-convex) (<FIG>-<NUM>) with clear apertures of <NUM> and <NUM>, respectively; downsizing the emerging fluorescence beam being collected by the high-NA and low magnification objective lens by a factor of <NUM>, and thereby providing ~<NUM> focused spot diameter throughout the scanning range, which is small enough to be inside the photosensitive area of the PMT (<FIG>-<NUM>) (R10699, Hamamatsu, photosensitive area = <NUM> × <NUM><NUM>). A band pass filter (FF01-<NUM>/<NUM>-<NUM>-D, Semrock) is placed before the PMT photocathode in order to ensure detection of Nav1. <NUM>-tdTomato two-photon fluorescence signal. For current to voltage conversion, signal from the PMT is passed through a transimpedance amplifier (C6438-<NUM>, Hamamatsu), whose output is digitized using AlazarTech ATS9440 digitizer with <NUM>-bit resolution.

As illustrated in <FIG>, a resonant scanning and galvanometer scanning mirrors (tagged as <FIG>-<NUM> and <FIG>-<NUM>, respectively) are separated by distance of <NUM>, resulting in non-identical performance in the X and Y directions, due to the fact that, both the mirrors cannot be equidistant from the scan lens (<FIG>-<NUM>). For an optimized design, a complete 3D simulation of the raster scanning system is performed using ZEMAX, simultaneously configuring different scanning angles of the resonant and galvanometer scanning mirrors for X and Y directions, respectively (i.e., <NUM><NUM> and ±<NUM><NUM> off-axis configurations (over the scan lens) in X and Y directions with respect to the optical-axis). The system is optimized at <NUM> considering the high-NA and low magnification objective lens (<FIG>-<NUM>, Olympus-XLUMPlanFl, 20X, <NUM>. 95W) as a paraxial lens with EFL of <NUM>. Performance of the optical system further depends on the size of input laser beam, with the fact that, for larger input beam diameter required for filling the back aperture of the objective lens for maximizing excitation-NA, the optical aberrations caused by the scan lens (<FIG>-<NUM>) and dedicated tube lens (<FIG>-<NUM>) become significant particularly for larger scanning angle and the overall performance gets degraded. Therefore, in order to assess the real performance of the scanning system, an input beam diameter of <NUM> (i.e., minimum size of the <NUM> resonant scanning mirror) is used while performing the simulation.

The data acquisition system <NUM>, being provided with control-electronics is depicted in <FIG>. In the data acquisition system <NUM>, a transimpedance amplifier (<FIG>-<NUM>) is used for current to voltage conversion of the output signal from the PMT (<FIG>-<NUM>). Output from the amplifier is digitized using a digitizer ATS9440 from AlazarTech (<FIG>-118a). A controlling card PCIe-<NUM> from National Instrument (<FIG>-118b) is used for synchronization of the slow Y-axis with the fast X-axis. A resonant scanning mirror controller (<FIG>-<NUM>) and a galvanometer scanning mirror controller (<FIG>-<NUM>) (electronic driver boards) are used for controlling the resonant scanning mirror (<FIG>-<NUM>) and galvanometer scanning mirror (<FIG>-<NUM>), respectively. Both elements <FIG>-118a and <FIG>-118b receive the sync signal (a <NUM> digital signal, each edge representing a change in motion-direction of the resonant scanning mirror) from the resonant scanning mirror controller (<FIG>-<NUM>). Element <FIG>-118b has control over the amplitude of resonant scanning mirror (<FIG>-<NUM>), through its controller unit (<FIG>-<NUM>). A <NUM>-bit Digital to Analog Converter (DAC) (<FIG>-<NUM>) is used to convert <NUM>-bit digital data words generated by <FIG>-118b (calculated and commanded by the control and acquisition software <FIG>-<NUM>) into voltage and to provide that specific voltage on to the galvanometer scanning mirror controller (<FIG>-<NUM>), producing specific orientation! angle of the slow Y-axis mirror (<FIG>-<NUM>). Element <FIG>-<NUM> is a custom developed C++ based GPU-accelerated control and acquisition software which has control over the elements: <FIG>118a, 118b, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

