Multifocal plane scanning using time-delay integrated imaging

Abstract

An imaging system for capturing spatial images of a biological tissue sample may include an imaging chamber configured to hold the biological tissue sample placed within the imaging system; a light source configured to illuminate the biological tissue sample to activate one or more fluorophores in the biological tissue sample; a time delay and integration (TDI) imager having multiple partitions, the multiple partitions may be configured to simultaneously capture images at multiple different depths within the biological tissue sample during a single scan by the TDI imager; and a controller configured to cause the TDI imager to scan the biological tissue sample.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 395,258, entitled "MULTI-FOCAL-PLANE SCANNING USING TIME DELAY INTEGRATION IMAGING," filed August 4, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes as if fully set forth herein.

[0002] This disclosure generally describes capturing multiplexed spatial images of a biological tissue sample. More specifically, this disclosure describes a camera configuration that captures multiple focal planes during a single scan. [Background technology]

[0003] Spatial biology is the study of cellular and subcellular environments across multiple dimensions. Spatial biology tools can be used to determine which cells are present in a tissue sample, where those cells are located within the tissue sample, their biomarker co-expression patterns, and how those cells are organized and interacting within the tissue sample. To visualize and quantify biomarker expression, specimen slides containing the tissue sample can be prepared in a variety of imaging workflows that can be performed to generate comprehensive images of cellular and subcellular organization, yielding single-cell resolution. The resulting images can reveal how cells interact and organize within the tissue sample.

[0004] Capturing these complex images of the cellular environment is sometimes referred to as spatial omics. Highly multiplexed, high-resolution spatial omics is rapidly becoming an essential tool in understanding disease and other biological conditions. This type of analysis typically involves hundreds of complex factors, variables, and processes. Integrated solutions may combine imaging and process control methods into a single machine for performing spatial omics. However, generating a complete spatial image of a tissue sample that accurately represents the sample's volume requires multiple individual imaging scans of the sample. This large number of scans required for a complete imaging analysis severely limits the system's throughput. Therefore, improvements in the art are needed. Summary of the Invention

[0005] In some embodiments, an imaging system for capturing a spatial image of a biological tissue sample may include an imaging chamber configured to hold the biological tissue sample placed within the imaging system; a light source configured to illuminate the biological tissue sample to activate one or more fluorophores in the biological tissue sample; a time delay and integration (TDI) imager having multiple partitions, wherein the multiple partitions may be configured to simultaneously capture images at multiple different depths within the biological tissue sample during a single scan by the TDI imager; and a controller configured to cause the TDI imager to scan the biological tissue sample.

[0006] In some embodiments, a method of capturing a spatial image of a biological tissue sample may include mounting the biological tissue sample in an imaging chamber of an imaging system; directing light from a light source to illuminate an area on the biological tissue sample to activate one or more fluorophores in the biological tissue sample; and scanning the biological tissue sample with a time delay and integration (TDI) imager comprising multiple partitions, wherein the multiple partitions may be configured to simultaneously capture images at multiple different depths within the biological tissue sample during the scan.

[0007] In some embodiments, the imaging system may include a time delay and integration (TDI) imager having multiple partitions, where the multiple partitions may be configured to simultaneously capture images at multiple different depths within a volume during a single scan by the TDI imager.

[0008] Any of the embodiments may implement one or more of the following features in any combination, without limitation: The TDI imager may be tilted at an angle relative to the biological tissue sample such that focal planes of the multiple partitions correspond to multiple different depths within the biological tissue sample. The multiple partitions on the TDI imager may be physically separated by spaces between the multiple partitions. The multiple partitions on the TDI imager may be separated by rows of covered pixels. The multiple different depths within the biological tissue sample may include multiple different depth ranges within the biological tissue sample. One of the multiple partitions on the TDI imager may correspond to one of multiple different depth ranges, the partition including multiple pixel rows, each corresponding to a different depth within the depth range. Data received from multiple pixel rows may be combined using focus-drilling combination to generate an image for the depth range. Data received from multiple pixel rows may be combined using focus-drilling combination to generate an image for the depth range. The depth range may be between about 250 nm and about 750 nm. The thickness of the biological tissue sample may be between about 2 μm and about 10 μm. Images of the biological tissue sample may be generated from each of the multiple partitions. The TDI imager may be tilted at an angle relative to the volume so that the focal planes of the multiple partitions correspond to multiple different depths within the volume, and the angle may be adjustable to fine-tune the focal planes to the multiple different depths within the volume. The system may include a glass cover over the TDI, the glass cover may include multiple sections corresponding to the multiple partitions, and depending on the thickness of the multiple sections of the glass cover, the focal planes of the multiple partitions may be located at multiple different depths within the volume. The system may include a lens in front of the TDI, the lens may include multiple sections corresponding to the multiple partitions, and depending on the thickness of the multiple sections of the lens, the focal planes of the multiple partitions may be located at multiple different depths within the volume.The partitions of the TDI imager may have different heights relative to one another, and the different heights may cause focal planes of the partitions to be located at different depths within the volume. The volume may include a biological tissue sample. The system may include a lens in front of the TDI, and the lens may include a wedge shape, and the wedge shape may cause focal planes of the partitions to be located at different depths within the volume.

[0009] An additional understanding of the nature and advantages of various embodiments may be realized by reference to the remaining portions of the specification and the drawings, in which like reference numerals are used to refer to like components throughout the several views. In some instances, a sub-label is associated with a reference numeral to indicate one of multiple similar components. When a reference numeral is referred to without specifying an existing sub-label, it is intended to refer to all of such multiple similar components. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 illustrates a high-resolution biometric imaging system according to some embodiments. [Figure 2] 1 is a flow diagram of a process for capturing an aerial image of a sample, according to some embodiments. [Figure 3] FIG. 1 illustrates how an imaging system captures multiple images illuminated by different wavelengths, according to some embodiments. [Figure 4] FIG. 1 illustrates a TDI camera that may be used in an imaging system, according to some embodiments. [Figure 5] FIG. 1 illustrates a TDI imager comprising a row of imaging pixels divided into multiple partitions, according to some embodiments. [Figure 6] 1A-1C illustrate configurations for a TDI imager to simultaneously capture multiple image slices of a tissue sample during a single scan, according to some embodiments. [Figure 7] 1A-1C illustrate a close-up view of a single partition and corresponding focal plane within an image slice, according to some embodiments. [Figure 8] FIG. 1 illustrates a TDI imager with a stepped profile, according to some embodiments. [Figure 9] 10A-10C illustrate how a TDI imager can be configured to capture image slices at multiple depths using a cover or lens with sections of different thicknesses, according to some embodiments. [Figure 10] 1A-1C illustrate a lens or glass cover having a wedge shape, according to some embodiments. [Figure 11] FIG. 1 illustrates a flow diagram of a method for capturing an aerial image of a biological tissue sample, according to some embodiments. [Figure 12] FIG. 1 illustrates an exemplary computer system upon which various embodiments may be implemented. DETAILED DESCRIPTION OF THE INVENTION

