Methods and apparatus for scattering-based light sheet microscopy

Abstract

Scattering-based light sheet microscopy devices, systems and methods are described that can produce images of the human tissue in vivo and at low cost. An example device includes a spatially incoherent light source that illuminates a collimation lens, and an aperture positioned to receive collimated light from the collimation lens and to produce a line illumination beam. The aperture has an opening with a rectangular cross-section, where a length of the rectangular cross-section is at least twice its width, and a ratio of numerical apertures in two different orthogonal directions of the aperture is at least 1 to 2. The device also includes a first objective lens to receive the line illumination beam for illuminating an area of a sample with focused light, and a second objective lens to receive light from the sample, and to direct the received light for detection by an imaging sensor.

Description

METHODS AND APPARATUS FOR SCATTERING-BASED LIGHT SHEET MICROSCOPYSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under Grant No. EB030079 awarded by National Institutes of Health. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATION(S)

[0002] This application claims priority to the provisional application with serial number 63 / 558,505 titled “METHODS AND APPARATUS FOR SCATTERING-BASED LIGHT SHEET MICROSCOPY,” filed February 27, 2024. The entire contents of the above noted provisional application are incorporated by reference as part of the disclosure of this document.TECHNICAL FIELD

[0003] The technology described in this patent document relates to scattering-based microscopy methods, devices and systems.BACKGROUND

[0004] In vivo microscopy visualizes microscopic details of intact tissues non-invasively and can aid disease diagnosis by visualizing cellular morphologic changes associated with the disease. Existing clinically viable in vivo microscopes such as reflectance confocal microscopy (RCM) and Optical coherence tomography (OCT) have several shortcomings and challenges that include, among others, RCM’s high cost and small field of view (FOV), and OCT’s high cost and inadequate resolution. Therefore, there is a need for improved in vivo microscopy methods and devices that overcome the deficiencies of prior systems.SUMMARY

[0005] The disclosed embodiments relate to scattering-based light sheet microscopes that, among other features and benefits, can provide microscopic images of the human tissue in vivo and can be produced at low cost. The disclosed example embodiments leverage the use of an incoherent line light source with a rectangular aperture to generate light sheet illumination and detect the scattered light from the illuminated tissue plane to generate images. The use of an incoherent line light source and rectangular aperture provides a small illumination light sheet width over a large field of view, while reducing the speckle noise and shadow artifacts.

[0006] An example scattering-based light sheet microscopy device includes a spatially incoherent light source positioned to provide illumination to a collimation lens, an aperture positioned to receive collimated light from the collimation lens and to produce a line illumination beam, the aperture having an opening with a rectangular cross-section, wherein a length of the rectangular cross-section is at least twice a width of the rectangular cross-section, wherein a ratio of numerical apertures in two different orthogonal directions associated with the aperture is at least 1 to 2. The scattering-based light sheet microscopy device also includes a first objective lens positioned to receive the line illumination beam to illuminate an area of a sample with focused light, and a second objective lens positioned to receive light from the area of the sample in response to illumination by the focused light, and to direct the received light for detection by an imaging sensor.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1 illustrates an example configuration of an existing scattering-based light sheet microscopy (sLSM) system.

[0008] FIG. 2 illustrates an sLSM configuration in accordance with an example embodiment.

[0009] FIG. 3A illustrates example side profiles associated with the rectangular aperture ofFIG. 2.

[0010] FIG. 3B illustrates different aspects associated with the disclosed embodiments, where having a larger numerical aperture along the line helps reduce the shadow artifacts.

[0011] FIG. 4 illustrates example configurations for producing a low-coherence line illumination in accordance with some embodiments.

[0012] FIG. 5 illustrates an sLSM configuration in accordance with another example embodiment.

[0013] FIG. 6A illustrates an sLSM configuration in accordance with still another example embodiment.

[0014] FIG. 6B illustrates a phase plate that can elongate the point spread function in accordance with an example embodiment.

[0015] FIG. 7 illustrates an sLSM configuration in accordance with yet another example embodiment.

[0016] FIG. 8 illustrates an sLSM configuration in accordance with another exampleembodiment.

[0017] FIG. 9 illustrates an sLSM configuration in accordance with another example embodiment.

[0018] FIG. 10 illustrates an sLSM configuration in accordance with another example embodiment.

