Kilohertz volumetric imaging of in VIVO dynamics using squeezed light field microscopy

Abstract

A modality for compressed light field measurement to capture 3D fluorescent signals at kilohertz volume rate, is described. This modality, termed Squeezed Light field Microscopy (SQLFM), acquires the light field with an array of rotated 2D sub-aperture images. An anamorphic relay system applies anisotropic scaling, effectively "squeezing" the image along one axis. This allows the camera sensor to detect the light field with a letterbox-shaped region of interest (ROI). Each squeezed sub-aperture image is complementary to the others, facilitating successful 3D reconstruction from compressed measurement while preserving the nominal FOV and spatial resolution. SQLFM can achieve over a fivefold increase in acquisition rate compared to traditional LFM.

Description

KILOHERTZ VOLUMETRIC IMAGING OF IN VIVO DYNAMICS USING SQUEEZED LIGHT FIELD MICROSCOPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. provisional patent application serial number 63 / 560,611 filed on March 1 , 2024, incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under HL165318, and NS128488 awarded by the National Institutes of Health. The Government has certain rights in the invention.NOTICE OF MATERIAL SUBJECT TO COPYRIGHT PROTECTION

[0003] A portion of the material in this patent document may be subject to copyright protection under the copyright laws of the United States and of other countries. The owner of the copyright rights has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the United States Patent and Trademark Office publicly available file or records but otherwise reserves all copyright rights whatsoever. The copyright owner does not hereby waive any of its rights to have this patent document maintained in secrecy, including without limitation its rights pursuant to 37 C. F. R. § 1 .14.BACKGROUND

[0004] 1. Technical Field

[0005] This technology pertains generally light-field microscopy (LFM) techniques and more particularly to an apparatus and methods for compressed light-field measurements, termed Squeezed Light field Microscopy (SQLFM), to capture 3D fluorescent signals at a kilohertz volume rate for use with precision surgical procedures.

[0006] 2. Background

[0007] High-speed fluorescence microscopy has been playing an indispensable role in revealing the dynamic interplay and functionality among cells in their native environment. Continuous improvements in fluorescent markers allow many transient biological processes, such as the blood flow and neural action potentials, to become trackable demanding microscopy with an ever higher spatiotemporal resolution.

[0008] Traditional three-dimensional (3D) imaging tools, such as confocal microscopy, light sheet microscopy, and two-photon microscopy, rely heavily on scanning to acquire a volumetric image. Despite advancements in beam shaping, remote refocusing mechanisms, detector array and detection geometry, there persists an inherent trade-off between temporal resolution, the 3D field-of-view (FOV), and spatial resolution in these techniques. This constraint marks a significant challenge to obtain optimal performance across a large 3D field of view (FOV) for robust ultrafast detection exceeding kilohertz (kHz).

[0009] Computational imaging mitigates this trade-off by encoding highdimensional information, such as depth, time, and spectra, into two- dimensional (2D) multiplexed camera measurements. Among these techniques, light-field microscopy (LFM) excelled in various biological applications, including observation of neural activity in freely moving animals and visualization of hemodynamics in the brain and heart. By simultaneously collecting the spatial and angular information of light rays, LFM enables volumetric reconstruction post hoc from snapshot measurements. Without scanning, the sensor bandwidth becomes the primary bottleneck for LFM 3D imaging speed. While modem scientific Complementary Metal-Oxide Semiconductor (sCMOS) sensors typically offer a full framerate lower than 100 Hz, increasing the imaging speed can be achieved by reading out only selected low-format regions of interest (ROI). However, this approach comes at the cost of sacrificing either the spatial and / or angular components associated with the field of view (FOV) and axial resolution.

[0010] The integration of ultra-high-speed cameras and event cameras holds promise for providing higher bandwidths to LFM. However, their currentlimitations in sensitivity and noise performance present challenges, especially for photon-starved applications like imaging genetically encoded voltage indicators (GEVIs) . On the other hand, the compressibility of fourdimensional (4D) (two spatial dimensions plus two angular dimensions) light fields has been leveraged for compressive detection. Coded masks and random diffusers are employed to modulate and integrate the spatio-angular components originally recorded by distinct pixels. Sparse nonlocal measurements can also be utilized across different angular views to acquire light fields with sensors of arbitrary formats.

[0011] Nevertheless, as compressive imaging relies on sparsity priors and optimization algorithms for signal recovery from the sub-Nyquist measurement, the performance is prone to degradation in challenging scenarios. These methods are primarily validated on photographic scenes and biological samples with relatively long exposure time. Their robustness and effectiveness in kilohertz microscopy with extremely low photon budget such as voltage imaging remain elusive.

[0012] Accordingly, there is a need for improved systems and methods for imaging that will address these challenges.BRIEF SUMMARY

[0013] An apparatus and methods for high-speed light-field compressed light field imaging, termed Squeezed Light field Microscopy (SQLFM) are presented, which allows the capture of 3D fluorescent signals at kilohertz volume rates in a highly data-efficient manner. The SQLFM apparatus acquires the light field with an array of rotated 2D sub-aperture images. An anamorphic relay system applies anisotropic scaling, effectively "squeezing" the image along one axis. This allows the camera sensor to detect the light field with a letterbox-shaped region of interest (ROI).

[0014] Leveraging the row-by-row readout architecture of modern CMOS sensors, it is possible to achieve over a fivefold increase in acquisition rate compared to traditional light-field microscopy systems. Each squeezed subaperture image complements the others, facilitating high-fidelity, robust 3D reconstruction from compressed measurements. By calculating the product ofspace-bandwidth product (SBP) and volume rate, SQLFM has been shown to measure approximately 7.3 gigavoxels per second, making it one of the fastest 3D fluorescent microscopes in the art.

[0015] The apparatus enables tracking high speed motion and millisecond fluorescent blinks across a 0550 / zm x 300 / zm FOV. Furthermore, it was shown that such high framerate could be exploited to enhance spatial resolution by combining multi-layer scanning light sheets. This allows imaging densely labeled structures that are challenging with conventional LFM systems and illustrated with imaging contracting myocardium in zebrafish, at 4800 frames per second (equivalently 300 vps).

[0016] The SQLFM apparatus and methods were demonstrated by 1 ) capturing flowing red blood cells in free swinging tails of embryonic zebrafish at 1 ,000 volumes per second (vps); 2) ex vivo voltage imaging in dissected leech ganglia at 800 vps; and 3) in vivo voltage imaging in hippocampus from behaving mice at 800 vps. The SQLFM system enabled the tracking of highspeed cellular motion across a 550 pm FOV within a 300 pm depth range. It allows detection of millisecond membrane action potentials and subthreshold oscillations in 3D space over extended time periods in awake, free-behaving animals. Furthermore, it was shown that the high framerate of SLIM could be exploited to enhance axial resolution when combined with multi-layer scanning light-sheet microscopy. This allows for imaging densely labeled structures, previously challenging with LFM, such as contracting myocardium in a zebrafish, at 4,800 frames per second (fps), leading to a volume rate of 300 vps.

[0017] Further aspects of the technology described herein will be brought out in the following portions of the specification, wherein the detailed description is for the purpose of fully disclosing preferred embodiments of the technology without placing limitations thereon.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The technology described herein will be more fully understood by reference to the following drawings which are for illustrative purposes only:

[0019] FIG. 1 is a schematic diagram of the SQLFM detection systemaccording to one embodiment of the technology.

[0020] FIG. 2 is a schematic diagram of the optical transformation in SQLFM including image rotation and squeezing. With a dove prism / lenslet array and an anamorphic relay, the apparatus acquires a snapshot acquisition of a series of rotated sub-aperture images with anisotropic scaling. SQLFM system performs sparse and compact sampling of the light field and reconstructs the signals by finding complementary information from each subaperture image.

[0021] FIG. 3A is a schematic side view of a scanning plane illumination configuration according to one embodiment of the technology.

[0022] FIG. 3B is a schematic side view of a static volume illumination configuration according to one embodiment of the technology.