[0011] FIG. 1 illustrates a high-resolution biological imaging system 100 according to some embodiments. The imaging system 100 may be configured to combine multiple imaging workflows into a single process to perform automated spatial analysis of tissue samples. The imaging system 100 may include multiple imaging chambers 108, 110, each configured to perform an individual imaging operation on a different tissue sample. The fluidic system 102 may provide integrated fluidic control for supplying multiple fluorophores and / or other fluids to the imaging chambers 108, 110 during the imaging process. Different fluorophores and reagents may be loaded into reservoirs of the fluidic system 102 so that these fluids can be automatically supplied to the imaging chambers 108, 110 as needed during the imaging process. In the context of the present disclosure, the fluorophores may be attached to one or more binding reagents that specifically interact with one or more analytes in the tissue sample. Exemplary binding reagents include nucleic acid probes, proteins (such as antibodies and antibody derivatives), and aptamers. Thus, when fluorophores are referred to in this disclosure, it should be understood that those fluorophores may be present as components of one or more binding reagents or may be attached to one or more binding reagents.

[0012] The imaging system 100 may include a computer system including one or more processors, one or more memory devices, and instructions stored on the one or more memory devices that cause the imaging system 100 to perform imaging operations on tissue samples in the imaging chambers 108, 110. Thus, each of the operations of the imaging processes described herein may be represented by instructions stored on one or more memory devices.

[0013] In an exemplary imaging workflow, a user or an automated process may load a tissue sample onto a slide and load the slide into the imaging chamber 108. Fluids may then be automatically pumped into the imaging chamber 108 after the tissue sample in the imaging chamber 108 is fixed. For example, fluids may be pumped into the imaging chamber 108 to wash the tissue and / or remove previous fluids or fluorophores that may be present in the imaging chamber 108. New fluids or fluorophores may be automatically supplied from the fluidics system 102 as specified by instructions executed by the controller. Generally, these “fluids” may more specifically include stains, probes, and other biological labels. During a typical cycle, one or more fluorophores configured to attach to cells in the tissue sample to visually highlight different features in the sample may be pumped into the imaging chamber 108. The sample in the imaging chamber 108 may then be illuminated using a corresponding laser wavelength to excite the fluorophores, and a camera may capture an image of the illuminated sample. The fluorophores may be matched with different laser wavelengths configured to illuminate that particular fluorophore.

[0014] After the imaging process is complete, the controller may convert the raw images from the system into RNA or protein spots. These RNA or protein spots may be visualized as cell type clusters highlighted by different fluorophores. Multiple images may then be merged for multi-omic analysis of the tissue sample. Software tools provided by the controller of the imaging system 100 may provide different visualization, data filtering, and analysis tools for examining the images.

[0015] Although imaging system 100 is described herein as a fully integrated solution combining control processing, image capture, and fluidics into a single, integrated system, other embodiments may use a distributed system to some degree. Separating portions of an integrated system into distributed subsystems may be advantageous when using techniques described below to increase imaging speed. For example, fluid operations and imaging operations need not be integrated into a single, integrated tool. Multiple fluid chambers may be connected to a single, standalone imaging tool using a robot or human to transfer materials back and forth between the multiple fluid chambers and the single, standalone imaging tool. Therefore, the term “imaging system” should be broadly interpreted to encompass both fully integrated and distributed systems.

[0016] FIG. 2 illustrates a process flow diagram 200 for capturing a multi-omic image of a sample, according to some embodiments. As described above, this process may include loading a tissue sample onto a substrate, such as a coverslip or slide, and securing the tissue sample inside one of the imaging chambers of the imaging system (202). Note that multiple stations within the image processing system 100 may operate independently and simultaneously. For example, the imaging chamber 108 may capture an image of the sample while another station exchanges fluid with the tissue sample. Some embodiments may also include a photobleaching station. The imaging system 100 may then supply fluids from the fluidics system 102 into the imaging chamber 108 (204). These fluids may contain fluorophores configured to attach to specific cell or tissue types to be highlighted in the resulting image.

[0017] To capture an image with a high enough resolution to visualize individual cells in detail, images of the sample may be captured in stages. For example, instead of capturing a single image of the sample, the field of view of the camera of the imaging system 100 may be reduced to increase resolution. Multiple images of the sample may then be captured and stitched together to form the overall image. For example, the overall image 250 may consist of multiple sub-images, each of which may be captured by the camera at high resolution. Each of the images may correspond to one field of view of the image. Thus, the process may include sequentially capturing field images using the camera (206) and then moving the camera field of view to subsequent positions with adjacent fields of view to prepare the camera for the subsequent step (208). At each field of view position, the process may repeat (207) by capturing images at different focal planes and / or using different wavelengths of light, thereby capturing multiple images at each position. This process may be repeated until the individual field of view images capture the overall image 250 of the sample.

[0018] To capture the overall image 250, the camera's field of view may be moved in a pattern over the tissue sample. For example, a first field of view 252 may be captured (206), and then the camera may move (208) to a second field of view 254, which may be sequential and / or adjacent to the first field of view 252, along a grid pattern. This process may be repeated for each field of view within the sample until the overall image 250 is captured. Note that the grid pattern shown in FIG. 2 is by way of example only and is not intended to be limiting. Other embodiments may move horizontally, vertically, diagonally, and / or along any other pattern that may be used to capture individual field images that may be combined into the overall image 250. In some embodiments, the individual fields of view may overlap, while in other embodiments, the individual fields of view may not overlap.

[0019] Multiple global images 250 of a tissue sample may be captured to highlight different features in the tissue sample for global multi-omic analysis. Thus, after capturing a global image 250 for a particular fluorophore or set of fluorophores, the process may be repeated for the same tissue sample containing a different fluorophore or set of fluorophores. For example, the previous fluorophores may be pumped out of the imaging chamber 108, a wash or rinse agent may be pumped through the imaging chamber 108 to clean the tissue sample, and a new set of fluorophores may be pumped into the imaging chamber 108 for the next image (204). Each global image captured using different fluorophores combined in a multi-omic analysis may be referred to as an "imaging cycle" or "hyb," where "hyb" is short for "hybridization" in "fluorophore labeling hybridization cycle." Typically, each sample may undergo multiple hybs using different fluorophores. For example, some embodiments may capture two, three, four, five, or six or more full images of the sample (corresponding to the number of unique fluorophores), thereby repeating the cycle (204) multiple times. When the desired number of images of the sample have been captured, the sample may be removed (210) from the imaging chamber 108. A new sample may then be added (202) to the imaging chamber 108, and the imaging process may be repeated.