[0019] FIG. 11 illustrates an sLSM configuration in accordance with another example embodiment.

[0020] FIG. 12 illustrates an sLSM configuration in accordance with another example embodiment.

[0021] FIG. 13 illustrates an sLSM configuration in accordance with another example embodiment.DETAILED DESCRIPTION

[0022] As noted earlier, RCM is a clinically viable in vivo microscopy technique; it is a widely evaluated method for guiding diagnosis of skin diseases. One challenge of RCM is the small field of view (FOV). Conventional RCM uses a high-numerical aperture (NA) 0.8) objective lens to achieve high lateral (1.25 pm) and axial resolution (5 pm), resulting in a small FOV (500 - 750 pm) and posing challenges in imaging the entire suspicious tissue region. Another challenge of conventional RCM is that it typically provides en face images of the tissue at a given time. Acquiring cross-sectional images of the tissue can be beneficial for certain diseases, such as anal and cervical precancers, where cellular morphologic change as a function of tissue depth is used as one of the diagnostic criteria. In addition, conventional RCM devices are expensive (> $65,000) due to the use of high-cost beam scanning devices and their associated electronics.

[0023] Optical coherence tomography (OCT) is another widely evaluated in vivo microscopy technology for diseases diagnosis. OCT also has the challenge in achieving high resolution and large field of view simultaneously. Conventional OCT has a relatively large FOV, approximately 2 - 3 mm, but its resolution is around 10 - 20 pm, which is suitable for imaging architectural features rather than cellular and sub-cellular features. High-resolution, large-FOV OCT technologies have been recently developed. However, the use of an expensive broadband coherent light source likely increases the device cost.

[0024] Light sheet microscopy (LSM) is a microscopy technology mainly used in basic life science research. LSM uses separate optical paths for illumination and detection, where the illumination optics mainly determine the axial resolution, and the detection optics determine the lateral resolution. In many LSMs, the illumination path and detection path are perpendicular to each other, and they are at ±45° with respect to the tissue surface, which produces cross- sectional images of the tissue. Most of the previous LSM devices are used for fluorescence imaging with exogenous contrast agents, which limits their useability for in vivo imaging due to the limited availability of clinically viable exogenous contrast agents. We recently developed scattering-based LSM (sLSM) that uses intrinsic scattering signal to generate microscopy images, which makes LSM imaging of intact human tissues in vivo feasible. sLSM can achieve a high lateral resolution (e.g., 1 - 2 pm) and large FOV (e.g., 2 - 3 mm) by using a detection objective lens with a moderate NA (e.g., < 0.4). It can also achieve a high axial resolution (e.g., 5 - 10 pm) over a large illumination depth of focus. Another potential advantage of sLSM is that the device cost can be low due to the use of a moderate-NA objective lens, an inexpensive light source such as an LED, and a standard CMOS sensor rather than an expensive scientific CMOS sensor.

[0025] An example of an existing sLSM configuration is illustrated in FIG. 1. Laser light is collimated by a collimating lens, is provided to a cylindrical lens, to a rectangular aperture, and onto the sample (tissue) by the focusing lens. Light from the tissue is received by the objective lens, then by a tube lens that directs the light to the imaging sensor. An immersion medium is often used to provide index matching. The bottom panels of FIG. 1 illustrate side and front views of the illumination path. However, the existing sLSM setups, such as those show in FIG. 1, suffer from two issues - speckle noise and shadow artifacts - which can significantly degrade the image quality and make it challenging to appreciate cellular morphologic details. Speckle noise is caused by the random interference of scattered coherent light and appear as high-contrast random granular patterns in the image. Shadow artifacts are dark stripes generated when the straight light sheet is partially blocked or scattered by dense or highly scattering structures within the tissue. The previous sLSM setups use a cylindrical lens to generate a light sheet by focusing the incoming laser light in one axis only, thereby creating a line of light, which, when projected, forms a thin sheet of light. However, the use of laser light with high spatial coherence causes noticeable speckle noise even when a relatively large bandwidth of 40 nm is used for the sLSM.Further, the illumination optics in the existing configurations that use a cylindrical lens result in parallel illumination with a small angular divergence along the light sheet width direction, which makes the shadow artifacts prominent.