[0023] FIG. 4 is a detailed side view multi-plane scanning light sheet illumination configuration according to one embodiment of the technology. Dual scanning light sheet replaces the flood illumination (i.e., illuminating the entire sample volume). In synchrony with the light sheet, the camera captures multiple frames at different scanning positions, each reconstructing two layers of the entire volume. By combining all measurements in one scan cycle, a 3D volume is synthesized for that time point.

[0024] FIG. 5A is a schematic diagram of detailed designs of the Dove prism- lenslet array and holder according to an embodiment of the technology.

[0025] FIG. 5B is a detailed front view of the Dove prism portion of the array and a detailed side view of a single prism.

[0026] FIG. 5C is a detailed front view of the spherical lenslet portion of the array that is aligned with the Dove prism and a detailed side view of a single lenslet.

[0027] FIG. 6 Schematic of a SQLFM microscopy system according to an alternative embodiment of the technology.DETAILED DESCRIPTION

[0028] Referring more specifically to the drawings, for illustrative purposes, apparatus and methods for squeezed light field microscopy (SQLFM) are generally shown. Several embodiments of the technology are describedgenerally in FIG. 1 to FIG. 6 to illustrate the characteristics and functionality of the apparatus and methods. It will be appreciated that the methods may vary as to the specific steps and sequence and the systems and apparatus may vary as to structural details without departing from the basic concepts as disclosed herein. The method steps are merely exemplary of the order that these steps may occur. The steps may occur in any order that is desired, such that it still performs the goals of the claimed technology.

[0029] Turning now to FIG. 1 , one basic structure of the SQLFM system and apparatus 10 is generally illustrated schematically. In this embodiment of the imaging system 10, the sample 12 is viewed by an objective 14. The system is compatible with different illumination modes and can be configured for widefield illumination, that is illustrated with behaving mice imaging, and selective volume imaging that is illustrated with zebra fish and leech ganglion imaging described below.

[0030] In the configuration of FIG. 1 , light from the objective 14 is preferably passed through an emission filter 16, a first achromatic doublet lens 18 and field aperture 20 to a second achromatic doublet lens 22.

[0031] Optical transformation comprises image rotation and squeezing that is performed by a dove prism / lenslet array 24 and an anamorphic relay 26. The input scene is imaged by a combined modules of an array of dove prisms and lenslets 24 and an anamorphic relay module 26. Each dove prism within the array 24 is rotated at a distinct angle relative to its optical axis. This arrangement gives rise to an array of perspective images, each rotated at twice the angle of its corresponding dove prism's rotation, all converging at an intermediate image plane situated behind the lenslets. Subsequently, these rotated perspective images are further processed through an anamorphic relay system 26 consisting of a third achromatic doublet 30 and two cylindrical achromatic doublets 32, 34 with orthogonal optical axes. This relay system module 26 imparts anisotropic scaling to the image array, where the images experience de-magnification (0.2X) along one spatial axis while preserving the original magnification along the orthogonal direction.

[0032] Finally, the rescaled image array is acquired by a 2D camera 28 as a full frame 36 with a squeezed sensor image plane called the squeezed frame38. The acquired read out of only pixel rows that receive light signals are referred to as an active readout RO I 40.

[0033] One advantage of employing squeezed optical mapping is improved readout speed. Modern CMOS sensors are equipped with parallel analog-to- digital converters (ADCs) for each column of pixels, ensuring consistent frame rates regardless of the number of pixel columns being readout. The frame rate is, therefore, solely determined by and inversely proportional to the number of pixel rows being readout. For example, on the Kinetix sCMOS developed by Teledyne, using a ROI of 200x3200 pixels allows SQLFM to capture a 19 subaperture image array at 1 ,326 fps and 7,476 fps in 16-bit and 8-bit mode, respectively. In contrast, the full-frame mode achieves frame rates of only 83 fps (16-bit) and 500 fps (8-bit).

[0034] The forward model of SQLFM is illustrated in FIG. 2. Similar to Fourier LFM (FLFM), SQLFM can be conceptualized as a tomographic system, where each sub-aperture image is essentially a parallel projection of a 3D volume along a line of sight at the sub-aperture’s view angle. However, unlike FLFM, where these sub-aperture images are directly captured by a 2D camera, SQLFM applies in-plane rotation and vertical scaling operations to these images before recording.

[0035] As shown in FIG. 2, Dove prisms 42 of the array are oriented and aligned with the lenslets 44 of the array, providing various rotation angles uniformly distributed in 180 degrees. Each dove prism 42 within the array 24 is rotated at a distinct angle relative to its optical axis. This arrangement gives rise to an array of perspective images, each rotated at twice the angle of its corresponding dove prism's rotation, all converging at an intermediate image plane 46 situated behind the lenslets 44. The images 12’ from each of the lenslets 44 are present on the plane 46 as shown in FIG. 2.

[0036] To leverage full horizontal resolution, the anamorphic relay module 26 of a third doublet 30 and two orthogonally placed cylindrical doublets demagnifies the intermediate image 46 five times vertically while maintaining 1 :1 relay horizontally. The output from the anamorphic relay 26 is a squeezed image 12” on the squeeze frame 48. One direct benefit of a squeezed frame 48 is readout speed.

[0037] In the 3D spatial frequency space, the Fourier spectrum of a SQLFM sub-aperture image manifests as a 2D elliptical slice (Fourier Slice Theorem). The short axis of this ellipse corresponds to the low-resolution sampling along the squeezing direction. By using an array of sub-aperture images rotated at complementary angles, SQLFM fills in the missing high-frequency information. This process results in a synthesized power spectrum with a bandwidth that approximates that of the original un-squeezed image.

[0038] In addition, the rotation angles of sub-aperture images may be carefully crafted to maximize the horizontal projections of their 3D point spread functions (PSFs). In other words, when imaging a 3D object, the sub-aperture images of SQFLM exhibit lateral disparity shifts due to their view angle difference. While the entire set of rotation angles are sampled uniformly from 0 to 180 degrees, it is possible to optimize the rotation angle assignment to each sub-aperture image to align its disparity shift with the un-squeezed spatial axis ( / .e., camera pixel row direction), thereby maximizing the samplings of disparity and consequently enhancing the axial resolution. With this approach, some sub-apertures may not receive their optimal angles. The sub-apertures in the outer region can be prioritized, as they play a key role in determining the system’s axial resolution. This forward model can be further extended to wave optics by using a sum of 2D convolutions between the sample sliced at each depth and the corresponding sub-aperture PSF. Through an iterative deconvolution algorithm, SQLFM reconstructs the 3D fluorescence distribution by fusing all sub-aperture images.

[0039] The SQLFM system can also be adapted with different illumination configurations depending on the application of the microscopy. For example, as shown in FIG. 3A, a scanning light sheet configuration 50 using a laser source 52 providing high power (up to 500 mW) can be used. This configuration has a maneuverable galvo mirror 54 and light sheet module 56 to produce light sheet illumination through the media 60 for observation by the objective 62 and camera. The light sheet 58 is controlled by the galvo-mirror 54, and the camera’s exposure is synchronized in a complete scan for synthetic volumetric illumination.

[0040] Alternatively, a selective volume illumination configuration 64 can beused in applications needing suppression of fluorescence out of the 3D ROI as shown in FIG. 3B. The Static LED configuration 64 uses an ultra-low-noise LED 66 with a beam 70 that has been shaped by lenses and an adjustable slit 68 that is aligned with the conjugate plane of the slit under the detection objective 74 to illuminate targets 72. Selective illumination, implemented with either a scanning light sheet or slit-confined LED, suppresses fluorescence outside of the imaging volume.

[0041] The widefield illumination configuration 50 shown in FIG. 3A was illustrated with behaving mice imaging and the selective volume illumination 64 was illustrated with zebrafish and leech ganglion imaging.