[0020] At each field of view image location, the sample may be illuminated with different wavelengths of light (e.g., different colors configured to illuminate different fluorophores in the sample), and therefore, multiple images may be captured at each location at different wavelengths. Additionally, the sample itself may be adjusted axially to capture multiple images at different Z-depth levels, resulting in a three-dimensional image slice of the tissue sample. As used herein, the term Z-depth may refer to the distance along the focal line of the camera, and in some instances may also be perpendicular to the surface of the tissue sample. The tissue sample under analysis is a three-dimensional volume at different Z-depths within a layer of cells (i.e., different distances from the camera or lens within the volume of the tissue sample). Thus, to capture a three-dimensional representation of the tissue sample, the imaging system 100 may capture complete images at different Z-depths by adjusting the focal length of the camera. For example, some embodiments may slice a volume of a tissue sample at 0.5 μm intervals (i.e., taking images at or near -1.0 μm, -0.5 μm, 0.0 μm, 0.5 μm, and 1.0 μm along the Z axis). This range may represent, for example, all slices within a single layer of cells, which may be approximately 10 μm to approximately 30 μm thick. While this process provides high-resolution, multi-omic image data, it can also be quite time-consuming. For example, 28 scans of the tissue sample may be used to capture images at seven different Z depths containing four different fluorophores. Each movement from one field of view to the next involves significant overhead, increasing the time required to capture each image. The process may involve moving the sample laterally so that the camera captures a new field of view, which may take time to acquire a new image, physically moving the sample using piezo motors or stage motors, configuring the laser and corresponding filter for the desired wavelength, stabilizing the camera, focusing the camera, and / or other operations.Combining these different factors together results in relatively long overall imaging times. For example, in an exemplary embodiment using a camera with a 40x objective and a 30x30 grid of field images to cover a sample, each hyb can take approximately 10 hours to complete. A typical 4-hyb session can take a total of 30-40 hours to complete. While lowering the camera resolution increases the field of view and reduces the total number of field images required, this also negatively impacts the quality of the resulting images. This significant time requirement presents a technical challenge in the field of biospatial omics.

[0021] Some embodiments may reduce the overhead of moving the imaging camera's complete field of view and instead use a time-delay integration (TDI) camera. Rather than moving between different fields of view, the TDI camera may be used to continuously scan the tissue sample along a row. The laser beam projected onto the imaging sample may be shaped to approximately coincide with the TDI image scan line. Switching to a TDI camera may improve many of the error sources and overhead challenges listed above. TDI scanning allows for continuous scan image collection that averages out many non-uniformities in the scan direction. This reduces the system's susceptibility to many different error sources, including illumination non-uniformities, pixel-to-pixel non-uniformities (and imperfections) in the image sensor, and / or lens aberrations. TDI scans may be stitched two-sided instead of four-sided, and scanning under constant acceleration may reduce acceleration force ripple that causes vibration of the tissue sample. Finally, the overhead due to mechanical movements can be significantly reduced, while a system with 100 fields (10 x 10 squares), 4 colors, and 5 foci requires only 195 overhead events [e.g., (9 scans x 4 colors + 3 color changes) x 5 foci].

[0022] FIG. 3 illustrates how imaging system 100 captures multiple images illuminated by different wavelengths, according to some embodiments. In this example, each region of a tissue sample may be illuminated by four different wavelengths. A first laser shot 302 may illuminate a region of tissue sample 304 with a first wavelength 303. Reflected or fluorescent light from first laser shot 302 may be recorded by camera 308 after passing through a first filter 306 configured to pass a first wavelength range 307. Note that first wavelength range 307 may be slightly higher than first wavelength 303 from first laser shot 302, and thus the excitation wavelength of first laser shot 302 may be blocked by first filter 306. For example, a fluorophore may be activated at a given wavelength, and the activated fluorophore may emit light within a wavelength range higher than the activation wavelength. Similarly, a second laser shot 312 may illuminate an area of the tissue sample 304 at a second wavelength 313. Reflected light or fluorescence from the second laser shot 312 may be recorded by the camera 308 after passing through a second filter 316 configured to pass a second wavelength range 317. A third laser shot 322 may illuminate an area of the tissue sample 304 at a third wavelength 323. Reflected light or fluorescence from the third laser shot 322 may be recorded by the camera 308 after passing through a third filter 326 configured to pass a third wavelength range 327. A fourth laser shot 532 may illuminate an area of the tissue sample 304 at a fourth wavelength 333. Reflected light or fluorescence from the fourth laser shot 332 may be recorded by the camera 308 after passing through a fourth filter 336 configured to pass a fourth wavelength range 337. The images captured by the camera 308 from each laser shot may be stitched together to form a complete image of the tissue sample, each illuminated by a different wavelength.

[0023] Typically, for each wavelength, a complete set of field images can be captured at each field position before moving to the next position. Time is required between image captures at each wavelength to change filter wheels, allow the filter wheels to stabilize, and move motors to account for wavelength-dependent focal plane shifts. Thus, the total time to image the tissue sample increases with each additional desired wavelength.

[0024] FIG. 4 illustrates a TDI camera 402 that may be used in an imaging system, according to some embodiments. The TDI camera 402 may include a charge-coupled device (CCD) or a CMOS photon-detecting device as an image sensor for capturing an image. For example, the TDI camera 402 may include a scan line 404 of individual CCD pixels in a horizontal configuration as shown in FIG. 4. Note that only a single partition of pixels is shown in this scan line 404 for clarity; however, this is not intended to be limiting. As discussed and shown below, the TDI camera 402 may include multiple horizontal rows of pixels organized into one or more partitions.

[0025] The operation of the TDI camera 402 may be contrasted with the operation of a conventional camera described above. As described above, a conventional camera may capture a single field of view, then move to another non-overlapping field of view before capturing the next image. Returning briefly to FIG. 2 , the field of view 252 may include a horizontal grid of individual pixels within the field of view 252, with each individual pixel simultaneously capturing an image when the camera shot is acquired. In contrast, the TDI camera 402 may use a scan line 404 of individual pixel rows. The TDI camera 402 may sequentially scan vertically across the image. The movement of the tissue sample and / or the TDI camera 402 may be synchronized so that an image is captured at each pixel step. The final horizontal line of pixels in the scan line 404 may accumulate and average the individual pixels to output an average reading for that scan position. The entire image may then be assembled from equally spaced lines throughout the linear field of view of the scan line 404. It should be noted that the terms "horizontal" and "vertical" are used merely to indicate orthogonal directions as shown in FIG. 4, and these terms are not intended to be limiting to any particular orientation.

[0026] The scan line 404 need not extend the entire horizontal length of the image. Instead, multiple vertical "columns" may be captured using multiple vertical successive scans. For example, to capture the entire image 450, the scan line 452 may continuously scan down a first vertical column 462 of the imaging area. When the scan of the first vertical column 462 is completed, the scan line 452 of the TDI camera may be repositioned over a second vertical column 464, and the scan line 452 may then continuously scan down the second vertical column 464. These vertical columns may be stitched together to form the entire image 450 of the tissue sample.