[0026] FIG. 2 illustrates an sLSM configuration in accordance with an example embodiment. Light from a low-coherence line source (or a line source module) is collimated by a collimation lens and illuminates a rectangular aperture. An objective lens with a moderate NA (e.g., lower than 0.4, or in some cases 0.3) focuses the illumination light onto the tissue through an immersion medium and generates light sheet illumination. The immersion medium (e.g., water) is provided for index matching between the interfaces. The light sheet plane can be at 45° angle with respect to the tissue surface. The light scattered from the tissue is collected and collimated by another objective that can be oriented at 90° angle with respect to the illumination path. The collimated light is focused onto an imaging sensor by a tube lens.

[0027] The rectangular aperture has a narrow dimension along the z-direction and a wide dimension along x-direction, as shown in FIG. 3A. The narrow aperture thickness along the z- axis produces low NA (e.g., less than 0.1) and long depth of focus (e.g., greater than 150 pm) along the y-direction, which forms a thin light sheet illumination. The shape of the light sheet is determined by the convolution of the image of the line light source on the tissue and the point spread function (PSF) of the illumination optics. The desired light sheet z-direction thickness and depth of focus can be achieved by optimizing the dimensions of the aperture along the x- and z- directions. For example, the aperture width (along the z-axis) and length (along the x-axis) may be selected to produce an NA ratio of l-to-3 to l-to-4. In some implementations, the NA ratio (in the two directions) can be l-to-2. The longer aperture dimension along the x-axis produces a moderate illumination NA on the tissue along the x-axis (e.g., < 0.4) and a larger angular divergence along the x-axis than the angular divergence along the z-axis. In this way, the light rays that traverse through the aperture can illuminate the object from multiple angles along the x- axis and shadow artifacts can be reduced. In particular, the prior systems that use a cylindrical lens, produce a small angular divergence along the light sheet width direction (x-direction); as a result, opaque features in the tissue can block the light from reaching feature that lie below the opaque elements. This problem is alleviated in the disclosed embodiments due to the large angular divergence of the beam along the x-axis.

[0028] FIG. 3B further illustrates the above aspect of the disclosed embodiments, wherehaving a larger numerical aperture along the line illumination direction (x-direction in FIG. 3A) helps reduce the shadow artifacts. As shown in panel (A), when the illumination numerical aperture is small, a highly-scattering object (shown as the optically-dense particle) blocks a large portion of the illumination beam from propagating deeper into the tissue. This makes the image exhibit vertical dark lines, which is termed shadow artifacts (see panel (B)’s dark vertical streaks). When the illumination numerical aperture is larger, such as that shown in panel (C), even when a highly-scattering object is present, the tissue portion underneath the highly- scattering object can be still illuminated. This greatly reduces the shadow artifacts, shown as the lack of dark vertical streaks in panel (D).

[0029] A low-coherence line illumination can produce low speckle noise. FIG. 4 illustrates example configurations for producing a low-coherence line illumination in accordance with some embodiments. In panel (A) light from an extended light source is coupled to a narrow slit. The light source can be, for example, one or more LEDs, arc lamps, or other light sources with a low coherence. As such, a laser light source is not used, which can further reduce the cost of the device. A focusing module can be added to increase the coupling efficiency, as shown in panel (B). A non-imaging waveguide, such as a multimode fiber, can be used to deliver the light to the slit, as shown in panel (C). The low-coherent line illumination can also be generated by a pixelated display, such as organic LED (OLED) and liquid crystal display (LCD), as shown panel (D).

[0030] FIG. 5 illustrates an sLSM configuration in accordance with another example embodiment. In the configuration of FIG. 5, an autofocusing module, such as a voice coil motor or a linear motorized stage, is used to adjust the focus of the objective lens (on detection side). The illumination light sheet plane should coincide with the detection plane. Errors in aligning the optics or the tissue refractive index variation can make the illumination light sheet and detection plane out of alignment. In order to compensate for this misalignment, the autofocusing module can be used to match the detection focal plane and the illumination light sheet. The autofocusing can be based on the cellular morphologic details, with the goal of increasing the sharpness of cellular features. For example, one or more images of the sample can be taken and analyzed to determine whether a desired level of image sharpness is achieved. If image does not have the desired sharpness, the autofocusing mechanism can move the objective to obtain the desired sharpness. This can be an iterative process, in which a processor coupled to the imaging sensorcan analyze the image(s) and provide the feedback to the voice coils or linear motorized stage to move the objective lens. The remaining elements of FIG. 5 provide similar functionalities, as explained earlier in this document.

