Time-delayed integrated acquisition for spatial genomics imaging

Abstract

An imaging system for capturing a spatial omic image of a biological tissue sample may include an imaging chamber configured to secure a biological tissue sample placed within the imaging system, a time delay and integration (TDI) imager having at least one scan line, a light source configured to illuminate an area on the biological tissue sample being captured by the TDI imager, and a controller configured to cause the TDI imager to scan the biological tissue sample using one or more TDI scans of 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 / 393,458, entitled "TIME DELAY INTEGRATION ACQUISITION FOR SPATIAL GENOMICS IMAGING," filed July 29, 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-omic images of biological tissue samples. More specifically, this disclosure describes camera and light configurations that reduce capture times in biological imaging systems. [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 pictures of the cellular environment is sometimes called 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 tool for performing spatial omics. Summary of the Invention

[0005] In some embodiments, an imaging system for capturing spatial omic images of a biological tissue sample may include an imaging chamber configured to secure a biological tissue sample placed within the imaging system, a Time Delay and Integration (TDI) imager having at least one scan line, a light source configured to illuminate an area on the biological tissue sample being captured by the TDI imager, and a controller configured to cause the TDI imager to scan the biological tissue sample using one or more TDI scans of the biological tissue sample.

[0006] In some embodiments, a method for capturing a spatial omic image of a biological tissue sample may include securing 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, and causing a time delay and integration (TDI) imager to scan the biological tissue sample, which may be illuminated by the light from the light source, using one or more TDI scans of the biological tissue sample.

[0007] In some embodiments, an imaging system for capturing spatial omic images of a biological tissue sample may include an imaging chamber configured to secure a biological tissue sample placed within the imaging system, a time delay and integration (TDI) imager, a light source configured to illuminate an area on the biological tissue sample being captured by the TDI imager with a plurality of different wavelengths, and a controller configured to cause the TDI imager to simultaneously capture a plurality of different images of the biological tissue sample, each of the plurality of different images may correspond to one of a plurality of different wavelengths.

[0008] Any of the embodiments may implement some or all of the following features in any combination, without limitation: The controller may cause the TDI imager to scan the biological tissue without requiring mechanical repositioning, stabilization, or focusing between captured scan lines within a column. The light source may provide light to the biological tissue sample through a fiber optic line that may project light onto a substantially circular illumination area on the biological tissue sample. The light source may provide light to the biological tissue sample through an aperture having a rectangular shape. The aperture may project a rectangular illumination area onto a rectangular field of view of the TDI imager on the biological sample. The light source may provide light to the biological tissue sample through an aperture having a shape that matches the shape of the field of view of the TDI imager. The light source may provide light to the biological tissue sample through a fiber optic line having an oval shape. The system may include a fiber optic bundle including multiple fiber optic lines, each configured to provide a different wavelength. Each of the multiple fiber optic lines may have an oval or rectangular shape. Each of the multiple fiber optic lines may be positioned to direct light to a different portion of the biological tissue sample. The system / method may assemble evenly spaced lines from a plurality of scan line arrays from the TDI imager. The light from the light source may include multiple different wavelengths that may be simultaneously projected onto the biological tissue sample. The system / method may use multiple filters before the TDI imager to filter out the different wavelengths. The system / method may use a beam splitter before the TDI imager to filter out the different wavelengths. The TDI imager may include multiple scan lines, each of which may capture one of multiple different images. The TDI imager may include multiple individual TDI cameras, each of which may be aimed at a different location on the biological tissue sample to simultaneously capture the different images. The system may include a filter wheel before the TDI imager, which may include multiple filters corresponding to the different wavelengths. The light source may include a broad-spectrum light source that combines the different wavelengths onto a single illumination area.

[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 a multi-omic image of a sample, according to some embodiments. [Figure 3] FIG. 1 illustrates a TDI camera that may be used in an imaging system, according to some embodiments. [Figure 4A] FIG. 1 illustrates an illumination region for a conventional camera, according to some embodiments. [Figure 4B] FIG. 1 illustrates an illumination area for a TDI camera according to some embodiments. [Figure 4C] FIG. 10 illustrates an illumination area where unused light area is minimized by apertures, according to some embodiments. [Figure 4D] FIG. 10 illustrates an illumination area where unused light area is minimized by shaping the laser beam, according to some embodiments. [Figure 5] FIG. 1 illustrates how a conventional camera system can capture multiple images illuminated by different wavelengths, according to some embodiments. [Figure 6] FIG. 1 illustrates an imaging system configuration that allows for capturing multiple wavelengths during a single pass through a tissue sample, according to some embodiments. [Figure 7]1A-1C illustrate different configurations of filters for simultaneous wavelength capture, according to some embodiments. [Figure 8] FIG. 10 is a diagram showing a configuration in which a beam splitter is used to separate different wavelength images. [Figure 9] 1 is a flow diagram of a method for capturing a spatial omic image of a biological tissue sample. [Figure 10] 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.