[0027] The use of the TDI camera 402 is a significant technological improvement over other cameras for scanning tissue samples. The TDI camera 402 may capture each vertical scan row continuously, eliminating the need to mechanically reposition the sample, stabilize, focus, and prepare for each individual field of view capture. Instead, the TDI camera 302 may move vertically at a constant speed and continuously scan to accumulate reflected light or fluorescent signals from the tissue sample. The only repositioning that needs to be done to the TDI camera 402 may be between each of the vertical row captures.

[0028] As mentioned above, generating a complete volumetric image of a biological tissue sample typically involves repeated imaging of the sample using different combinations of light sources and color filters, as well as imaging at multiple focal planes within the tissue sample volume. This generates images at multiple focal planes to generate a multi-slice volume image, much like a plenoptic camera, light field camera, or 3D confocal microscope. Ideally, each individual slice should image all of the fluorophores across a depth range within a volume image slice of the sample while avoiding imaging fluorophores in adjacent volume image slices. For example, the thickness of a typical biological tissue sample may be between about 2 μm and about 10 μm. Imaging slices across the volume may be taken at regular intervals, such as every 0.5 μm (e.g., images may be recorded at -1.0 μm, -0.5 μm, 0.0 μm, 0.5 μm, 1.0 μm, etc.). Some embodiments may capture images at specific Z depths within these depth ranges, while other embodiments may capture images representing an average of increasing depths across the depth range in each of these image slices, as will be described in more detail below.

[0029] FIG. 5 illustrates a TDI imager 500 with a row of imaging pixels divided into multiple partitions, according to some embodiments. Conventional TDI imaging would scan the entire biological sample n times to acquire n images at n focal depth locations within n volume slices. For example, when dividing a tissue sample into five volume slices, the TDI imager would complete five complete scans, each using a different focal plane of the TDI imager, and this would be repeated for each fluorophore color. These focal planes would be located near the center of each of the volume image slices. The TDI imager 500 illustrated in FIG. 5 divides a horizontal pixel row into multiple partitions 502. Each horizontal pixel row within each of the multiple partitions 502 may function independently as a separate TDI sub-imager. In some commercial implementations, the TDI imager 500 may include a different color filter placed over each of the partitions 502. For example, some existing TDI imagers may include a set of three-band RGB color-segmented TDI partitions, with each partition configured to capture a different wavelength range. In the example of Figure 5, the TDI imager may include seven partitions 502, each of which may independently capture one field of view of the scanned image.

[0030] Prior to the present disclosure, conventional use of multi-partition TDI imagers, such as the TDI imager 500 shown in FIG. 5, was primarily used to capture different wavelengths of light in a field of view common to all of the partitions 502. In other words, each of the partitions 502 was configured to capture an image at a common focal plane. While the color filters for each partition 502 may be different, the focal plane of the image received at each partition was the same.

[0031] The embodiments described herein may configure the TDI imager 500 to capture images at different focal planes corresponding to different volume depth ranges or image slices within the tissue sample. By independently moving the focal plane of each of the partitions 502 of the TDI imager, each partition 502 may be configured to capture a different image slice within the tissue sample. The TDI imager 500 may then simultaneously scan images at different depth slices within the tissue sample's volume. For example, instead of requiring seven separate complete scans of the tissue sample to acquire images at seven different image slices within the sample's volume, a single scan of the sample may simultaneously capture images at each of the seven image slice depths. This represents a significant improvement in the total time required to image a tissue sample. As mentioned above, previously, capturing images at seven different volume depths using a combination of four different fluorophores required 28 complete scans of the tissue sample. Configuring the partitions 502 of the TDI imager 500 to simultaneously capture all of the image slice depths during a single scan may reduce the total number of scans from 28 to four.

[0032] FIG. 6 illustrates a configuration for a TDI imager 600 to simultaneously capture multiple image slices of a tissue sample during a single scan, according to some embodiments. One way to configure the TDI imager 600 is to position the TDI imager 600 at a tilt angle 603 relative to the surface of the tissue sample 605. By tilting the TDI imager 600, the focal plane 610 of each partition 602 may also be tilted so that the focal plane 610 of each partition 602 penetrates to different depths within the tissue sample 605. By controlling the tilt angle 603, the corresponding focal plane 610 of each of the partitions 602 of the TDI imager 600 may be aligned with the boundaries of different desired image slices within the volume of the tissue sample. Thus, the tilt angle 603 may be selected based on the thickness of the tissue sample 605, the total number of desired image slices, and / or the total number of partitions 602. Alternatively, some embodiments may mount the TDI sensor vertically (ie, in a flat position perpendicular to the optical axis) and then tilt the lens.

[0033] By appropriately adjusting the tilt angle 603, each of the partitions 602 may be configured to image all of the volume within the corresponding image slice while virtually eliminating imaging of portions of the volume outside the corresponding image slice. As shown in FIG. 6 , the field of view for each of the partitions 602 may be aligned with the boundaries of the image slice within the tissue sample 605 to control the depth range imaged by each partition 602. By tilting the focal plane as shown in FIG. 6 , each partition 602 of the TDI imager 600 may completely image one of the different corresponding volume slices without overlapping with adjacent image slices. Note that this implementation does not require modification of the architecture of the TDI imager 600. Instead, a commercial multi-partition TDI imager 600 or lens may be tilted according to the tilt angle 603 within an imaging system assembly to create multi-depth image capture capabilities.

[0034] In addition to initially adjusting the tilt angle 603, some embodiments may allow for fine tuning the placement of the focal plane 610 by adjusting the tilt angle 603. For example, software / hardware controls may be provided that allow for adjusting the tilt angle 603 of the TDI imager and / or lens to move the focal plane 610 within the sample. This ability to fine tune the tilt angle 603 provides this embodiment with an advantage over other embodiments.

[0035] It should be noted that the present disclosure uses a number of partitions and image slices, such as five or seven partitions, by way of example only. These examples of partitions and image slices are not intended to be limiting. Other embodiments may use more or fewer partitions and / or image slices without limitation. For example, some embodiments may use three partitions in the TDI imager, corresponding to three image slices in the tissue. Other embodiments may use two, four, six, eight, nine, ten, or more partitions, each with a corresponding number of image slices in the tissue. It should also be noted that for clarity, the following figures may omit the lenses themselves from the illustrations. However, it should be understood that in an actual implementation, lenses would be placed between the TDI imager and the tissue sample. Furthermore, the dotted lines from the TDI partitions to the image slices in the tissue sample do not represent exact ray traces, as the lenses would modify these optical paths.

[0036] Figure 7 shows a close-up view of a single partition 702 and corresponding focal plane 710 within an image slice 712, according to some embodiments. The figure illustrates how a single partition 702 can be composed of multiple individual horizontal pixel rows. As a cross-sectional view of a single partition 702, the figure shows a cross-section of the individual pixel rows within the partition 702. Note that 17 pixel rows are used in Figure 7 merely as an example and are not intended to be limiting. Embodiments of a TDI imager may include partitions containing any number of pixel rows, such as 32 rows, 64 rows, 128 rows, 256 rows, etc.