[0042] Light field microscopy, including SQLFM, is constrained by the trade-off between spatial resolution and imaging volume coverage. Its application is potentially hindered by the resolving capability and reconstruction artifacts, and it prefers objects with high sparseness. LFM with scanning illumination and detection have been proposed to reduce the background noise and enhance spatial performance.

[0043] Referring also to FIG. 4, another embodiment of high-speed 3D imaging with scanning multi-plane illumination 80 is shown schematically. A dual light sheet configuration is shown with an upper light sheet 84 and a lower light sheet 86 separated by a focal plane 88. The pair of light sheets 84, 86 in the water media 92 provides a scan range 90 beneath the detection objective 82. The SQLFM’s kilohertz frame rate was shown to provide high flexibility in a scanning scheme.

[0044] As seen in FIG. 4, a dual light sheet illumination may be implemented together with a sawtooth function by the galvo-mirror to scan. Instead of synchronizing with the entire scan range, the camera can be actuated at a higher rate so that each frame 94 captured one subset of layers of the fluorescent signals. The light sheet 84, 86 introduces significant improvement in optical sectioning capability. The SQLFM system, on the other hand, supports simultaneous multi-plane illumination and maintains high speed after scanning. This was demonstrated by imaging beating zebrafish heart at 300 vps by scanning light sheet at 300 Hz and synchronized with camera recording at 4800 fps in 8-bit speed mode. This allowed the reconstruction ofthe heart with 30 planes across 200 pm depth range and with enhanced spatial resolution and contrast.

[0045] The embryonic zebrafish myocardium 7g(cmlc:m Cherry) has been challenging to LFM due to densely labeled muscle tissue. SQLFM with scanning illumination can resolve the heart chamber clearly and outline the time-dependent dimension with sufficient speed to detect variation beat to beat.

[0046] FIG. 5A to FIG. 5C illustrates an example of detailed designs of the prism, lenslet, and holders of the apparatus. FIG. 5A shows the design of the dove prism holder 102 and dove prism array 106 coupled with the lenslet holder 104 and lenslet array 108 orienting the arrays. FIG. 5B shows a detailed cross-section of the prism holder 102 and prism array 106 oriented with the lenslet holder 104 and lenslet array 108. A front view of the coupled prism 102 and lenslet 108 holders and oriented prisms 110 and prism array 106 of different rotations are shown in FIG. 5B. Side views of a single Dove prism 102 and single lenslet 114 are shown in FIG. 5B and FIG. 5C respectively. It can be seen that the prism holder 102 allows the parallel orientation of each prism to be determined and fixed at different rotations. Similarly, the lenslet holder 108 properly orients the parallel linear orientation of the dove prisms 102 with each of the lenslets 114.

[0047] One embodiment of the anamorphic relay system preferably comprises a spherical achromat doublet (ACT508-250-A, Thorlabs) and two orthogonally oriented cylindrical achromat doublets (ACY254-250-A, ACY254- 50-A, Thorlabs). The back focal planes of two cylindrical lenses are colocated, producing image with anisotropic scaling factor. A sCMOS camera (Kinetix, Teledyne) captures the final image, with a 320 x 3200 px ROI covering all 29 sub-aperture images, or a 200 x 3200 px ROI for 19 subaperture images. The maximal readout speeds for two ROIs are 830 fps and 1328 fps in 16-bit dynamic range mode, and 5000 fps and 8000 fps in 8-bit speed mode. The 29 sub-aperture configuration collects around 50% more light than the 19 sub-aperture one.

[0048] Turning now to FIG. 6, an alternative embodiment of the light field microscopy system 120 is shown schematically. A selective volume side-illumination setup illustrated. In this embodiment, the illumination is produced with a laser beam 122 providing a beam to a preferably 50:50 beamsplitter 124 that splits the beam 122 into two arms. The first arm is directed to a mirror 126 and a first cylindrical lens 128. The second arm is directed to a first mirror 132 to a second mirror 134 to a second cylindrical lens 136. The beam emitted from the first cylindrical lens 128 is directed to one side of a knife edge mirror 130. The beam from the second cylindrical mirror 134 is directed to a second side of the knife edge mirror 130. The resulting combined beams are passed through an achromatic doublet 138 to a galvo mirror 140. The galvo mirror 140 can direct a controlled beam through a scan lens 142, a tube lens 144 to an illumination objective 146 to provide illumination 148 to a target.

[0049] The imaging subsystem has an oblique imaging objective 150 that directs image data through an optional long pass filter 152 and a first achromat doublet 154 and a second achromatic doublet 156. The beam from the second achromat doublet 156 is then directed to the Dove prism lenslet array 158. The data from the Dove prism-lenslet array 158 is directed through an anamorphic relay subsystem 160 to a camera 166. The camera 166, objectives, 146, 150, galvo mirror 140, laser 122 and other controllable elements of the system may be controlled by a computer controller with programming, storage and display. Computations and image creation and display can also be performed by computer processors and programming.

[0050] Image formation and reconstruction with the SQLFM illumination and imaging system begins with the selection and adaptation of the system for use on a particular target of interest. This includes the selection of the illumination subsystem approach and processing.

[0051] The light field of the fluorescent sample is acquired by dividing the objective’s back pupil with a lenslet array and recording a group of subaperture images. Depending on their sub-aperture locations, they display disparity, that is the distinct displacement shown by the same signal. After calibrating the displacement at every axial position, the formation of each subaperture image can be modeled as a sum of laterally shifted depth slices. The shifting operator is replaced with a convolution with PSF to account forboth the diffraction and displacement. A dove prism is a truncated right-angle prism that is used to rotate the incident beam. The rotation of the prism around its longitudinal axis causes the beam to rotate at twice the rate of the prism’s rotation. By placing a dove prism array in the infinity space between the objective and lenslet array, varying in-plane rotations can be applied to sub-aperture images. Finally, the anamorphic relay subsystem is used to introduce anisotropic scaling to the image array. This subsystem can demagnify (squeeze) the image in the direction perpendicular to the camera read-out axis while maintaining the original scale in the other direction. This one-axis scaling and the aforementioned in-plane rotation are both directly applied to the 3D fluorescent image in the model.

[0052] Given a 3D fluorescence distribution 0(x,y,z) and system PSF, the formation of camera measurement I(x,y, v) can be modeled as:where v is the index of sub-aperture, 0 represents the 2D convolution, / ?(v) applies sub-aperture-dependent rotation from dove prism, and S introduces image scaling from the anamorphic relay system.

[0053] The volume reconstruction algorithm was derived from Richardson- Lucy deconvolution. Based on the forward model of Eq. (1 ), the 3D fluorescence distribution 0(x,y,z) is iteratively solved from the camera measurements I(x,y,v) and empirical point spread functions PSF. This can be accomplished with computer program instructions (software) for implementing this process.

[0054] The rotation angles and image scaling factors have been pre-calibrated as known priors in the reconstruction. The measurement patch I(x,y, v) is cropped from the raw sensor image according to the center location of each sub-aperture image. The methods measure the point spread functions by scanning a sub-diffraction fluorescent bead at different axial positions using a motorized translation stage. The PSF is assumed to be spatially invariant within each sub-aperture image.

[0055] With this implementation, each measurement patch I(x,y,v) has a resolution of 301 x 61 px. The numbers of channels (v) and axial slices (z)are configured based on the targeted framerate and depth range / step size. For example, with 19 sub-aperture measurements to reconstruct a volume of 305 x 305 x 151 px, the deconvolution takes around 30 seconds via 8 iterations using a desktop computer with a modest GPU (Nvidia RTX 3070).

[0056] Accordingly, SQLFM presents a snapshot 3D detection apparatus and method address pressing need for high-speed volumetric microscopy operating at kilohertz speeds. This is accomplished by capturing a condensed (squeezed) representation of the original light field using a compact ROI on the sensor. The sampling strategy relies on the principle that the inherent spatio-angular correlation in the light field can be exploited to recover signals from compressive measurements. Validations across a range of applications, including hemodynamics, neural imaging, cardiovascular imaging and bacteria dynamics, demonstrate SQLFM’s versatility and robustness.