The developed control and acquisition software (<FIG>-<NUM>) is a multi-threaded application written in C++ and C# (using Visual Studio <NUM>, Microsoft Corporation, Redmond, WA, USA), integrating C/ C++ support from AlazarApi and NIDAQmx providing control over ATS9440 (from AlazarTech) (<FIG>-118a) and PCIe-<NUM> (from National Instruments) (<FIG>-118b), respectively. The digitizer ATS9440 (<FIG>-118a) enables simultaneous sampling of <NUM> channels at up to a sampling rate of <NUM> samples per second with <NUM>-bit resolution, further providing dual-port memory support, enabling simultaneous data transfer to the host memory during ongoing data acquisition. For high-speed processing of acquired data, NVIDIA CUDA (Compute Unified Device Architecture) (version: <NUM>) accelerated OpenCV (library for image processing) (version: <NUM>. <NUM>) is utilized by the custom developed control and acquisition software. A computer with Intel® Core™ i7-9800X processor and Nvidia Quadro RTX <NUM> graphics card is used to run the control and acquisition software.

The custom developed control and acquisition software further implicates a multithreaded control algorithm for synchronization of the slow Y-axis with the fast X-axis, without sending external electrical frame-trigger signals after completion of each frame. A <NUM>-bit DAC module (Model <NUM> from Cambridge Technology) (<FIG>-<NUM>) is interfaced with MicroMax™ Series <NUM> (driver module for galvanometer scanning mirror from Cambridge Technology) (<FIG>-<NUM>) for precise movement of the slow Y-axis. For sending the <NUM>-bit data word to the <NUM> DAC module (<FIG>-<NUM>), a controlling card PCIe-<NUM> (National Instruments, <FIG>-118b) (with <NUM> digital I/O pins) is directly interfaced to the computer motherboard, thereby enabling direct control over the slow Y-axis motion from the control and acquisition software. A background thread continuously monitors the line trigger signal (sync signal) from the resonant scanner and produces the <NUM>-bit data words, i.e., the angle-positioning-steps to the DAC module making use of <NUM>-bit resolution. A frame rate of ~<NUM> fps is achieved with single-frame pixel number of <NUM> × <NUM> (× <NUM> channels), i.e., <NUM>,<NUM> (× <NUM> channels) voxels at a sampling rate of <NUM> samples per second, including real-time storage of the acquired data in <NUM>-bit format with <NUM>-bit resolution, reaching the maximum frame rate limited by the resonant scanner frequency, confirming the robustness of slow Y-axis synchronization.

Referring to <FIG>, <FIG> and <FIG>, the essence of the preferred embodiment of the invention is summarized as follows. The invention provides a large-angle optical raster scanning system <NUM> for high-speed deep tissue imaging, being provided with field of view (FOV) of at least one square millimeter with sub-femtoliter effective 3D resolution resolved by Nyquist-exceeded synchronized sampling, comprising:.

According to the large-angle optical raster scanning system <NUM> of the invention, the data acquisition system <NUM> enables synchronized sampling with a sampling rate equal to the repetition rate Rn ≥ An × Nn of the nth pulsed laser, i.e., the one pulsed laser source, or the highest repetition rate pulsed laser source, in case of one, or more pulsed laser source(s), respectively, with each sampling event synchronized to each optical pulse, thereby achieving a Nyquist-exceeded (exceeding Nyquist Criterion) pixel number to resolve micro-optical resolution across horizontal FOV of ><NUM> without shrinking down the FOV size.

In the large-angle optical raster scanning system of the invention, the high-NA and low magnification objective lens <NUM> is ><NUM> in the numerical aperture and ≤20X in an effective magnification.

As provided, the frequency of the resonant scanning mirror <NUM> is at least <NUM>, and the resonant scanning mirror <NUM> provides a clear aperture of <NUM> × <NUM>, which is overfilled with the input one or more laser beams to maximize a scanning beam size.

In the invention, a first pulsed laser source operates at a repetition rate of <NUM> centered at <NUM> and a second pulsed laser source operates at a repetition rate of <NUM> centered at <NUM>.

According to the invention, the dedicated tube lens <NUM> comprises the three plano-convex lenses combined together, each having an effective focal length of <NUM>, resulting in a combined effective focal length of <NUM>, and providing a large clear aperture of ><NUM> in diameter for supporting large scanning angle by the resonant scanning mirror and the galvanometer scanning mirror.