[0037] Each individual pixel row within partition 702 has a focal plane corresponding to a different Z depth level within image slice 712. Thus, even if the movement of the tissue sample relative to the TDI camera is parallel, a single partition of the TDI imager may scan image slice 712 at progressively successive depths. This configuration enhances the ability to accurately image the entire image slice without negatively impacting imaging. For example, if the focal planes of all horizontal pixel rows within partition 702 were the same focal plane, the resulting image may miss some fluorophores that are within image slice 712 but not at the exact depth of the focal plane of partition 702. However, by tilting the TDI imager, and consequently each pixel row within each partition, pixels may be aligned to many different focal planes within each image slice, thereby capturing fluorophores located anywhere within the depth range of the image slice. This provides a more complete and accurate view of the tissue sample volume.

[0038] Partition 702 may continue to function as a conventional TDI imager, in which case, as the imager or tissue sample moves, signals received from previous rows may be aggregated with signals received from the current row, etc. Thus, the aggregated image produced by partition 702 will aggregate signals across the entire depth range of image slice 712 to produce a single image representing the entire depth range of image slice 712. This combination of individual pixel rows at different depths is referred to herein as "focal drilling." In the example described above, the slice spacing (0.5 μm) is approximately equal to the expected nominal focal drilling amplitude (0.5 μm), which allows the multi-partition TDI sensor to capture multiple image slices in a single scan, with each slice being combined with a focal drilling combination to more uniformly capture all fluorophores within that slice. Seven different volume slices, corresponding to seven different partitions on the TDI sensor, may each be focal drilled to more effectively capture all fluorophores within that slice. Furthermore, all seven volume slices may be captured in a single scan.

[0039] FIG. 7 further illustrates how an image from one image slice 712 can be separated from images generated by adjacent image slices. First, using focus drilling combination of rows at different focal planes within a partition produces a process window with a steep attenuation gradient. Instead of a low gradient or a gradually decaying gradient at the edge of the process window, focus drilling combination produces a process window with a somewhat "top hat" shape, with an adjustable width (i.e., adjustable relative to the image slice thickness in the Z-depth direction) depending on the degree of focus drilling. In effect, this steep attenuation at the edge of the process window tends to isolate one image slice from another, and there is a large stable region within the process window for capturing a volume image. This produces more discrete volume image slices with highly controllable thickness compared to previous techniques.

[0040] The presence of a fluorophore at the boundary between two image slices can result in overlapping images of that fluorophore in both adjacent image slices. If unwanted double imaging of fluorophores in adjacent volume slices creates a significant problem, having a small separation between partitions on the TDI imager can mitigate this issue. This can be done by designing the partitions to have a physical separation between them, or, when working with OEM sensors, by patterning a black matrix resist over the array that blocks certain rows of TDI sensors. This partition separation approach will reduce the focus drilling amplitude below the volume slicing pitch and the guard band against overlapping fluorophore imaging in adjacent image slices.

[0041] For example, some configurations may use physical separation or space to isolate one image slice from an adjacent image slice. In the example TDI imager shown in Figure 5, partitions 502 on the TDI imager 500 are physically separated by spaces between the partitions 502. For example, horizontal pixel rows within each partition may be closely spaced next to each other, while the spacing between horizontal pixel rows in adjacent partitions may be much larger (e.g., 5 times, 10 times, etc.) than the row spacing within the partition.

[0042] Instead of physically separating the partitions, partitions may be formed within the TDI imager array by covering one or more rows of horizontal pixels to create the partitions. For example, dark photoresist may be placed over one or more rows of pixels to separate the rows from one another and form partitions within the TDI imager array. For example, FIG. 7 shows pixels 704 and 706 at the edges of partition 702, where pixels 704 and 706 are darkened, blocked, or otherwise obscured or omitted from partition 702 to isolate the image from adjacent image slices.

[0043] FIG. 8 illustrates a TDI imager 800 with a stepped profile, according to some embodiments. Instead of configuring the TDI imager so that the focal planes of the partitions are tilted at an angle relative to the volume to correspond to different depths within the volume, other embodiments may use alternative configurations of the TDI imager or other system components to produce a similar effect. For example, FIG. 8 uses a TDI imager in which multiple partitions 802 each have a different height within the device itself. To adjust the relative heights between these partitions, steps of filler material may be formed on the imager substrate below the imaging pixels. The height difference between the partitions 802 may correspond to the relative thicknesses of the image slices. Moreover, each of the partitions may correspond to a different depth range within different image slices.

[0044] In contrast to configurations with tilted TDI imagers, stepped TDI imagers may generate a single focal plane at the same Z depth within the tissue sample for each horizontal pixel row within each partition. Therefore, it is not necessary to use focal drilling coupling of pixel rows; instead, these embodiments may generate an image with a focal plane centered at a single location within the depth range of the image slice, as shown in FIG. 8 . These embodiments may use custom TDI imagers built specifically for tissue samples with known or predictable thicknesses. As described below, a glass cover section or lens section above the imager may be used to fine-tune the focal plane depth of each partition 802.

[0045] 9 illustrates how a TDI imager can be configured to capture image slices at multiple depths using a cover or lens with sections of different thicknesses, according to some embodiments. Instead of adjusting the angle or contour of the TDI imager 902, the TDI imager 902 can be oriented parallel to the tissue sample 906 so that each of the partitions of the TDI imager 902 is at the same level relative to the tissue sample. This would normally only produce a focal plane for each partition at the same Z depth, allowing image capture at a single image slice at a time. However, by incorporating a layer 904 in front of the TDI imager 902, the focal distance from each partition can be adjusted to correspond to a different image slice within the tissue sample 906.

[0046] For example, layer 904 may be implemented as a glass cover on TDI imager 902. The glass cover may include multiple sections corresponding to multiple partitions on TDI imager 902. The sections on the glass cover may have different thicknesses and may be adjusted for each partition to center the corresponding focal plane of the partition below it in one of the image slices. Similarly, layer 904 may be implemented as a lens in front of TDI imager 902. Sections with different thicknesses may be implemented in the lens to center the focal plane of the partition below it in the various image slices.

[0047] FIG. 10 illustrates a wedge-shaped lens or glass cover according to some embodiments. Instead of using discrete stepped sections with discrete thicknesses, a continuous wedge-shaped layer 1004 may be placed in front of the TDI imager 1002. The layer 1004 may be implemented as a glass cover on the TDI imager 1002 and / or as a lens in front of the TDI imager 1002. Note that the wedge-shaped lens or cover produces an effect similar to tilting the TDI imager. Specifically, the focal plane of each partition increases stepwise for each horizontal pixel row in the image slice. This allows the focus drilling combination of pixel rows described above to be implemented with this configuration. For example, the angle of the wedge-shaped lens or cover may be similar to the tilt angle 603 of the TDI sensor in FIG. 6.