[0057] SQLFM’s kilohertz 3D imaging speed, rarely provided by existing methods and often entailing significant design tradeoffs and hardware requirements, presents new opportunities to investigate millisecond-scale dynamics in emerging fields such as voltage imaging. It is universally adaptable to the vast majority of CMOS sensors, which generally allow for higher frame rates at reduced readout pixel rows.

[0058] While kilohertz speeds are a milestone for 3D fluorescent microscopy, SQLFM can theoretically achieve tens of and even hundreds of thousands of volumes per second with current high-speed cameras. It mitigates the tradeoff between speed and other sensor characteristics, allowing us to prioritize cameras with higher sensitivity, lower noise, and other favorable attributes.

[0059] The SQLFM system offers a snapshot acquisition that effectively addresses the trade-off between pixel exposure time and volumetric frame rate encountered in conventional scanning-based 3D optical microscopy techniques. This unique approach gives SQLFM distinct advantages in terms of photon efficiency and signal-to-noise ratio (SNR), especially beneficial for high-speed imaging of weak fluorescence.

[0060] The SQLFM method compresses images only along the vertical axis and redistributes the information to the horizontal axis, which retains full sampling power through image rotation. As a result, SQLFM can reconstructthe FOV of a FLFM that occupies the full sensor area, achieving comparable spatial resolution, provided the sample's sparsity allows. This design is specifically tailored to utilize a low-format rectangular sensor, marking a fundamental departure from existing compressive light field imaging. The latter retrieves light fields at the same or lower resolutions than the multiplexed measurement and suffers from a linear reduction in FOV and pixels as it crops the sensor ROI. Moreover, SQLFM does not require multiple shots or learning on a sparse basis prior to reconstruction. It also shows excellent scalability in various ROI sizes.

[0061] It has been shown that multi-sheet scanning offers a solution by trading imaging speed for improved optical sectioning, extending SQLFM’s applicability to densely-labeled tissue imaging. The refocusing capability within a largely extended depth of field makes SQLFM compatible with various 3D illumination structures.

[0062] The technology described herein may be better understood with reference to the accompanying examples, which are intended for purposes of illustration only and should not be construed as in any sense limiting the scope of the technology described herein as defined in the claims appended hereto.

[0063] Example 1

[0064] In order to demonstrate the functionality of the apparatus and methods, a light field microscope was built employing selective volume illumination. Two setups were built to demonstrate SQLFM’s applications across a wide range of high-speed biological processes. A widefield epi-illumination setup was used for mouse imaging through cranial windows, while a selective volume illumination setup was designed for small animals like zebrafish larvae and dissected leech ganglia, where the target of interest can be accessed via side illumination.

[0065] The detection setup featured a 20X water-dipping objective (N20X- PFH, Olympus XLUMPLFLN 20X, 1.0 NA). A 4F relay system (AC508-180-A, AC508-200-A, Thorlabs) formed a conjugate plane of the objective’s back pupil, accommodating a customized dove prism and a spherical lenslet array. The dove prism (aperture length: 1.3 mm, material: H-K9L, that was fabricatedby Changchun Sunday Optics), was positioned anteriorly to the plano-convex lenslet (aperture diameter: 1.3 mm, focal length: 36 mm, material: PMMA, fabricated in-house). Each pair generated a rotated sub-aperture image with a magnification of 3.6X and NA of 0.065. In total, 29 pairs were utilized and securely housed in a 3D-printed mechanical holder at selected orientations.

[0066] The designs of the prism, lenslet, and array holder shown in FIG. 5A through FIG. 5C were constructed. The anamorphic relay system comprised a spherical achromat doublet (ACT508-250-A, Thorlabs) and two orthogonally oriented cylindrical achromat doublets (ACY254-250-A, ACY254-50-A, Thorlabs). The back focal planes of two cylindrical lenses were colocated, producing an image with anisotropic scaling factor. A sCMOS camera (Kinetix, Teledyne) captured the final images, with a 320 x 3200 px ROI covering all 29 sub-aperture images, or a 200 x 3200 px ROI for 19 subaperture images. The maximal readout speeds for two regions of interest were 830 fps and 1328 fps in the 16-bit dynamic range mode, and 5000 fps and 8000 fps in the 8-bit speed mode. The 29 sub-aperture configuration collected around 50% more light than the 19 sub-aperture configuration.

[0067] The illumination sources included blue and green continuous lasers (MBL-FN-473-500mWand MGL-lll-532-300mW, CNI Laser) and an ultra-low- noise blue LED (Pritzmatix). For the scanning light sheet setup, a knife-edge mirror (MRAK25-G01 , Thorlabs) was used to combine two beams with adjustable spacing and a galvo-mirror (GVS011 , Thorlabs) to scan them together as illustrated in FIG. 6. Planar illumination was formed perpendicular to the detection axis by a cylindrical lens and a dry objective (RMS4X-PF, Olympus 4X, 0.13 NA). A sawtooth function was used to drive the galvo- mirror.

[0068] In the synthetic volume illumination configuration, one light sheet beam is blocked. The camera was triggered at the beginning of sawtooth waveform and exposed for the entire scan. In scanning plane illumination configuration, two light sheet beams are used and the camera is triggered multiple times during a scan. The static LED setup shares the same illumination objective and perpendicular geometry. A Koehler illumination system was built that used an adjustable slit for the field aperture. The conjugate plane of the slitwas relayed under the detection system and the slit controlled the depth range of the beam. The LED provided ultra-stable illumination power and thus suppressed noise from the excitation source during the voltage imaging experiments.

[0069] Example 2

[0070] To further demonstrate the functionality of the apparatus and methods, flowing red blood cells in an embryonic zebrafish were imaged. Transgenic zebrafish lines Tg(gata1a:dsRed), Tg(flk:mCherry), and Tg(cmlc:GFP) were used to image blood cells, endothelial cells, and myocardium, respectively. Embryonic fish were maintained at three days post-fertilization in a standard E3 medium, which was supplemented with extra 1 -phenyl 2-thiourea (Sigma Aldrich) to inhibit melanogenesis on day one.

[0071] For brain hemodynamics and cardiac imaging, the larvae were anesthetized with tricaine (3-aminobenzoic acid ethyl ester, Sigma Aldrich) and immobilized in 1 % low-melting-point agarose inside a fluorinated ethylene propylene tube before imaging.

[0072] For tail experiments, the larvae were first positioned on cover glass before the heads were fixed by 3% low-melting-point agarose. Immediately after the agarose solidified, the sample was immersed in a water chamber. The imaging started after visually confirming the unconstrained movement of the tail.

[0073] Experimental characterization of blood flow in living organisms provides valuable insights into local metabolism, vascular development, and disease states. Using fluorescently labeled blood cells, various imaging methods have been demonstrated in single-cell velocimetry, such as in the larval zebrafish heart and tail. However, these methods are often limited to 2D imaging or are restricted by a limited volumetric frame rate, which hinders the detection of fast flow and necessitates sedation of the animal to reduce motion artifacts.

[0074] By comparison, the SQLFM system can be used to capture the fastcirculating red blood cells (RBCs) in a zebrafish at a kilohertz volumetric rate, both with and without sedation. Transgenic zebrafish embryos expressing DsRed in red blood cells (RBCs) were imaged at three days post fertilization(dpf). The zebrafish brain was excited using light-sheet-synthesized volumetric illumination and the fluorescence was recorded using SQLFM with 19 sub-aperture images at 1 ,000 frames per second. The reconstruction revealed the 3D distribution of RBCs and allowed cell tracking over time.

[0075] Two separate recordings from the dorsal and ventral views were taken, each visualizing RBCs at representative time points and the vasculature network by maximum intensity projection (MIP) throughout all frames. The flowing velocity was pulsatile temporally and varied spatially in the aorta and vein. The tracking revealed the velocity distribution in 3D and highlights vessels with a high-speed flow of up to 6 mm / s. The kilohertz imaging capability of SQLYM, seized the transient motion at a millisecond time scale, effectively eliminating the motion blur and enabled robust cell tracking compared to a lower imaging rate.