[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. Fluid may then be automatically pumped into the imaging chamber 108 after fixing the tissue sample in the imaging chamber 108. For example, some fluid 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. 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, and a camera may capture an image of the illuminated sample. The fluorophores may be matched to different laser wavelengths configured to illuminate that particular fluorophore.

[0014] After the imaging process is complete, the controller may convert raw images from the system into RNA spots. These RNA 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] FIG. 2 shows a flow diagram 200 of a process for capturing multi-omic images 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 slide (202) inside one of the imaging chambers of the imaging system. Note that multiple imaging chambers may operate independently and simultaneously within the same imaging system 100. For example, one imaging chamber 108 may capture an image of the sample while another imaging chamber 110 replaces the fluid within the imaging chamber 110. The imaging system 100 may then supply fluids (204) from the fluidics system 102 into the imaging chamber 108. These fluids may contain fluorophores configured to attach to specific cell or tissue types to be highlighted in the resulting image.

[0016] 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 an 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 involve sequentially capturing field images using the camera (206) and then moving the camera to a subsequent position with adjacent fields of view to prepare the camera for the subsequent step (208). This process may be repeated until the individual field images capture the overall image 250 of the sample.

[0017] To capture the overall image 250, the camera may move its field of view 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 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.

[0018] 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 fluorophore may be pumped out of the imaging chamber 108, a cleaning or rinsing 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." Typically, each sample may undergo multiple hybs using different fluorophores. For example, some embodiments may capture two, three, four, five, or six or more global images of a sample, 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.

[0019] While this process provides high-resolution, multi-omic image data, it is also time-consuming. At each field of view image location, the sample may be illuminated by different laser wavelengths (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. After capturing an image at each field of view location, the process involves laterally shifting the sample so that the camera captures the new field of view location, which may require time to acquire a new image. This may include moving the sample laterally, physically moving the sample using piezo-motors or stage motors, configuring the laser and corresponding filters for the desired wavelengths, stabilizing the camera, focusing the camera, and / or other operations. Combining these different factors together contributes to a relatively long overall imaging time. For example, in an exemplary implementation using a camera with a 40x objective and a 30x30 grid of field of view images to cover the sample, each hybrid may take approximately 10 hours to complete. A typical 4hyb 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.

[0020] To address these and other technical challenges, embodiments described herein may employ several technical improvements to imaging systems to significantly reduce overall imaging time. A time-delay integration (TDI) camera may be used to continuously scan the tissue sample along a row without moving between different fields of view. A laser beam projected onto the imaging sample may be shaped to approximately coincide with the TDI image scan line. Some embodiments may simultaneously illuminate different locations of the tissue sample captured by individual TDI cameras or portions of a single TDI camera with different wavelengths. As described below, these improvements, alone or in combination, help dramatically improve imaging times for imaging systems.

[0021] FIG. 3 illustrates a TDI camera 302 that may be used in an imaging system, according to some embodiments. The TDI camera 302 may include a charge-coupled device (CCD) as an image sensor for capturing an image. For example, the TDI camera 302 may include a scan line 304 of individual CCD pixels in a horizontal configuration as shown in FIG. 3. Note that only a single line of pixels is shown in the scan line 304 for clarity; however, this is not intended to be limiting. As will be discussed and shown later, the TDI camera 302 may include multiple horizontal rows of pixels.

[0022] The operation of the TDI camera 302 may be contrasted with the operation of the conventional camera described above. As noted 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 202, with each individual pixel simultaneously capturing an image when acquiring a camera shot. In contrast, the TDI camera 302 may use scan lines 304 of individual pixels. The TDI camera 302 may sequentially scan vertically across the image. The entire image may then be assembled from equally spaced lines throughout the linear field of view of the scan lines 304. Note that the terms “horizontal” and “vertical” are used merely to indicate orthogonal directions as shown in FIG. 3 and are not intended to be limiting to any particular direction.

[0023] The scan line 304 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 350, the scan line 352 may continuously scan down a first vertical column 362 of the imaging area. When the scan of the first vertical column 362 is completed, the scan line 352 of the TDI camera may be repositioned over a second vertical column 364, and the scan line 352 may then continuously scan down the second vertical column 364. These vertical columns may be stitched together to form the entire image 350 of the tissue sample.