[0048] FIG. 11 shows a flowchart 1100 of a method for capturing an aerial image of a biological tissue sample, according to some embodiments. The method may be performed by the imaging system 100 described above. For example, each of the steps of the method may be implemented by a set of instructions executed by a controller of the imaging system. The controller may include one or more processors that execute instructions stored on one or more memory devices to perform the operations described below. For example, the imaging system controller may be configured (or programmed) to cause the TDI imager to scan the biological tissue sample. An example of a computer system that may be used as the controller is described below in FIG. 12.

[0049] The method may include mounting 1102 a biological tissue sample in an imaging chamber of an imaging system. The tissue sample may include any type of biological material and may be mounted on a slide, coverslip, or other transparent surface. As described above, one or more fluorophores may be added to the imaging chamber and mixed with the tissue sample.

[0050] The method may further include directing light from a light source to illuminate an area on the biological tissue sample (1104) to activate one or more fluorophores in the biological tissue sample. As described above in FIG. 3, specific wavelengths of light may be generated from a laser or other light source to activate specific fluorophores in the sample. In some embodiments, multiple wavelengths may be provided at once, or multiple wavelengths may be provided sequentially, so that specific fluorophores can be highlighted in each image. Activated fluorophores may return wavelengths of light that fall within a range higher than the activation wavelength of the corresponding fluorophore.

[0051] The method may further include scanning 1106 the biological tissue sample with a TDI imager having multiple partitions. The multiple partitions may be configured to simultaneously capture images at multiple different depths within the biological tissue sample during the scan. For example, the TDI imager may be positioned at an oblique angle relative to the tissue sample so that the focal plane of each partition falls within a different depth range of the tissue sample. Alternatively, as described above in FIGS. 6-10, the TDI imager may be fabricated with a stepped profile, or a lens or glass cover may be used to shift the focal plane of each partition to a different image slice of the sample. As mentioned above, some configurations may allow for focal drilling coupling of individual pixel rows within the partitions.

[0052] It should be understood that the specific steps illustrated in FIG. 10 provide a particular method for capturing a spatial image of a biological tissue sample according to various embodiments. Other sequences of steps may be performed according to alternative embodiments. For example, alternative embodiments may perform the steps outlined above in a different order. Moreover, individual steps illustrated in FIG. 10 may include multiple sub-steps that may be performed in various sequences consistent with the individual steps. Furthermore, additional steps may be added or removed depending on the particular application. Furthermore, many variations, modifications, and alternatives are within the scope of the present disclosure.

[0053] The above examples refer to a biological tissue sample as the volume imaged by the imaging system. However, these techniques may be extended to other transparent volumes that do not necessarily contain biological tissue. Specifically, each of the above techniques may be used to image any transparent volume at multiple focal planes during a single scan of the TDI imager.

[0054] 12 illustrates an exemplary computer system 1200 on which various embodiments may be implemented. System 1200 may be used to implement any of the computer systems described above, including the controller of imaging system 100. As shown in this figure, computer system 1200 includes a processing unit 1204 that communicates with several peripheral subsystems via a bus subsystem 1202. These peripheral subsystems may include a processing acceleration unit 1206, an I / O subsystem 1208, a storage subsystem 1218, and a communication subsystem 1224. Storage subsystem 1218 includes a tangible computer-readable storage medium 1222 and a system memory 1210.

[0055] Bus subsystem 1202 provides a mechanism for allowing the various components and subsystems of computer system 1200 to communicate with each other as intended. While bus subsystem 1202 is shown diagrammatically as a single bus, alternative embodiments of the bus subsystem may utilize multiple buses. Bus subsystem 1202 may be any of several types of bus structures, including a memory bus or memory controller, a peripheral bus, and a local bus, using any of a variety of bus architectures. For example, such architectures may include an Industry Standard Architecture (ISA) bus, a MicroChannel Architecture (MCA) bus, an Enhanced ISA (EISA) bus, a Video Electronics Standards Association (VESA) local bus, and a Peripheral Component Interconnect (PCI) bus, which may be implemented as a mezzanine bus manufactured to the IEEE P1386.1 standard.

[0056] Processing unit 1204, which may be implemented as one or more integrated circuits (e.g., conventional microprocessors or microcontrollers), controls the operation of computer system 1200. Processing unit 1204 may include one or more processors. These processors may include single-core or multi-core processors. In certain embodiments, processing unit 1204 may be implemented as one or more independent processing units 1232 and / or 1234, each including a single or multi-core processor. In other embodiments, processing unit 1204 may be implemented as a quad-core processing unit formed by integrating two dual-core processors into a single chip.

[0057] In various embodiments, processing unit 1204 may execute various programs in response to program code and may maintain multiple programs or processes running simultaneously. At any given time, some or all of the program code being executed may reside in processor 1204 and / or storage subsystem 1218. Through appropriate programming, processor 1204 may provide the various functions described above. Computer system 1200 may further include a processing acceleration unit 1206, which may include a digital signal processor (DSP), a special purpose processor, and / or the like.

[0058] The I / O subsystem 1208 may include user interface input devices and user interface output devices. User interface input devices may include keyboards, pointing devices such as mice or trackballs, touchpads or touchscreens integrated into displays, scroll wheels, click wheels, dials, buttons, switches, keypads, audio input devices including voice command recognition systems, microphones, and other types of input devices. In addition, the user interface input devices may include voice recognition sensing devices that allow a user to interact with a voice recognition system (e.g., Siri® Navigator) via voice commands.

[0059] User interface input devices may further include, but are not limited to, three-dimensional (3D) mice, joysticks or pointing sticks, gamepads, and graphic tablets, as well as audio / visual devices such as speakers, digital cameras, digital video cameras, portable media players, webcams, image scanners, fingerprint scanners, barcode reader 3D scanners, 3D printers, laser range finders, and gaze tracking devices. In addition, user interface input devices may include medical imaging input devices such as, for example, computed tomography, magnetic resonance imaging, position emission tomography, and medical ultrasound devices. User interface input devices may also include audio input devices such as, for example, MIDI keyboards, digital musical instruments, and the like.

[0060] User interface output devices may include a display subsystem, indicator lights, or non-visual displays such as audio output devices. The display subsystem may be a cathode ray tube (CRT), a flat panel device, such as a flat panel device using a liquid crystal display (LCD) or a plasma display, a projection device, a touch screen, etc. In general, use of the term "output device" is intended to include all possible types of devices and mechanisms for outputting information from computer system 1200 to a user or to another computer. For example, user interface output devices may include, but are not limited to, various display devices that visually convey text, graphics, and audio / video information, such as monitors, printers, speakers, headphones, automobile navigation systems, plotters, voice output devices, modems, etc.

[0061] Computer system 1200 may include a storage subsystem 1218 that contains software elements, which are currently shown as located in system memory 1210. System memory 1210 may store program instructions loadable and executable on processing unit 1204, as well as data generated during the execution of these programs.