[0076] The speed advantage of the SQLYM system was further demonstrated by imaging the free-moving tail of a zebrafish without sedation. The embryo was mounted on a cover glass with its head restrained using agarose while allowing the tail to move freely in the water. The SQLYM system captured the high-frequency tail swings without any motion blur, maintaining its capability to track individual RBCs and revealing the compound movement that combines oscillation vertical to the tail plane and normal progression along the vessels.

[0077] Example 3

[0078] The performance of the SQLFM system and methods was further demonstrated with the optical recording of membrane action potentials in medicinal leech ganglia.

[0079] The development of voltage imaging has enabled neuroscientists to examine neural dynamics in large cell populations with high spatio-temporal resolution. However, it has long been a challenge to capture the voltage signal in vivo across a large volume due to the extremely fast transients and low signal-to-noise ratio. With the millisecond-temporal resolution, SQLFM can detect exact spike timings across a large 3D neural network. It paves the way for a precise mapping of the interaction of neuronal components, and to clarify the mechanisms underlying sensory processing and behavioral generation.

[0080] Medicinal leeches (Hirudo verbana) were housed in an artificial pond water maintained at 15°C. Thereafter, an adult leech was anesthetized in ice- cold leech saline and an individual segmental ganglion (M10 or M11 ) was dissected out. The ganglion was pinned down ventral side up on a rectangular-shaped flat substrate made of Polydimethylsiloxane (PDMS) (Sylgard 184, Dow Coming).

[0081] After removing the sheath that covers the ganglion, a voltage-sensitive dye (FluoVolt, ThermoFisher) bath-loaded using a peristaltic pump. The sample was placed under the detection objective of the SLIM system for imaging. In swim experiments, the entire nervous system was dissected out, except for the cephalic ganglia. Segmental ganglion M10 or M11 was desheathed as before. Additionally, the dorsal posterior nerves (DP1 ) of ganglion M13 or M14 were exposed for extracellular stimulation and recording with a suction electrode. Nerve stimulation in these caudal ganglia is a well- established method for eliciting fictive swimming.

[0082] As a demonstration, a voltage-sensitive dye (FluoVolt, F10488, Thermo Fisher Scientific) was loaded to a dissected ganglion from a medicinal leech. Using SQLFM, the fluorescent signals were recorded with 29 sub-aperture images at 800 Hz under the illumination of an ultra-low-noise LED.

[0083] Concurrently, an intracellular microelectrode was introduced for simultaneous electrophysiological stimulation and recording. Glass microelectrodes (20-50 MQ) were filled with a recording solution of 3 M potassium acetate and 60 mM potassium chloride. After penetrating the membrane of a cell of interest, small negative holding currents were injected to ensure stability. Intracellular electrophysiology used Neuroprobe amplifiers (Model 1600; A-M systems). Membrane voltage and electrode current were digitized along with the camera trigger signal at 10 kHz using a 16-bit data acquisition board (Nl USB-6002; National Instruments). A camera was used that triggers as time stamps to align recorded frames with electrophysiological data. Extracellular electrophysiology used a custom-built differential amplifier that allowed for rapid switching between stimulation and recording.

[0084] After image reconstruction, corrections for sample movement were made by running a 2D registration and demotion between adjacent framesusing a modified version of SWiFT-IR67. The 3D ROIs were then manually defined for each neuron. The optical readout FFtt was calculated by averaging the pixel intensities in the ROI and normalized by the temporal baseline: FF=(FFtt-FFo) / FFo, where FFQ is the temporal mean value. To detect spikes from the optical signal, the trace FF was detrended by subtracting its median-filtered version (window size, 50 ms). It was then binarized by a Schmitt trigger, and a peak detection was performed to locate the voltage spikes.

[0085] Timing and waveforms of neuronal action potentials were adequately sampled from the reconstructed 3D image sequence. The data was further processed by correcting motion drift, manually choosing an area of interest on each cell, and averaging pixels from the corresponding cell membrane. The resultant time-lapse fluorescence intensities at selected neurons were imaged and evaluated. The SQLFM measurements were shown to agree with the electrophysiological record in quantitative detail, including the reduction of spike amplitude when strong depolarizing currents were applied. Agreement of SQLFM measurement to electrode readout was verified and high correlation between stimulation current and spike firing rate was observed.

[0086] In a separate experiment, a train of electrical pulses was used to simulate a dorsal posterior (DP) nerve root of midbody ganglion (M13), which mimics a touch to the body wall in an intact leech to elicit fictive swimming. Using the SQLFM system, the selected midbody ganglion 10 (M10) was imaged from the dorsal side at an 800 Hz volume rate under the same illumination conditions as the previous demonstration. The nerve signal was simultaneously recorded through the suction microelectrode, which showed rhythmic dorsal motor neuron bursts characteristic of swimming.

[0087] After manually selecting and averaging 3D region of interest for each cell, the optical fluorescence signals at selected motor neurons (dorsal and ventral inhibitory and excitatory motor neurons DI-1 , DE-3, and VE-4.) and pressure sensitive cell (P2) were assessed. The rhythmic activity characteristic of swimming (1 Hz to 1 ,5Hz) was clearly observed in all motor neurons, consistent with previous work. To characterize how cells participated in generating the swim rhythm, the magnitude and phase of coherence werecalculated for each cell in the swimming oscillation band with respect to the extracellular recording.

[0088] SQLFM measurements matched well with the oscillatory behavior of the neurons, including the overall coherence phase distribution of all cells in dorsal side and four pairs of specific motor neurons, D 1-1 , DE-3, VI-2 and VE- 4, were very regular in their location and indeed overlapped in the measured and predicted phase maps.

[0089] Example 4

[0090] By combining the system and methods with closed-loop tracking and a translational stage, SQLFM’s high-speed volumetric imaging is capable of studying hemodynamics under natural conditions during locomotor behavior. The performance of the SQLFM system in awake, behaving mice was illustrated with voltage imaging of the hippocampus.

[0091] Mice were anaesthetized with isoflurane (5% for induction, 1-2% (v / v) for maintenance). The depth of anesthesia was monitored continuously and adjusted when necessary. After induction of anesthesia, the mice were fitted into a stereotaxic frame (Kopf), with their heads secured by blunt ear bars and their noses placed into an anesthesia and ventilation system (David Kopf Instruments). The body temperature was kept at 37 °C with a feedback- controlled heating pad (Harvard Apparatus). Mice were administered 0.05 ml lidocaine (2%; Akorn) subcutaneously as a local anesthetic before surgery. The surgical incision site was cleaned three times with 10% povidone-iodine and 70% ethanol. After removing the scalp and clearing the skull of connective tissues, a hole was drilled above the virus injection location. Then, GEVIs were injected to the CA1 of the hippocampus, with coordinates ML: ±1.8mm, AP: -2mm, DV: -1.3mm from Bregma. One Chrna2-Cre+ mouse was injected with Cre-dependent GEVI pAce (AAV-DJ-CAG-DIO-pAce-kv2.1 ; titer, 2.6x1012viral genomes per mL), allowing for imaging of neuron populations expressing nicotinic acetylcholine receptor alpha2, a specific marker for oriens lacunosum-moleculare (OLM) interneurons.

[0092] Another wild-type mouse was injected with a cocktail of the GEVI pAce (AAV9-EF1a-DIO-pAce-Kv-WPRE, titer, 2.1x1013viral genomes per mL) and a principal-cell specific Cre promoter (pAAV1-CamKII-Cre, Addgene, 105558;titer, 1 .9x1013viral genomes per mL) allowing for imaging of excitatory pyramidal neurons. Dura over the exposed brain surface was removed and the cortical tissue above the dorsal CA1 was carefully aspirated using a 27- gauge blunt needle. Buffered artificial cerebrospinal fluid (7.888 g NaCI, 0.372 g KCI, 1.192 g HEPES, 0.264 g CaCI2, 0.204 g MgCI2per 1000 mL milipore water) was constantly applied throughout the aspiration to prevent desiccation of the tissue. The aspiration ceased after partial removal of the corpus callosum and bleeding terminated, at which point a 3-mm titanium ring with a glass coverslip attached to its bottom was implanted into the aspirated area and its circular flange was secured to the skull surface.