[0024] The use of the TDI camera 302 is a significant technological improvement over other cameras for scanning tissue samples. The TDI camera 302 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 302 may be between each of the vertical row captures. Thus, the use of the TDI camera 302 increases the speed at which a complete image of the tissue sample can be captured, reducing the total time from over 35 hours to approximately 5 hours when combined with other improvements described below.

[0025] FIG. 4A illustrates an illumination area 402 for a conventional camera, according to some embodiments. As described above, a conventional camera may capture an individual field of view 404 of a tissue sample. Within the field of view 404, a plurality of individual pixels arranged in a rectangular grid may each simultaneously capture a portion of the field of view 404. Prior to capturing the image, the tissue sample may be illuminated by a laser using a specified wavelength. This wavelength may be configured to illuminate a type of fluorophore present on the tissue sample. Light from the laser may be supplied from the laser through a fiber optic line to an imaging chamber. Conventionally, the fiber optic line has a circular shape, so the output of light directed onto the tissue sample may have an approximately circular shape. As shown in FIG. 4, this may result in an illumination area 402 that is approximately circular to encompass the camera's field of view 404.

[0026] Because the circular shape of the illumination area 402 does not exactly match the rectangular shape of the field of view 404, there may be unused light areas 406 around the edges of the field of view 404. However, the unused light areas 406 are relatively small compared to the size of the field of view 404 and the total illumination area 402.

[0027] FIG. 4B illustrates an illumination area 412 for a TDI camera, according to some embodiments. As mentioned above, the scan line 414 may include a small number of horizontal rows of pixels, as shown in FIG. 4B. However, it should be noted that the shape of the illumination area 412 still corresponds to the circular output of a conventional fiber optic line that delivers laser light to the tissue sample. Because the shape of the illumination area 412 is significantly larger than and different from the shape of the scan line 414, the unused light area 416 is significantly larger than when using a conventional camera. It has been found that in some situations, overexposing the tissue sample with wasted light from the unused light area 416 can be harmful. For example, fluorophores in the unused light area 416 may be damaged, causing images of those fluorophores to be washed out or have lower contrast. Additionally, reflections of light from the unused light area 416 may interfere when capturing an image using the scan line 414. Therefore, some embodiments may be further configured to minimize the unused light area 416 using methods described below.

[0028] FIG. 4C illustrates an illumination area 422 in which an aperture minimizes unused light area 426, according to some embodiments. This configuration continues to use the circular light output from a conventional fiber optic line. However, instead of outputting an entire circular light pattern onto the tissue sample, an aperture 428 may be used to shape the output light. In this example, the aperture may block the top and / or bottom of the output of the fiber optic line so that the illumination area 422 is more rectangular in shape. Thus, this embodiment may generally be characterized as providing light to the tissue sample with a shape substantially similar to the shape of the TDI camera's scan line 424, which in this particular example is rectangular in shape. Note that other camera fields of view having different shapes may be used, and so this example is not intended to be limiting.

[0029] The overall shape of the illuminated area 422 on the tissue sample may be configured to substantially match the shape of the scan line 424 of the TDI camera, although some embodiments may include a buffer area of light around the scan line 424. In the example of FIG. 4C , there is still an unused area of light 426 around the scan line 424. Some embodiments may minimize this unused area of light 426, while other embodiments may tolerate it such that the illuminated area 422 is larger than the area of the scan line 424. For example, the unused light area 426 may be between about 5% and 10%, between about 10% and 15%, between about 15% and 20%, between about 20% and 25%, between about 25% and 30%, between about 30% and 35%, between about 35% and 40%, between about 40% and 45%, between about 45% and 50%, between about 50% and 55%, between about 55% and 60%, between about 60% and 65%, between about 65% and 70%, between about 70% and 75%, between about 75% and 100%, or more than 100% larger than the area of the scan line 424.

[0030] FIG. 4D shows an illumination area 432 in which unused light area 436 is minimized due to the shape of the fiber optic line, according to some embodiments. Instead of using a circular fiber optic line, some embodiments may shape the fiber optic line to be more oval or rectangular in shape to better match the shape of the scan line 334. In this example, an oval fiber optic line may produce an illumination area 432 that more closely matches the shape of the scan line 334, as shown in FIG. 4D. This configuration minimizes unused light area 436 around the scan line 334, while also minimizing wasted light power from the laser. Instead of simply blocking the light power using an aperture, as shown in FIG. 4C, the light power may be concentrated onto the area of the scan line 334 by shaping the fiber optic line.