[0031] FIG. 6A illustrates an sLSM configuration in accordance with another example embodiment. In the configuration of FIG. 6A, a phase plate is placed between the detection objective lens and tube lens to provide an extended depth of focus for the detection optics. When the illumination light sheet plane and the detection focal plane are tilted or when the two planes do not coincide with each other due to alignment error or variability of the tissue refractive index, the extended depth of focus maintains a high lateral resolution of the detection optics at the tissue location illuminated by the light sheet. This is further illustrated in the bottom two panels of FIG. 6A, where the PSF of the configurations with and without the phase plate are compared. As illustrated, the elongated PSF due to the inclusion of the phase plate extends the depth of focus, which can tolerate a larger tilt or misalignment between the detection plane and the illumination light sheet. In one example, the phase plate is configured to produce 3X elongation of the PSF along the axial direction. Elongation of the PSF is often accompanied by the undesirable effect of producing side lobes in the PSF. In one example, the phase plate is configured to produce a side lobe that is 10% or less of the main lobe. The remaining elements of FIG. 6A provide similar functionalities, as explained earlier in this document.

[0032] FIG. 6B shows an example of a phase plate that can elongate the PSF. Panel (A) shows the schematic of an example plate that is made of a transparent resin with the refractive index of 1.5. The phase plate has two different heights with the height difference of 405 nm. When the phase plate is located at the back focal plane of an objective lens with the focal length of 9.26 mm, the focused PSF on the front focal plane on the tissue is elongated along the axial direction (see panel (B)). In this particular example, the depth of focus (defined by the axial range that produces over 0.8 for the center intensity) is elongated from 2.8 pm to 11.9 pm when the wavelength of 405 nm is used, as illustrated in panel (C).

[0033] FIG. 7 illustrates an sLSM configuration in accordance with another example embodiment. A freeform optical element (e.g., a freeform prism in the FIG. 7) is used between the detection objective lens and the tube lens. In the configuration of FIG. 7, the immersion medium is not used, which can simplify the implementation, but at the same time, it can introduce aberrations (e.g., astigmatism) in the system. The freeform prism can be configured tocompensate for the aberration introduced by the index mismatch between the tissue and air when index-matching gel is difficult to apply and therefore is not used. While the configuration of FIG. 7 shows a freeform prism, it is understood that the freeform optical component does not need to be shaped as a prism, and other shapes can be constructed to compensate for the aberration. The remaining elements of FIG. 7 provide similar functionalities, as explained earlier in this document.

[0034] FIG. 8 illustrates an sLSM configuration in accordance with another example embodiment. In the configuration of FIG. 8, a mirror and an imaging window are used before the illumination objective to fold the illumination light path and make a more compact device structure. The imaging window provides an interface to the immersion medium (since the illumination side objective is now moved to the interior of the microscope). Small lenses (e.g., having a diameter of 10 mm or smaller) can be used to further reduce the form factor of the system to a scale that is applicable for handheld operation (e g., having an outer diameter of less than 35 mm). The remaining elements of FIG. 8 provide similar functionalities, as explained earlier in this document.

[0035] FIG. 9 illustrates an sLSM configuration in accordance with another example embodiment. In the configuration of FIG. 9, a mirror, an imaging window, and a phase plate are used to provide a compact form factor and provide extended depth of focus to compensate for the alignment error. The remaining elements provide similar functionalities, as explained earlier in this document.

[0036] FIG. 10 illustrates an sLSM configuration in accordance with another example embodiment. In the configuration of FIG. 10, a mirror and a freeform prism are used to provide compact form factor and aberration compensation when index matching gel is not used. The remaining elements of FIG. 10 provide similar functionalities, as explained earlier in this document.

[0037] FIG. 11 illustrates a sLSM configuration in accordance with another example embodiment. In the configuration of FIG. 11, the microscope is implemented as part of an endoscope to image luminal organs. The extended light source outside the endoscope is coupled by an optical fiber and directed to a narrow slit inside the endoscope. Illumination light sheet is generated by miniature lenses and aperture and the light path is folded by the mirror to form a compact structure. The system is packaged inside an index-matching imaging window, whichdirectly touches the tissue. Index matching epoxy is used to provide solid and nonflowing indexmatching medium at the tissue interface. For example, the refractive index of the index matching material can be between 1.34 to 1.4. The scattered light is captured by an objective lens and focused onto an imaging sensor. Another mirror (mirror 2) is used to fold the detection light path to form a compact structure. The remaining elements of FIG. 11 provide similar functionalities, as explained earlier in this document.