[0093] A custom-made lightweight metal head holder (headbar) was attached to the skull posterior to the implant. Cyanoacrylate glue and black dental cement (Ortho-Jet, Lang Dental) were used to seal and cover the exposed skull. During recovery mice were administered carprofen (5 mg per kg of body weight) for 3 days as a systemic analgesic and amoxicillin antibiotic (0.25 mg / ml in drinking water) through the water supply for 7 days.

[0094] Neuronal activity in the CA1 of the hippocampus was monitored through an implanted cranial window in mice expressing genetically encoded voltage indicator (GEVI) pAce. Mice were imaged continuously at 800 Hz for three minutes on a treadmill set-up that used an optical rotary encoder to track movement.

[0095] Conventional widefield microscopes suffer from a shallow depth of focus and have difficulty imaging axially distributed neuron population. In contrast, SQLFM provides a volumetric mapping of signals, allowing for simultaneous optical measurement of neurons at different depths. After image reconstruction and motion correction, membrane-potential traces from multiple neuronal sources exhibiting strong speed-related action potential modulation across the imaged volume were extracted. The relative fluorescence change over the three-minute recording was calculated, both in signal-to-noise ratio (SNR) and in AFF / FFo, where SLIM’s millisecond temporal resolution provided sufficient sampling on the rising and falling slopes of transient spikes.

[0096] Due to photobleaching, the amplitude of the spike waveform exhibited a gradual decay, but the SNR maintained around five across the entire recording. SQLFM also detected subtle subthreshold membrane potential oscillations. The observed signals predominantly showed significant frequency components in the 4 Hz to 10 Hz band, likely originating from theta oscillations commonly found in the hippocampus.

[0097] The examination over the inactive neurons and background revealed the absence of spikes and subthreshold oscillations, further confirming the fidelity of observed signals. By correlating the time-dependent firing rate of each neuron with locomotion speed, it was observed that the majority of neurons were positively modulated by locomotion speed which is consistent with previous findings.

[0098] Additionally, the SQLFM’s compressed measurement offers a unique advantage over alternative methods, providing highly efficient data bandwidth and making it more accessible for long-term 3D voltage imaging across large volumes. Overall, SQLFM enabled 3D voltage imaging in neuron populations distributed across large volumes, with the potential to elucidate network dynamics and interactions between different cell types across layers.

[0099] Example 5

[0100] To further demonstrate the functionality of the imaging system, the beating of an embryonic zebrafish heart was imaged with scanning multisheet illumination. Although light field microscopy (LFM) techniques, including SQLFM, offer the ability to numerically refocus to specific depths, they typically lack intrinsic optical sectioning capability. Its application is potentially hindered by the spatial resolution and reconstruction artifacts, and it favors objects with high sparseness. Here, it was shown that SQLFM can be combined with scanning multi-sheet illumination. The synergy enables high- contrast 3D imaging of densely-labeled fluorescent objects.

[0101] A dual-light-sheet illumination module was constructed and scanned the beams using a galvo-mirror driven by a sawtooth function. Rather than synchronizing the camera exposure with the entire scan range as in previous experiments, the camera was operated at a higher rate, allowing each frame to capture a subset of depth layers of the fluorescent object (FIG. 3A and FIG.4). This approach significantly suppresses out-of-focus light and improves axial resolution in the reconstruction, as shown on fluorescent beads and zebrafish vasculature networks (7g(flk:m Cherry)).

[0102] On the other hand, SQLFM offers an ultra-high framerate and supports simultaneous multi-plane detection. These features enable SQLFM to maintain a high-volume rate even within this scanning scheme.

[0103] The acquisition scheme was illustrated by imaging a beating zebrafish heart (7g(cmlc2:GFP)) at 300 vps. This was achieved by scanning the dual light sheets at 300 Hz, synchronized with camera recording at 4,800 fps in an 8-bit speed mode. This setup allowed the reconstruction of the heart with 30 planes across 200 pm depth range with the microstructures like ventricular trabeculation clearly delineated. The enhanced spatial resolution and contrast offer the potential for accurate segmentation of the heart chamber’s geometry, facilitating cardiac studies, such as regional myocardial contractility analysis and computational fluid dynamics (CFD) for hemodynamic forces simulation. While current LFM cardiac imaging is mostly demonstrated on sparse markers like cardiomyocyte nuclei and blood cells, SQLFM, with scanning multi-sheet illumination, proved effective in resolving the densely labeled muscle tissue. It provides high 3D imaging speed to capture the beating heart in real time and outlines the time-dependent chamber dimension to detect beat-to-beat variations.

[0104] Embodiments of the technology of this disclosure may be described herein with reference to flowchart illustrations of methods and systems according to embodiments of the technology. Embodiments of the technology of this disclosure may also be described with reference to procedures, algorithms, steps, operations, formulae, or other computational depictions, which may be included within the flowchart illustrations or otherwise described herein. It will be appreciated that any of the foregoing may also be implemented as computer program instructions. In this regard, each block or step of a flowchart, and combinations of blocks (and / or steps) in a flowchart, as well as any procedure, algorithm, step, operation, formula, or computational depiction can be implemented by various means, such as hardware, firmware, and / or software including one or more computer programinstructions embodied in computer-readable program code. As will be appreciated, any such computer program instructions may be executed by one or more computer processors, including without limitation a general purpose computer or special purpose computer, or other programmable processing apparatus to produce a machine, such that the computer program instructions which execute on the computer processor(s) or other programmable processing apparatus create means for implementing the function(s) specified.

[0105] Accordingly, blocks of the flowcharts, and procedures, algorithms, steps, operations, formulae, or computational depictions described herein support combinations of means for performing the specified function(s), combinations of steps for performing the specified function(s), and computer program instructions, such as embodied in computer-readable program code logic means, for performing the specified function(s). It will also be understood that each block of the flowchart illustrations, as well as any procedures, algorithms, steps, operations, formulae, or computational depictions and combinations thereof described herein, can be implemented by special purpose hardware-based computer systems which perform the specified function(s) or step(s), or combinations of special purpose hardware and computer-readable program code.

[0106] Furthermore, these computer program instructions, such as embodied in computer-readable program code, may also be stored in one or more computer-readable memory or memory devices that can direct a computer processor or other programmable processing apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory or memory devices produce an article of manufacture including instruction means which implement the function specified in the block(s) of the flowchart(s). The computer program instructions may also be executed by a computer processor or other programmable processing apparatus to cause a series of operational steps to be performed on the computer processor or other programmable processing apparatus to produce a computer- implemented process such that the instructions which execute on the computer processor or other programmable processing apparatus providesteps for implementing the functions specified in the block(s) of the flowchart(s), procedure (s) algorithm(s), step(s), operation(s), formula(e), or computational depiction(s).

[0107] It will further be appreciated that the terms "programming" or "program executable" as used herein refer to one or more instructions that can be executed by one or more computer processors to perform one or more functions as described herein. The instructions can be embodied in software, in firmware, or in a combination of software and firmware. The instructions can be stored locally to the device in non-transitory media, or can be stored remotely such as on a server, or all or a portion of the instructions can be stored locally and remotely. Instructions stored remotely can be downloaded (pushed) to the device by user initiation, or automatically based on one or more factors.

[0108] It will further be appreciated that as used herein, the terms controller, microcontroller, processor, microprocessor, hardware processor, computer processor, central processing unit (CPU), and computer are used synonymously to denote a device capable of executing the instructions and communicating with input / output interfaces and / or peripheral devices, and that the terms controller, microcontroller, processor, microprocessor, hardware processor, computer processor, CPU, and computer are intended to encompass single or multiple devices, single core and multicore devices, and variations thereof.