[0031] In addition to using a TDI camera and shaping the illumination area to coincide with the scan line of the TDI camera, some embodiments may further improve the throughput of the imaging system by optimizing how multiple images of the same area can be captured using different illumination wavelengths.

[0032] FIG. 5 illustrates how a conventional camera system, according to some embodiments, can capture multiple images illuminated with different wavelengths. In this example, each region of a tissue sample may be illuminated with four different wavelengths. A first laser shot 502 may illuminate a region of a tissue sample 504 with a first wavelength 503. Reflected light or fluorescence from the first laser shot 502 may be recorded by a camera 508 after passing through a first filter 506 configured to pass a first wavelength range 507. Note that the first wavelength range 507 may be slightly higher than the first wavelength 503 from the first laser shot 502. Similarly, a second laser shot 512 may illuminate a region of the tissue sample 504 with a second wavelength 513. Reflected light or fluorescence from the second laser shot 512 may be recorded by a camera 508 after passing through a second filter 516 configured to pass a second wavelength range 517. A third laser shot 522 may illuminate an area of the tissue sample 504 at a third wavelength 523. Reflected light or fluorescence from the third laser shot 522 may be recorded by the camera 508 after passing through a third filter 526 configured to pass a third wavelength range 527. A fourth laser shot 532 may illuminate an area of the tissue sample 504 at a fourth wavelength 533. Reflected light or fluorescence from the fourth laser shot 532 may be recorded by the camera 508 after passing through a fourth filter 536 configured to pass a fourth wavelength range 537. The images from each laser shot captured by the camera 508 may be stitched together to form a complete image of the tissue sample, each illuminated by a different wavelength.

[0033] 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, stabilize the filter wheels, move motors to account for wavelength-dependent focal plane shifts, etc. Thus, the total time to image the tissue sample increases significantly with each additional desired wavelength. The embodiments described herein improve this process by capturing multiple wavelengths at once using a camera and tissue sample.

[0034] FIG. 6 illustrates an imaging system configuration 600 that enables capturing multiple wavelengths during a single pass through a tissue sample, according to some embodiments. Instead of a single fiber optic line supplying a single wavelength, a fiber optic bundle 602 may supply four individual fiber optic lines 603. Each of the individual fiber optic lines 603 may be shaped into an oval, rectangular, or other shape configured to match the size / shape of a corresponding scan line of a TDI camera. In the example of FIG. 6, the individual fiber optic lines 603 may have an oval shape and may be stacked to form a vertically configured fiber optic bundle 602 such that corresponding illumination areas on the tissue sample are arranged vertically one after the other as shown in FIG. 6. Alternatively, instead of shaping the fiber optic line itself, some embodiments may shape the circular, square, or other shaped output beam of the fiber optic line. For example, the output beam may be shaped into an oval shape before or after directing light into or out of the fiber optic line.

[0035] Each of the fiber optic lines 603 may individually provide a different wavelength corresponding to a different fluorophore in the tissue sample. Some embodiments may sequentially illuminate the tissue sample during the TDI image acquisition process. For example, four illumination regions corresponding to the fiber optic lines 603 may be sequentially illuminated and scanned vertically downward across the tissue sample 604 along with corresponding scan lines of the TDI camera. Additionally, a filter 606 may be positioned and configured to remove portions of the reflected or fluorescent light from the tissue sample 604 based on the wavelength of the fiber optic line 603. Exemplary configurations of the filter 606 are described in detail below.

[0036] The system may further include a TDI camera 608 with four TDI sensors. For example, the TDI camera 608 may include four individual scan lines arranged vertically as shown in FIG. 6 . Each of the scan lines may be configured to scan an area on the tissue sample 604 illuminated by a corresponding light from one of the fiber optic lines 603. Thus, each of the fiber optic lines 603 may be aligned with and correspond to a respective one of the scan lines 609 of the TDI camera 608. The filter 606 may further include a plurality of individual filters aligned with the light path between the fiber optic lines 603 and the scan lines 609 of the TDI camera 608.

[0037] These embodiments allow for the simultaneous capture of multiple images, each with a different wavelength, during each vertical column scan of the tissue sample. Instead of using one of four individual passes through the tissue sample for each wavelength, these embodiments may capture some or all of the desired wavelengths in a single pass. This may reduce the total time by up to four times for four different wavelengths.