[0062] Depending on the configuration and type of computer system 1200, system memory 1210 may be volatile (such as random access memory (RAM)) and / or non-volatile (such as read-only memory (ROM), flash memory, etc.). RAM typically contains data and / or program modules that are immediately accessible to and / or currently being operated on and executed by processing unit 1204. In some implementations, system memory 1210 may include different types of memory, such as static random access memory (SRAM) or dynamic random access memory (DRAM). In some implementations, a basic input / output system (BIOS), containing the basic routines that help to transfer information between elements within computer system 1200, such as during start-up, may typically be stored in ROM. By way of example, and not limitation, system memory 1210 further illustrates application programs 1212, program data 1214, and operating system 1216; application programs 1212 may include client applications, a web browser, a mid-tier application, a relational database management system (RDBMS), etc. By way of example, operating system 1216 may include various versions of Microsoft Windows®, Apple Macintosh®, and / or Linux® operating systems, various commercially available UNIX® or UNIX-like operating systems (including, but not limited to, various GNU / Linux operating systems, Google Chrome® OS, etc.), and / or mobile operating systems such as iOS, Windows® Phone, Android® OS, BlackBerry® 10 OS, and Palm® OS operating systems.

[0063] The storage subsystem 1018 may also provide a tangible, computer-readable storage medium for storing the basic programming and data constructs that provide the functionality of some embodiments. Software (programs, code modules, instructions) that, when executed by a processor, provide the functionality described above may be stored in the storage subsystem 1218. These software modules or instructions may be executed by the processing unit 1204. The storage subsystem 1218 may also provide a repository for storing data used in accordance with some embodiments.

[0064] Storage subsystem 1200 may further include computer-readable storage medium reader 1220 that may further connect to computer-readable storage medium 1222. Together with, and optionally in combination with, system memory 1210, computer-readable storage medium 1222 may collectively represent remote, local, fixed, and / or removable storage devices and storage media for temporarily and / or more permanently containing, storing, transmitting, and retrieving computer-readable information.

[0065] The computer-readable storage medium 1222 containing the code or portions of code may further include any suitable medium, including, but not limited to, storage and communication media such as volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storing and / or transmitting information. This may include tangible computer-readable storage media such as RAM, ROM, Electronically Erasable Programmable ROM (EEPROM), flash memory or other memory technology, CD-ROM, digital versatile disk (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or other tangible computer-readable media. This may further include non-tangible computer-readable media such as data signals, data transmissions, or any other medium that can be used to transmit the desired information and that can be accessed by computing system 1000.

[0066] By way of example, computer-readable storage medium 1222 may include hard disk drives that read from or write to non-removable, non-volatile magnetic media, magnetic disk drives that read from or write to removable, non-volatile magnetic disks, and optical disk drives that read from or write to removable, non-volatile optical disks, such as CD-ROMs, DVDs, and Blu-Ray® disks or other optical media. Computer-readable storage medium 1222 may also include, but is not limited to, Zip® drives, flash memory cards, Universal Serial Bus (USB) flash drives, Secure Digital (SD) cards, DVD disks, digital video tapes, etc. The computer-readable storage media 1222 may also include flash memory-based solid-state drives (SSDs), enterprise flash drives, SSDs based on non-volatile memory such as solid-state ROM, SSDs based on volatile memory such as solid-state RAM, dynamic RAM, static RAM, DRAM-based SSDs, magnetoresistive RAM (MRAM) SSDs, and hybrid SSDs that use a combination of DRAM and flash memory-based SSDs. Disk drives and their associated computer-readable media may provide non-volatile storage of computer-readable instructions, data structures, program modules, and other data for the computer system 1200.

[0067] The communications subsystem 1224 provides an interface with other computer systems and networks. The communications subsystem 1224 serves as an interface for receiving data from other systems and transmitting data from the computer system 1200 to other systems. For example, the communications subsystem 1224 may enable the computer system 1200 to connect to one or more devices via the Internet. In some embodiments, the communications subsystem 1224 may include radio frequency (RF) transceiver components for accessing wireless voice and / or data networks (e.g., using cellular telephone technology, advanced data network technologies such as 3G, 4G, or EDGE (enhanced data rates for global evolution), WiFi (IEEE 802.11 family of standards, or other mobile communications technologies, or any combination thereof), global positioning system (GPS) receiving components, and / or other components. In some embodiments, the communications subsystem 1224 may provide a wired network connection (e.g., Ethernet) in addition to or instead of a wireless interface.

[0068] In some embodiments, the communications subsystem 1224 may also receive incoming communications in the form of structured and / or unstructured data feeds 1226, event streams 1228, event updates 1230, etc., on behalf of one or more users who may use the computer system 1200.

[0069] By way of example, the communications subsystem 1224 may be configured to receive data feeds 1226, such as web feeds such as Twitter® feeds, Facebook® updates, Rich Site Summary (RSS) feeds, and / or real-time updates from one or more third-party sources, in real time from users of social networks and / or other communications services.

[0070] Additionally, the communications subsystem 1224 may be further configured to receive data in the form of a continuous data stream, which may include an event stream 1228 of real-time events and / or event updates 1230, which may be continuous or infinite in nature without an explicit end. Examples of applications that generate continuous data may include, for example, sensor data applications, financial tickers, network performance measurement tools (e.g., network monitoring and traffic management applications), clickstream analysis tools, automobile traffic monitoring, etc.

[0071] The communications subsystem 1224 may further be configured to output structured and / or unstructured data feeds 1226, event streams 1228, event updates 1230, etc. to one or more databases that may be in communication with one or more streaming data source computers coupled to the computer system 1200.

[0072] Because the nature of computers and networks is constantly changing, the description of computer system 1200 shown in this figure is intended to be a specific example only. Many other configurations are possible, having more or fewer components than the system shown in the figure. For example, customized hardware may be used, and / or particular elements may be implemented in hardware, firmware, software (including applets), or a combination. Furthermore, connections to other computing devices, such as network input / output devices, may be used. Based on the disclosure and teachings provided herein, other ways and / or methods of implementing various embodiments should be apparent.

[0073] As used herein, the terms "about" or "approximately" or "substantially" may be interpreted to be within the range that one skilled in the art would expect in light of the specification.

[0074] In the above description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of various embodiments. It will be apparent, however, that some embodiments may be practiced without some of these specific details. In other instances, well-known structures and devices are shown in block diagram form.

[0075] The above description provides only exemplary embodiments, and the above description is not intended to limit the scope, applicability, or configuration of the present disclosure. Rather, the above description of various embodiments provides an enabling disclosure for implementing at least one embodiment. It should be understood that various changes can be made in the function and arrangement of elements without departing from the spirit and scope of the several embodiments as set forth in the appended claims.