[0038] FIG. 12 illustrates an sLSM configuration in accordance with another example embodiment. In the configuration of FIG. 12, large-volume, three-dimensional imaging of the luminal organ can be achieved by longitudinal scans and rotational scans. The endoscope images three-dimensional volume when the endoscope is pulled back by the longitudinal scanning mechanism. For example, after one longitudinal scan, the endoscope is rotated to another angle and another longitudinal scan is conducted. The combination of longitudinal and rotational scans enables the imaging of a large volume of the luminal organ, such as anal canal. The remaining elements of FIG. 12 provide similar functionalities, as explained earlier in this document.

[0039] FIG. 13 illustrates an sLSM configuration in accordance with another example embodiment. In the configuration of FIG. 13, a phase plate is used to enable extended depth of focus that compensates for alignment errors, as explained in connection with FIG. 5. The remaining elements of FIG. 13 provide similar functionalities, as explained earlier in this document. It should be noted that similar to the description in connection with FIG. 7, in the endoscope configurations, an optical element (e.g., a prism) can also be included for aberration compensation, which allows the index-matching material to be removed. In some embodiments, the endoscope configurations can have an autofocus mechanism, similar to the description in connection with FIG. 5.

[0040] In the disclosed embodiments, scattering contrast is used to visualize cellular details without the need of fluorescence labeling. The use of an objective lens with a moderate numerical aperture (NA) (<0.4) can generate high resolution and large field of view and reduce the cost and complexity of the microscopy optics. The use of incoherent line light source and rectangular aperture can generate light sheet illumination for optical sectioning and provide high image quality by reducing speckle noise and shadow artifacts.

[0041] The disclosed technology enables design and implementation of low-cost, small formfactor light sheet microscopy devices for diagnosing disease of human organs in vivo and freshlyexcised specimens ex vivo, including, but not limited to, anus, esophagus, stomach, duodenum, and colon. The microscope device can be used in a wide range of clinical settings (including primary-care clinics, surgical suites, and under-resourced remote hospitals without pathology services), as well as for training and educational purposes (e.g., real-time microscopic feedback for technically challenging procedures).

[0042] One aspect of the disclosed technology relates to a scattering-based light sheet microscopy device that includes a spatially incoherent light source positioned to provide illumination to a collimation lens, an aperture positioned to receive collimated light from the collimation lens and to produce a line illumination beam, the aperture having an opening with a rectangular cross-section, wherein a length of the rectangular cross-section is at least twice a width of the rectangular cross-section, wherein a ratio of numerical apertures in two different orthogonal directions associated with the aperture is at least 1 to 2. The scattering-based light sheet microscopy device also includes a first objective lens positioned to receive the line illumination beam to illuminate an area of a sample with focused light, and a second objective lens positioned to receive light from the area of the sample in response to illumination by the focused light, and to direct the received light for detection by an imaging sensor.

[0043] In one example embodiment, the spatially incoherent light source comprises one or more of a light emitting diode or an arc lamp. In particular, the spatially incoherent light source excludes any laser light sources. In another example embodiment, the scattering-based light sheet microscopy device includes an index-matching material positioned at an interface of the first or second objective lenses for contacting the sample. In still another example embodiment, the spatially incoherent light source is part of a light module that includes one of (a) an extended light source and a slit, (b) an extended light source, a focusing module and a slit, (c) an extended light source, a non-imaging waveguide and a slit, or (d) a pixelated array configurable to produce a line illumination via a selected row or a selected column of the pixelated array.

[0044] According to another example embodiment, the scattering-based light sheet microscopy device includes an autofocus mechanism coupled to the second objective lens to move the second objective lens in a longitudinal direction in response to a signal indicative that one or more images obtained by the imaging sensor is not in focus. In yet another example embodiment, the scattering-based light sheet microscopy device includes a processor and a memory including instructions stored thereon, wherein the processor is coupled to the autofocus mechanism and tothe imagine sensor, wherein the instructions upon execution by the processor cause the processor to receive information corresponding to the one or more images obtained by the imagining sensor, to determine whether or not the one or more obtained images conform to a predetermined sharpness criterion, and to provide the signal to the autofocus mechanism to trigger a movement of the second objective lens. In some example embodiments, the autofocusing mechanism includes a voice coil motor or a linear motorized stage.