[0109] From the description herein, it will be appreciated that the present disclosure encompasses multiple implementations of the technology which include, but are not limited to, the following:

[0110] a Squeezed Light Field Microscopy (SQLFM) apparatus, the apparatus comprising: (a) an imaging objective; (b) a first lens; (c) a field aperture; (d) a second lens; (e) an array of Dove prisms configured to receive light from the field aperture; (f)a plurality of lenslets, each lenslet optically coupled to at least one Dove prism of the array of Dove prisms; and (g) an imaging module positioned after the array of lenslets, the module configured to image parallel beam projections from each lenslet.

[0111] The apparatus of any previous or following implementation, furthercomprising: a filter positioned between the objective and the first lens.

[0112] The apparatus of any previous or following implementation, wherein individual Dove prisms in the array of Dove prisms are fixed with an axial rotation of an angle from 0° to 180°.

[0113] The apparatus of any previous or following implementation, wherein the imaging module comprises: an anamorphic relay; and an electronic image sensor selected from the group of a charge-coupled device (CCD), InGaAs and a CMOS sensor.

[0114] The apparatus of any previous or following implementation, wherein the anamorphic relay of the imaging module comprises: an achromatic doublet; a first achromatic cylindrical doublet; and a second achromatic cylindrical doublet.

[0115] The apparatus of any previous or following implementation, further comprising a target illumination light source, wherein reflected light from a target is imaged by the imaging device.

[0116] The apparatus of any previous or following implementation, wherein the target illumination light source produces a wide field illumination, the light source comprising: a low noise light emitting diode; a first achromatic doublet; a second achromatic doublet; a third achromatic doublet; and an imaging objective.

[0117] The apparatus of any previous or following implementation, wherein the target illumination light source produces a multi-plane scanning light sheet illumination, the light source comprising: a beam splitter capable of dividing a beam into a first beam and a second beam; a laser configured to direct a beam to the beam splitter; a first mirror and a first cylindrical lens; a second and third mirrors and a second cylindrical lens; a knife edge mirror configured to receive the first and second beams and reflect a illumination beam; a achromat doublet; a galvo mirror; a scan lens; a tube lens; and an illumination objective positioned orthogonally to the imaging objective.

[0118] The apparatus of c any previous or following implementation, the imaging module further comprising: (a) a processor configured to control the lenses and imaging sensor; and (b) a non-transitory memory storing instructions executable by the processor; (c) wherein the instructions, whenexecuted by the processor, perform steps comprising: (i) acquiring a dataset of image data from an imaging device over time; and (ii) analyzing the acquired dataset.

[0119] The apparatus of any previous or following implementation, wherein the instructions when executed by the processor further perform steps comprising: (i) controlling the target illumination light source; and (ii) controlling the rate of image data acquisition.

[0120] A Squeezed Light Field Microscopy (SQLFM) system, the system comprising: (a) a target illumination subsystem; (b) an imaging subsystem, comprising: (i) an objective; (ii) a first lens; (iii) a field aperture; (iv) a second lens; (v) a Dove prism array optically coupled to an array of lenslets; (vi) an anamorphic relay; and (vii) an electronic image sensor.

[0121] The system of any previous or following implementation, wherein the target illumination subsystem produces a wide field illumination, the light source comprising: a low noise light emitting diode; a first achromatic doublet; a second achromatic doublet; a third achromatic doublet; and an imaging objective.

[0122] The system of any previous or following implementation, wherein the target illumination light source produces a multi-plane scanning light sheet illumination, the light source comprising: a beam splitter capable of dividing a beam into a first beam and a second beam; a laser configured to direct a beam to the beam splitter; a first mirror and a first cylindrical lens; a second and third mirrors and a second cylindrical lens; a knife edge mirror configured to receive the first and second beams and reflect a illumination beam; a achromat doublet; a galvo mirror; a scan lens; a tube lens; and an illumination objective positioned orthogonally to the imaging objective.

[0123] The system of any previous or following implementation, further comprising: a long pass filter positioned between the objective and the first lens.

[0124] The system of any previous or following implementation, wherein individual Dove prisms in the array of Dove prisms are fixed with an axial rotation of an angle from 0° to 180°.

[0125] The system of any previous or following implementation, wherein theanamorphic relay of the imaging subsystem comprises: an achromatic doublet; and a first achromatic cylindrical doublet; and a second achromatic cylindrical doublet.

[0126] The system of any previous or following implementation, wherein the electronic image sensor is selected from the group consisting of a charge- coupled device (CCD), InGaAs and a CMOS sensor.

[0127] The system of any previous or following implementation, the imaging subsystem further comprising: (a) a processor configured to control the lenses and imaging sensor; and (b) a non-transitory memory storing instructions executable by the processor; (c) wherein the instructions, when executed by the processor, perform steps comprising: (i) acquiring a dataset of image data from an imaging device over time; and (ii) analyzing the acquired dataset.

[0128] The apparatus of any previous or following implementation, wherein the instructions when executed by the processor further perform steps comprising: (i) controlling the target illumination light source; and (ii) controlling the rate of image data acquisition.

[0129] A Squeezed Light Field Microscopy (SQLFM) system, the system comprising: (a) a target illumination subsystem; (b) an imaging subsystem, comprising: (i) an objective; (ii) a long pass filter; (iii) a first lens; (iv) a field aperture; (v) a second lens; (vi) a Dove prism array optically coupled to an array of lenslets; (vii) an anamorphic relay; and (viii) an electronic image sensor; (c) a processing subsystem with a processor configured to control the lenses and imaging sensor; and (d) a non-transitory memory storing instructions executable by the processor; (e) wherein the instructions, when executed by the processor, perform steps comprising: (i) acquiring a dataset of image data from an imaging device over time; and (ii) analyzing the acquired dataset.

[0130] As used herein, the term "implementation" is intended to include, without limitation, embodiments, examples, or other forms of practicing the technology described herein.

[0131] As used herein, the singular terms "a," "an," and "the" may include plural referents unless the context clearly dictates otherwise. Reference to an object in the singular is not intended to mean "one and only one" unlessexplicitly so stated, but rather "one or more."

[0132] Phrasing constructs, such as “A, B and / or C”, within the present disclosure describe where either A, B, or C can be present, or any combination of items A, B and C. Phrasing constructs indicating, such as “at least one of” followed by listing a group of elements, indicates that at least one of these groups of elements is present, which includes any possible combination of the listed elements as applicable.

[0133] References in this disclosure referring to “an embodiment”, “at least one embodiment” or similar embodiment wording indicates that a particular feature, structure, or characteristic described in connection with a described embodiment is included in at least one embodiment of the present disclosure. Thus, these various embodiment phrases are not necessarily all referring to the same embodiment, or to a specific embodiment which differs from all the other embodiments being described. The embodiment phrasing should be construed to mean that the particular features, structures, or characteristics of a given embodiment may be combined in any suitable manner in one or more embodiments of the disclosed apparatus, system, or method.

[0134] As used herein, the term "set" refers to a collection of one or more objects. Thus, for example, a set of objects can include a single object or multiple objects.

[0135] Relational terms such as first and second, top and bottom, upper and lower, left and right, and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[0136] The terms "comprises," "comprising," "has", "having," "includes", "including," "contains", "containing" or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, apparatus, or system, that comprises, has, includes, or contains a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, apparatus, or system. An element proceeded by "comprises . . . a", "has . . . a", "includes . . . a", "contains . . . a" does not, without more constraints, preclude the existence of additional identical elements in the process,method, article, apparatus, or system, that comprises, has, includes, contains the element.