[0076] In the above description, specific details have been set forth to provide a thorough understanding of the embodiments. However, it will be understood that the embodiments may be practiced without these specific details. For example, circuits, systems, networks, processes, and other components may be shown as components in block diagram form so as not to obscure the embodiments in unnecessary detail. In other instances, well-known circuits, processes, algorithms, structures, and techniques may be shown without unnecessary detail so as to avoid obscuring the embodiments.

[0077] Furthermore, it should be noted that particular embodiments may be described as a process that is depicted as a flowchart, flow diagram, data flow diagram, structure diagram, or block diagram. While a flowchart may describe operations as a sequential process, many of the operations may be performed in parallel or simultaneously. Additionally, the order of operations may be rearranged. A process terminates when the operations of the process are completed, but may have additional steps not included in the diagram. A process may correspond to a method, a function, a procedure, a subroutine, a subprogram, etc. When a process corresponds to a function, the termination of the process may correspond to a return of the function to the calling function or the main function.

[0078] The term "computer-readable medium" includes, but is not limited to, portable or fixed storage devices, optical storage devices, wireless channels, and various other media capable of storing, containing, or carrying instructions and / or data. A code segment or machine-executable instructions may represent a procedure, a function, a subprogram, a program, a routine, a subroutine, a module, a software package, a class, or any combination of instructions, data structures, or program statements. A code segment may be coupled to another code segment or a hardware circuit by passing and / or receiving information, data, arguments, parameters, or memory contents. Information, arguments, parameters, data, etc. may be passed, forwarded, or transmitted via any suitable means including memory sharing, message passing, token passing, network transmission, etc.

[0079] Furthermore, the embodiments may be implemented in hardware, software, firmware, middleware, microcode, hardware description languages, or any combination thereof. When implemented in software, firmware, middleware, or microcode, the program code or code segments to perform the necessary tasks may be stored in a machine-readable medium. A processor may perform the necessary tasks.

[0080] In the foregoing specification, features have been described with reference to specific embodiments thereof, but it should be recognized that not all embodiments are limited thereto. Various features and aspects of the several embodiments may be used individually or jointly. Moreover, the embodiments may be utilized in any number of environments and applications other than those described herein without departing from the broader spirit and scope of the specification. Accordingly, the specification and drawings are to be regarded as illustrative and not restrictive.

[0081] Additionally, for illustrative purposes, the methods have been described in a particular order. It should be understood that in alternative embodiments, the methods may be performed in an order different from that described. It should also be understood that the methods described above may be performed by hardware components or embodied in a sequence of machine-executable instructions, which may be used to cause a machine, such as a general-purpose or special-purpose processor or general-purpose or special-purpose logic circuitry, programmed with the instructions to perform the method. These machine-executable instructions may be stored on one or more machine-readable media, such as a CD-ROM or other type of optical disk, floppy diskette, ROM, RAM, EPROM, EEPROM, magnetic or optical card, flash memory, or other type of machine-readable medium suitable for storing electronic instructions. Alternatively, the methods may be performed by a combination of hardware and software.

Claims

1. 1. An imaging system for capturing a spatial image of a biological tissue sample, comprising: an imaging chamber configured to hold a biological tissue sample placed within the imaging system; a light source configured to illuminate the biological tissue sample to activate one or more fluorophores in the biological tissue sample; a time delay and integration (TDI) imager comprising a plurality of partitions configured to simultaneously capture images at a plurality of different depths within the biological tissue sample during a single scan by the TDI imager; and a controller configured to cause the TDI imager to scan the tissue sample; An imaging system comprising:

2. 2. The imaging system of claim 1, wherein the TDI imager is tilted at an angle relative to the tissue sample such that focal planes of the partitions correspond to the different depths within the tissue sample.

3. The imaging system of claim 1 , wherein the partitions on the TDI imager are physically separated by spaces between the partitions.

4. The imaging system of claim 1 , wherein the plurality of partitions on the TDI imager are separated by rows of covered pixels.

5. The imaging system of claim 1 , wherein the plurality of different depths within the biological tissue sample comprises a plurality of different depth ranges within the biological tissue sample.

6. 6. The imaging system of claim 5, wherein one partition of the plurality of partitions on the TDI imager corresponds to one depth range of the plurality of different depth ranges, the partition including a plurality of pixel rows, each of the plurality of pixel rows corresponding to a different depth within the depth range.

7. 7. The imaging system of claim 6, wherein data received from the plurality of pixel rows is combined in a focus drilling combination to generate an image for the depth range.

8. 1. A method for capturing a spatial image of a biological tissue sample, comprising: mounting a biological tissue sample in an imaging chamber of an imaging system; directing light from a light source to illuminate an area on the biological tissue sample to activate one or more fluorophores in the biological tissue sample; scanning the biological tissue sample with a time delay and integration (TDI) imager comprising multiple partitions configured to simultaneously capture images at multiple different depths within the biological tissue sample during the scan; A method comprising:

9. 9. The method of claim 8, wherein the plurality of different depths within the biological tissue sample comprises a plurality of different depth ranges within the biological tissue sample, wherein one partition of the plurality of partitions on the TDI imager corresponds to one depth range of the plurality of different depth ranges, and wherein the partition comprises a plurality of pixel rows, each of the plurality of pixel rows corresponding to a different depth within the depth range.

10. 10. The method of claim 9, further comprising combining data received from the plurality of pixel rows with a focus drilling combination to generate an image for the depth range.

11. The method of claim 9, wherein the depth range is between about 250 nm and about 750 nm.

12. 9. The method of claim 8, wherein the thickness of the biological tissue sample is between about 2 μm and about 10 μm.

13. The method of claim 8 , further comprising generating an image of the tissue sample from each of the plurality of partitions.

14. A time delay and integration (TDI) imager comprising multiple partitions configured to simultaneously capture images at multiple different depths within a volume during a single scan by the TDI imager. An imaging system comprising:

15. 15. The imaging system of claim 14, wherein the TDI imager is tilted at an angle relative to the volume such that focal planes of the partitions correspond to the different depths within the volume, and the angle is adjustable to fine-tune the different depths within the volume.

16. 15. The imaging system of claim 14, further comprising a glass cover over the TDI, the glass cover including multiple sections corresponding to the multiple partitions, and thicknesses of the multiple sections of the glass cover position the focal planes of the multiple partitions at the multiple different depths within the volume.

17. 15. The imaging system of claim 14, further comprising a lens in front of the TDI, the lens including multiple sections corresponding to the multiple partitions, and thicknesses of the multiple sections of the lens position the focal planes of the multiple partitions at the multiple different depths within the volume.

18. 15. The imaging system of claim 14, wherein the partitions of the TDI imager have different heights relative to one another, the different heights causing the focal planes of the partitions to be located at the different depths within the volume.

19. The imaging system of claim 14 , wherein the volume comprises a biological tissue sample.

20. 15. The imaging system of claim 14, further comprising a lens in front of the TDI, the lens including a wedge shape such that the focal planes of the partitions are located at the different depths within the volume.

Images