[0045] In one example embodiment, the scattering-based light sheet microscopy device includes a tube lens for receiving light from the second objective lens and to direct the light in a direction of the imaging sensor, and a phase plate positioned between the second objective lens and the tube lens to increase a depth of focus of the scattering-based light sheet microscopy device. In another example embodiment, the phase plate is configured to extend a point spread function of the scattering-based light sheet microscopy device by a factor of about 3X. In still another example embodiment, the scattering-based light sheet microscopy device includes a tube lens for receiving light from the second objective lens and to direct the light in a direction of the imaging sensor, and a freeform optical element positioned between the second objective lens and the tube lens, wherein the freeform optical element is configured to compensate for at least a portion of aberrations present in the scattering-based light sheet microscopy device. In one example embodiment, the scattering-based light sheet microscopy device excludes any phase matching material at an interface between the first or the second objective lens for contacting the sample. In another example embodiment, the freeform optical element is a freeform prism.

[0046] In yet another example embodiment, the scattering-based light sheet microscopy device includes a folded illumination light path and comprises a mirror positioned after the first objective lens to receive light from the first objective lens and direct the received light to a window for illumination of the sample, wherein the window provides an interface to an index-matching material positioned to contact the sample. In another example embodiment, the scattering-based light sheet microscopy device includes the index-matching material that is positioned to contact the sample. In still another example embodiment, the scattering-based light sheet microscopy device: (a) includes a folded illumination light path and comprises a mirror positioned after the first objective lens to receive light from the first objective lens and to direct the received light in the direction of the sample, (b) excludes any index-matching material at an interface between the scattering-based light sheet microscopy device and the sample, (c) includes a tube lens forreceiving light from the second objective lens and to direct the light in a direction of the imaging sensor, and (d) includes a freeform optical element positioned between the second objective lens and the tube lens to compensate for at least a portion of aberrations present in the scattering-based light sheet microscopy device.

[0047] According to another example embodiment, the scattering-based light sheet microscopy device is at least partially incorporated into an endoscopic imaging device, and the scattering-based light sheet microscopy device includes: a folded illumination light path and comprises a first mirror positioned between the aperture and the first objective lens, and a folded detection optical path and comprises a second mirror positioned to receive light from the second objective lens and to direct the light received thereon in the direction of the imaging sensor. In one example embodiment, the spatially incoherent light source is positioned outside of a distal end of the endoscopic imaging device and is coupled to the distal end by an optical fiber that delivers light from the spatially incoherent light source to a slit that is housed within the distal end of the endoscopic imaging device. In yet other example embodiments, an index matching material is positioned at an interface associated with the first or the second objective lens and a transparent section of the distal end of the endoscopic imaging device that is configured to contact the sample, and a phase plate is positioned between the second objective lens and the second mirror to increase a depth of focus of the scattering-based light sheet microscopy device.

[0048] In some example embodiments, a freeform optical element is positioned between the second objective lens and the second mirror, wherein the freeform optical element is configured to compensate for at least a portion of aberrations present in the scattering-based light sheet microscopy device. In another example embodiment, the scattering-based light sheet microscopy device includes a longitudinal scanning mechanism and a rotational scanning mechanism coupled to the distal end of the endoscopic imaging device to allow the distal end to be moved in a longitudinal and a rotational direction, respectively. In still another example embodiment, the scattering-based light sheet microscopy device has a numerical aperture of less than 0.1 and a depth of focus of greater than 150 pm.

[0049] Another aspect of the disclosed embodiments relates to a scattering-based light sheet microscopy device that includes a rectangular aperture positioned to receive a line illumination beam, wherein the line illumination beam is collimated and is spatially incoherent, and wherein a ratio of numerical apertures in two different orthogonal directions associated with the rectangularaperture is at least 1 to 3. The scattering-based light sheet microscopy device also includes a first objective lens positioned to receive the line illumination beam after passing through the rectangular aperture and to direct the light receive thereon in a direction of an area of a sample, a second objective lens positioned to receive light from the area of the sample in response to illumination of the sample, and a tube lens positioned to receive light from the second objective lens and to direct the light received thereon in a direction of the imaging sensor.