[0137] As used herein, the terms "approximately", "approximate", "substantially", "substantial", "essentially", and "about", or any other version thereof, are used to describe and account for small variations. When used in conjunction with an event or circumstance, the terms can refer to instances in which the event or circumstance occurs precisely as well as instances in which the event or circumstance occurs to a close approximation. When used in conjunction with a numerical value, the terms can refer to a range of variation of less than or equal to ± 10% of that numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1 %, less than or equal to ±0.5%, less than or equal to ±0.1 %, or less than or equal to ±0.05%. For example, "substantially" aligned can refer to a range of angular variation of less than or equal to ±10°, such as less than or equal to ±5°, less than or equal to ±4°, less than or equal to ±3°, less than or equal to ±2°, less than or equal to ±1 °, less than or equal to ±0.5°, less than or equal to ±0.1 °, or less than or equal to ±0.05°.

[0138] Additionally, amounts, ratios, and other numerical values may sometimes be presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

[0139] The term "coupled" as used herein is defined as connected, although not necessarily directly and not necessarily mechanically. A device or structure that is "configured" in a certain way is configured in at least that way, but may also be configured in ways that are not listed.

[0140] Benefits, advantages, solutions to problems, and any element(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or element of the technology described herein or any or all the claims.

[0141] In addition, in the foregoing disclosure various features may be grouped together in various embodiments for the purpose of streamlining the disclosure. This method of disclosure is not to be interpreted as reflecting an intention that the claimed embodiments require more features than are expressly recited in each claim. Inventive subject matter can lie in less than all features of a single disclosed embodiment.

[0142] The abstract of the disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.

[0143] It will be appreciated that the practice of some jurisdictions may require deletion of one or more portions of the disclosure after the application is filed. Accordingly, the reader should consult the application as filed for the original content of the disclosure. Any deletion of content of the disclosure should not be construed as a disclaimer, forfeiture, or dedication to the public of any subject matter of the application as originally filed.

[0144] All text in a drawing figure is hereby incorporated into the disclosure and is to be treated as part of the written description of the drawing figure.

[0145] The following claims are hereby incorporated into the disclosure, with each claim standing on its own as a separately claimed subject matter.

[0146] Although the description herein contains many details, these should not be construed as limiting the scope of the disclosure, but as merely providing illustrations of some of the presently preferred embodiments. Therefore, it will be appreciated that the scope of the disclosure fully encompasses other embodiments which may become obvious to those skilled in the art.

[0147] All structural and functional equivalents to the elements of the disclosed embodiments that are known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the present claims. Furthermore, no element, component,or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. No claim element herein is to be construed as a "means plus function" element unless the element is expressly recited using the phrase "means for". No claim element herein is to be construed as a"step plus function" element unless the element is expressly recited using the phrase "step for".

Claims

CLAIMSWhat is claimed:1 . A Squeezed Light Field Microscopy (SQLFM) apparatus, the apparatus comprising:(a) an imaging objective;(b) a first lens;(c) a field aperture;(d) a second lens;(e) an array of Dove prisms configured to receive light from the field aperture;(f) a plurality of lenslets, each lenslet optically coupled to at least one Dove prism of said array of Dove prisms; and(g) an imaging module positioned after the array of lenslets, said module configured to image parallel beam projections from each lenslet.

2. The apparatus of claim 1 , further comprising: a filter positioned between the objective and the first lens.

3. The apparatus of claim 1 , wherein individual Dove prisms in the array of Dove prisms are fixed with an axial rotation of an angle from 0° to 180°.

4. The apparatus of claim 1 , wherein the imaging module comprises: an anamorphic relay; and an electronic image sensor selected from the group of a charge-coupled device (CCD), InGaAs and a CMOS sensor.

5. The apparatus of claim 4, wherein the anamorphic relay of the imaging module comprises: an achromatic doublet; a first achromatic cylindrical doublet; and a second achromatic cylindrical doublet.

6. The apparatus of claim 1 , further comprising: a target illumination light source, wherein reflected light from a target is imaged by the imaging device.

7. The apparatus of claim 6, wherein the target illumination light source produces a wide field illumination, the light source comprising: a low noise light emitting diode; a first achromatic doublet; a second achromatic doublet; a third achromatic doublet; and an imaging objective.

8. The apparatus of claim 6, wherein the target illumination light source produces a multi-plane scanning light sheet illumination, the light source comprising: a beam splitter capable of dividing a beam into a first beam and a second beam; a laser configured to direct a beam to the beam splitter; a first mirror and a first cylindrical lens; a second and third mirrors and a second cylindrical lens; a knife edge mirror configured to receive the first and second beams and reflect an illumination beam; an achromat doublet; a galvo mirror; a scan lens; a tube lens; and an illumination objective positioned orthogonally to the imaging objective.

9. The apparatus of claim 1 , said imaging module further comprising:(a) a processor configured to control said lenses and imaging sensor; and(b) a non-transitory memory storing instructions executable by the processor;(c) wherein said instructions, when executed by the processor, perform steps comprising:(i) acquiring a dataset of image data from an imaging device over time; and(ii) analyzing the acquired dataset.

10. The apparatus of claim 9, wherein said instructions when executed by the processor further perform steps comprising:(i) controlling the target illumination light source; and(ii) controlling the rate of image data acquisition.

11. A Squeezed Light Field Microscopy (SQLFM) system, the system comprising:(a) a target illumination subsystem;(b) an imaging subsystem, comprising:(i) an objective;(ii) a first lens;(iii) a field aperture;(iv) a second lens;(v) a Dove prism array optically coupled to an array of lenslets;(vi) an anamorphic relay; and(vii) an electronic image sensor.

12. The system of claim 11 , wherein the target illumination subsystem produces a wide field illumination, the light source comprising: a low noise light emitting diode; a first achromatic doublet; a second achromatic doublet; a third achromatic doublet; and an imaging objective.

13. The system of claim 11 , wherein the target illumination light source produces a multi-plane scanning light sheet illumination, the light source comprising: a beam splitter capable of dividing a beam into a first beam and a secondbeam; a laser configured to direct a beam to the beam splitter; a first mirror and a first cylindrical lens; a second and third mirrors and a second cylindrical lens; a knife edge mirror configured to receive the first and second beams and reflect an illumination beam; an achromat doublet; a galvo mirror; a scan lens; a tube lens; and an illumination objective positioned orthogonally to the imaging objective.

14. The system of claim 11 , further comprising: a long pass filter positioned between the objective and the first lens.

15. The system of claim 1 , wherein individual Dove prisms in the array of Dove prisms are fixed with an axial rotation of an angle from 0° to 180°.

16. The system of claim 11 , wherein the anamorphic relay of the imaging subsystem comprises: an achromatic doublet; a first achromatic cylindrical doublet; and a second achromatic cylindrical doublet.

17. The system of claim 11 , wherein the electronic image sensor is selected from the group consisting of a charge-coupled device (CCD), InGaAs and a CMOS sensor.

18. The system of claim 11 , said imaging subsystem further comprising:(a) a processor configured to control said lenses and imaging sensor; and(b) a non-transitory memory storing instructions executable by the processor;(c) wherein said instructions, when executed by the processor, perform steps comprising:(i) acquiring a dataset of image data from an imaging device over time; and(ii) analyzing the acquired dataset.

19. The system of claim 18, wherein said instructions when executed by the processor further perform steps comprising:(i) controlling the target illumination light source; and(ii) controlling the rate of image data acquisition.

20. A Squeezed Light Field Microscopy (SQLFM) system, the system comprising:(a) a target illumination subsystem;(b) an imaging subsystem, comprising:(i) an objective;(ii) a long pass filter(iii) a first lens;(iv) a field aperture;(v) a second lens;(vi) a Dove prism array optically coupled to an array of lenslets;(vii) an anamorphic relay; and(viii) an electronic image sensor;(c) a processing subsystem with a processor configured to control said lenses and imaging sensor; and(d) a non-transitory memory storing instructions executable by the processor;(e) wherein said instructions, when executed by the processor, perform steps comprising:(i) acquiring a dataset of image data from an imaging device over time; and(ii) analyzing the acquired dataset.

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