In one embodiment of the large-angle optical raster scanning system <NUM> of the invention, the scan lens <NUM> and the dedicated tube lens <NUM> with effective focal lengths of <NUM> and <NUM>, respectively, constitute a low magnification relay system with magnification of <NUM>, thereby providing a scanning angle of up to ~±<NUM><NUM> on the back aperture of the high-NA and low magnification objective lens with a scanning angle of up to ~±<NUM><NUM> over the scan lens, and hence the square and circular field of view (FOV) of up to <NUM> × <NUM><NUM> and <NUM> in diameter, respectively, but concurrently providing an enlarged beam size of ><NUM> (up to <NUM>) over the back aperture of the high-NA and low magnification objective lens (NA><NUM>), thereby providing high-resolution.

In a further embodiment of the large-angle optical raster scanning system <NUM> of the invention, an input beam at λ=<NUM> with a diameter of <NUM> and the high-NA and low magnification objective lens simulated as a paraxial lens, produce root mean square (RMS) wavefront errors (without defocus) and Strehl Ratios to be <<NUM>λ and ><NUM>%, respectively, for <NUM><NUM> and ±<NUM><NUM> off-axis configurations (over the scan lens) in X and Y directions, confirming a diffraction-limited performance at edge-centers of the FOV of <NUM> × <NUM><NUM>, indicating ><NUM>% of the FOV, i.e., π × <NUM><NUM> mm<NUM> = <NUM><NUM> circular-FOV out of <NUM> × <NUM><NUM> = <NUM><NUM> square-FOV, to be diffraction-limited (Maréchal Criterion).

In another embodiment of the invention, an input beam at λ=<NUM> with a diameter of <NUM> and the high-NA and low magnification objective lens simulated as a paraxial lens, produce RMS wavefront errors (without defocus) at a fixed image plane simultaneously for all configurations of <NUM><NUM> and ±<NUM><NUM> off-axis over the scan lens in both X and Y directions to be under <NUM>λ, concluding a low field curvature of the system.

Further, efficient collection of the fluorescence signal is achieved by a relay system with a demagnification factor of <NUM>, resulting in a ~<NUM> focused spot diameter to be inside a photosensitive area of the PMT.

According to the invention, a first turning mirror <NUM> and a second turning mirror <NUM> are optically coupled to the scan lens <NUM>, the dedicated tube lens <NUM> is optically coupled to the second turning mirror <NUM>, a third turning mirror <NUM> is optically coupled to the dedicated tube lens <NUM>, and the high-NA and low magnification objective lens <NUM> is optically coupled to the third turning mirror to achieve a portable form factor.

In addition, it is provided sequentially a dichroic beam splitter <NUM>, a bi-convex lens <NUM>, a fourth turning mirror <NUM>, a fifth turning mirror <NUM>, and a plano-convex lens <NUM> between the back aperture of the high-NA and low magnification objective lens <NUM> and the photomultiplier tube (PMT) <NUM>.

In the invention, to exceed the requirement for the Nyquist Criterion for complete FOV with a sub-micron lateral optical resolution, the data acquisition system provides capability of simultaneously sampling <NUM> channels at up to a <NUM> samples per second sampling rate, with ability of data acquisition, transfer, processing, previewing and storing of <NUM>-bit raw data with <NUM>-bit resolution for <NUM> channels simultaneously, and reaching a single-frame pixel number of <NUM> × <NUM> (× <NUM> channels), and leading to ~<NUM> Gigapixels per frame acquisition, while maintaining ~<NUM> fps.

Alternatively, an acquisition speed of the data acquisition system is maximized at <NUM> samples per second by means of the one pulsed laser source pulsing at <NUM> repetition rate, with synchronized sampling of <NUM> voxel per optical pulse, with the ability of scanning a <NUM> × <NUM> × <NUM><NUM> volume, with <NUM> × <NUM> × <NUM> (× <NUM> channels), i.e., <NUM> Giga-voxels, capturing ~<NUM> Terabyte of <NUM>-bit raw data with <NUM>-bit resolution in <<NUM> minutes at <NUM> Z-steps, and maintaining a Nyquist-exceeded voxel-size, a Nyquist-exceeded volume-scanning speed and a Nyquist-exceeded line-scanning speed of <<NUM> attoliter, ><NUM><NUM>/ ms and ><NUM>/ ms, while maintaining an effective pixel dwell time of <<NUM> ns, at up to an effective lateral resolution of <<NUM>.