[0050] It is understood that the various disclosed embodiments may be implemented individually, or collectively, in devices comprised of various components, including electronics hardware and / or software modules and optical components. These devices, for example, may comprise or use a processor, a memory unit, an interface that are communicatively connected to each other, and may range from desktop and / or laptop computers, to mobile devices and the like. The processor and / or controller can perform various disclosed operations based on execution of program code that is stored on a storage medium. The processor and / or controller can, for example, be in communication with at least one memory and with at least one communication unit that enables the exchange of data and information, directly or indirectly, through the communication link with other entities, devices and networks. The communication unit may provide wired and / or wireless communication capabilities in accordance with one or more communication protocols, and therefore it may comprise the proper transmitter / receiver antennas, circuitry and ports, as well as the encoding / decoding capabilities that may be necessary for proper transmission and / or reception of data and other information.

[0051] Various information and data processing operations described herein may be implemented in one embodiment by a computer program product, embodied in a computer- readable medium, including computer-executable instructions, such as program code, executed by computers in networked environments. A computer-readable medium may include removable and non-removable storage devices including, but not limited to, Read Only Memory (ROM), Random Access Memory (RAM), compact discs (CDs), digital versatile discs (DVD), etc. Therefore, the computer-readable media that is described in the present application comprises non-transitory storage media. Generally, program modules may include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Computer-executable instructions, associated data structures, and program modules represent examples of program code for executing steps of the methods disclosedherein. The particular sequence of such executable instructions or associated data structures represents examples of corresponding acts for implementing the functions described in such steps or processes.

[0052] Only a few implementations and examples are described and other implementations, enhancements and variations can be made based on what is described and illustrated in this patent document.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A scattering-based light sheet microscopy device, comprising: a spatially incoherent light source positioned to provide illumination to a collimation lens; an aperture positioned to receive collimated light from the collimation lens and to produce a line illumination beam, the aperture having an opening with a rectangular crosssection, wherein a length of the rectangular cross-section is at least twice a width of the rectangular cross- section, wherein a ratio of numerical apertures in two different orthogonal directions associated with the aperture is at least 1 to 2; a first objective lens positioned to receive the line illumination beam to illuminate an area of a sample with focused light; and a second objective lens positioned to receive light from the area of the sample in response to illumination by the focused light, and to direct the received light for detection by an imaging sensor.

2. The scattering-based light sheet microscopy device of claim 1, wherein the spatially incoherent light source comprises one or more of a light emitting diode or an arc lamp.

3. The scattering-based light sheet microscopy device of claim 1, wherein the spatially incoherent light source excludes any laser light sources.

4. The scattering-based light sheet microscopy device of claim 1, including an indexmatching material positioned at an interface of the first or second objective lenses for contacting the sample.

5. The scattering-based light sheet microscopy device of claim 1, wherein the spatially incoherent light source is part of a light module that includes one of:(a) an extended light source and a slit,(b) an extended light source, a focusing module and a slit,(c) an extended light source, a non-imaging waveguide and a slit, or(d) a pixelated array configurable to produce a line illumination via a selected row or a selected column of the pixelated array.

6. The scattering-based light sheet microscopy device of claim 1, comprising: an autofocus mechanism coupled to the second objective lens to move the second objective lens in a longitudinal direction in response to a signal indicative that one or more images obtained by the imaging sensor is not in focus.

7. The scattering-based light sheet microscopy device of claim 6, comprising: a processor and a memory including instructions stored thereon, wherein the processor is coupled to the autofocus mechanism and to the imagine sensor, wherein the instructions upon execution by the processor cause the processor to: receive information corresponding to the one or more images obtained by the imagining sensor, determine whether or not the one or more obtained images conform to a predetermined sharpness criterion, and provide the signal to the autofocus mechanism to trigger a movement of the second objective lens.

8. The scattering-based light sheet microscopy device of claim 6 or 7, wherein the autofocusing mechanism includes a voice coil motor or a linear motorized stage.

9. The scattering-based light sheet microscopy device of claim 1, comprising: a tube lens for receiving light from the second objective lens and to direct the light in a direction of the imaging sensor, and a phase plate positioned between the second objective lens and the tube lens to increase a depth of focus of the scattering-based light sheet microscopy device.