In still further an embodiment of the invention, an acquisition speed of the data acquisition system is maximized at <NUM> samples per second by means of the one pulsed laser source pulsing at <NUM> repetition rate, with synchronized sampling of <NUM> voxel per optical pulse, with the ability of scanning a <NUM> × <NUM> × <NUM><NUM> volume, with <NUM> × <NUM> × <NUM> (× <NUM> channels), i.e., <NUM> Tera-voxels, capturing -<NUM> Terabyte of <NUM>-bit raw data with <NUM>-bit resolution in <<NUM> minutes at <NUM> Z-steps, and maintaining a Nyquist-exceeded voxel-size, a Nyquist-exceeded volume-scanning speed and a Nyquist-exceeded line-scanning speed of <<NUM> attoliter, ><NUM><NUM>/ ms and ><NUM>/ ms, while maintaining an effective pixel dwell time of <<NUM> ns, at up to an effective lateral resolution of <<NUM>.

Still further, the data acquisition system <NUM> comprises a multithreaded control algorithm for synchronization of slow Y-axis scanning by the galvanometer scanning mirror (maintaining <NUM>-bit precision movement) with fast X-axis scanning by the resonant scanning mirror, without sending external electrical frame-trigger signals after completion of each frame, thereby reaching a resonant scanner limited frame rate of ~<NUM> fps with <NUM> × <NUM> (× <NUM> channels) voxels per frame. In addition, the data acquisition system <NUM> enables GPU-accelerated real-time calibrations to correct distortions along the fast X-axis caused by a nonlinear speed profile of the resonant scanning mirror.

According to the large-angle optical raster scanning system <NUM> of the invention, effective two-photon lateral and axial resolutions resolved by the full-field (without shrinking down the FOV) Nyquist-exceeded sampling are <<NUM> and <<NUM>, respectively, resulting in an effective 3D resolution of <<NUM> femtoliter, with a standard deviation and a standard error of the mean for the lateral resolution to be <<NUM> and <<NUM>, respectively and for the axial resolution to be <<NUM> and <<NUM>, respectively.

Claim 1:
A large-angle optical raster scanning system (<NUM>) for high-speed deep tissue imaging, being provided with field of view, hereinafter called FOV, of at least one square millimeter with sub-femtoliter effective 3D resolution resolved by Nyquist-exceeded synchronized sampling, comprising:
one or more, i.e., 1st to nth, pulsed laser source(s) (<NUM>) for emitting one or more laser beams with central wavelengths of λn and a Nyquist-exceeding repetition rate of Rn ≥ An × Nn for the nth pulsed laser source, where An is a Nyquist-limited sampling rate given by <NUM> times the horizontal FOV multiplied by a resonant scanner frequency divided by theoretical objective-limited lateral resolution, for resolving micro-optical resolution across a horizontal FOV of ><NUM>, and Nn is an integer ≥ <NUM> signifying a number of laser pulse(s) per voxel;
a resonant scanning mirror (<NUM>) optically coupled to the one or more pulsed laser source(s)(<NUM>) ;
a galvanometer scanning mirror (<NUM>) optically coupled to the resonant scanning mirror (<NUM>);
a scan lens (<NUM>) optically coupled to the galvanometer scanning mirror (<NUM>);
a dedicated tube lens (<NUM>), comprising three plano-convex lenses, each with an effective focal length of <NUM>, combined together and optically coupled to the scan lens (<NUM>);
a high-numerical aperture, hereinafter called high-NA, and low magnification objective lens (<NUM>) optically coupled to the dedicated tube lens (<NUM>) for raster scanning a volumetric tissue-sample and for collecting a sample-generated fluorescence signal which is guided to a photomultiplier tube, PMT, to produce an electrical signal; and
a data acquisition system (<NUM>) coupled to receive the electrical signal (<NUM>) from the PMT with each sampling event synchronized to each optical pulse either from the one pulsed laser source (<NUM>), or from the highest repetition rate pulsed laser source (<NUM>), in case of one, or more pulsed laser source(s) (<NUM>), respectively, wherein
the scan lens (<NUM>) and the dedicated tube lens (<NUM>) constitute a beam expander with low magnification, thereby maximizing the FOV, but concurrently providing an enlarged beam size over a back aperture of the high-NA and low magnification objective lens (<NUM>) to maintain high excitation-NA, and thereby high-resolution.