10. The scattering-based light sheet microscopy device of claim 9, wherein the phase plate is configured to extend a point spread function of the scattering-based light sheet microscopy device by a factor of about 3X.

11. The scattering-based light sheet microscopy device of claim 1, comprising: a tube lens for receiving light from the second objective lens and to direct the light in a direction of the imaging sensor, and a freeform optical element positioned between the second objective lens and the tube lens, wherein the freeform optical element is configured to compensate for at least a portion of aberrations present in the scattering-based light sheet microscopy device.

12. The scattering-based light sheet microscopy device of claim 11, wherein the scatteringbased light sheet microscopy device excludes any phase matching material at an interface between the first or the second objective lens for contacting the sample.

13. The scattering-based light sheet microscopy device of claim 11, wherein the freeform optical element is a freeform prism.

14. The scattering-based light sheet microscopy device of claim 1, wherein the scatteringbased light sheet microscopy device includes a folded illumination light path and comprises a mirror positioned after the first objective lens to receive light from the first objective lens and direct the received light to a window for illumination of the sample, and wherein the window provides an interface to an index-matching material positioned to contact the sample.

15. The scattering-based light sheet microscopy device of claim 14, including the indexmatching material positioned to contact the sample.

16. The scattering-based light sheet microscopy device of claim 1, wherein the scatteringbased light sheet microscopy device:includes a folded illumination light path and comprises a mirror positioned after the first objective lens to receive light from the first objective lens and to direct the received light in the direction of the sample, excludes any index-matching material at an interface between the scattering-based light sheet microscopy device and the sample, includes a tube lens for receiving light from the second objective lens and to direct the light in a direction of the imaging sensor, and includes a freeform optical element positioned between the second objective lens and the tube lens to compensate for at least a portion of aberrations present in the scattering-based light sheet microscopy device.

17. The scattering-based light sheet microscopy device of claim 1, wherein: the scattering-based light sheet microscopy device is at least partially incorporated into an endoscopic imaging device, the scattering-based light sheet microscopy device includes: a folded illumination light path and comprises a first mirror positioned between the aperture and the first objective lens, and a folded detection optical path and comprises a second mirror positioned to receive light from the second objective lens and to direct the light received thereon in the direction of the imaging sensor.

18. The scattering-based light sheet microscopy device of claim 17, wherein: the spatially incoherent light source is positioned outside of a distal end of the endoscopic imaging device and is coupled to the distal end by an optical fiber that delivers light from the spatially incoherent light source to a slit that is housed within the distal end of the endoscopic imaging device.

19. The scattering-based light sheet microscopy device of claims 17 or 18, comprising: an index matching material positioned at an interface associated with the first or the second objective lens and a transparent section of the distal end of the endoscopic imaging device that is configured to contact the sample, anda phase plate positioned between the second objective lens and the second mirror to increase a depth of focus of the scattering-based light sheet microscopy device.

20. The scattering-based light sheet microscopy device of claims 17 or 18, comprising: a freeform optical element positioned between the second objective lens and the second mirror, wherein the freeform optical element is configured to compensate for at least a portion of aberrations present in the scattering-based light sheet microscopy device.

21. The scattering-based light sheet microscopy device of claims 17 or 18, comprising: a longitudinal scanning mechanism and a rotational scanning mechanism coupled to the distal end of the endoscopic imaging device to allow the distal end to be moved in a longitudinal and a rotational direction, respectively.

22. The scattering-based light sheet microscopy device of claim 1 , wherein the scatteringbased light sheet microscopy device has a numerical aperture of less than 0.1 and a depth of focus of greater than 150 pm.

23. The scattering-based light sheet microscopy device, comprising: a rectangular aperture positioned to receive a line illumination beam, wherein the line illumination beam is collimated and is spatially incoherent, and wherein a ratio of numerical apertures in two different orthogonal directions associated with the rectangular aperture is at least 1 to 3; a first objective lens positioned to receive the line illumination beam after passing through the rectangular aperture and to direct the light receive thereon in a direction of an area of a sample; a second objective lens positioned to receive light from the area of the sample in response to illumination of the sample; and a tube lens positioned to receive light from the second objective lens and to direct the light received thereon in a direction of the imaging sensor.

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