[0038] While FIG. 6 shows four individual TDI camera scan lines that may be physically separate and distinct from one another, other embodiments may use a single TDI scan line into which individual pixel rows are subdivided. For example, a single TDI imager may include a set of horizontal scan lines. However, the filter 606 and optical fiber line 603 may be configured to reflect light onto different regions of the set of pixel rows in the scan line. For example, three or four of the pixel rows on the TDI camera may be assigned to one wavelength, while the next three or four pixel rows on the TDI camera may be assigned to the next wavelength, and so on. Software operating the TDI camera 608 may then subdivide these pixel rows when processing the image. Thus, instead of physically separating the scan lines on the TDI camera 608 as shown in FIG. 6, some embodiments may instead perform this separation of different wavelength images in software.

[0039] 7 illustrates different configurations of filters for simultaneous wavelength capture, according to some embodiments. In a first configuration 704, individual filters may be placed on the scan lines of the TDI camera itself. As described above, the TDI camera may include different scan lines for each different wavelength. The filters may be placed adjacent to the individual scan line pixels for each wavelength as part of the camera structure. Custom CMOS image sensors may also be used to integrate the filters on the camera.

[0040] In a second configuration 706, the individual filters may be fabricated together as a cover glass that is placed over the imager. For example, the cover may be placed over the TDI imager such that the area for each filter on the cover glass is in front of a corresponding scan line of the TDI imager.

[0041] The third arrangement 708 may co-locate filters at a location corresponding to the intermediate focal plane of the TDI imager on a "butcher block" filter. In some embodiments, the third arrangement 708 may be configured as a filter wheel.

[0042] 8 illustrates a configuration using a beam splitter to separate different wavelength images. Instead of using four different laser wavelengths to illuminate different areas on a tissue sample, some embodiments may illuminate the tissue sample using broad-spectrum light that combines each of the desired wavelengths into a single illuminated area on the tissue sample. Reflected or fluorescent light 800 from the tissue sample may then pass through a beam splitter 802. The beam splitter 802 may then separate the different wavelengths of the reflected or fluorescent light 802 and output them onto separate TDI imagers 806, 808, 810.

[0043] 9 shows a flowchart 900 of a method for capturing a spatial omic image of a biological tissue sample. The method may include securing (902) the biological tissue sample in an imaging chamber of an imaging system. The method may further include directing (904) light from a light source to illuminate an area on the biological tissue sample. The method may further include causing (906) a TDI imager to scan the biological tissue sample illuminated by the light from the light source using one or more TDI scans of the biological tissue sample. Each of these operations may be performed as described in detail above.

[0044] It should be understood that the specific steps illustrated in FIG. 9 provide a particular method for capturing a spatial omic 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. 9 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.

[0045] Each of the methods described herein may be implemented by a computer system. Each step of these methods may be performed automatically by the computer system and / or each step of these methods may be provided with user-involved input / output. For example, a user may provide input for each step of the method, and each of these inputs may be in response to requesting a particular output, which is generated by the computer system. Each input may be received in response to requesting a corresponding output. Furthermore, input may be received from a user, received as a data stream from another computer system, retrieved from a memory location, retrieved across a network, requested from a web service, and / or the like. Similarly, output may be provided to a user, provided as a data stream to another computer system, saved to a memory location, sent across a network, provided to a web service, and / or the like. In summary, each step of the methods described herein may be performed by a computer system and may include any number of inputs, outputs, and / or requests to and from the computer system, which may or may not involve a user. Steps that do not involve a user may be said to be performed automatically by the computer system without human intervention. Thus, in light of the present disclosure, it will be understood that each step of each method described herein may be modified to include inputs and outputs to and from a user, or may be performed automatically by a computer system without human intervention, in which case all decisions are made by a processor.Additionally, some embodiments of each of the methods described herein may be implemented as a set of instructions stored on a tangible, non-transitory storage medium to form a tangible software product.

[0046] 10 illustrates an exemplary computer system 1000 on which various embodiments may be implemented. System 1000 may be used to implement any of the computer systems described above. As shown in this figure, computer system 1000 includes a processing unit 1004 that communicates with several peripheral subsystems via a bus subsystem 1002. These peripheral subsystems may include a processing acceleration unit 1006, an I / O subsystem 1008, a storage subsystem 1018, and a communication subsystem 1024. Storage subsystem 1018 includes a tangible computer-readable storage medium 1022 and a system memory 1010.

[0047] Bus subsystem 1002 provides a mechanism for allowing the various components and subsystems of computer system 1000 to communicate with each other as intended. While bus subsystem 1002 is shown diagrammatically as a single bus, alternative embodiments of the bus subsystem may utilize multiple buses. Bus subsystem 1002 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.

[0048] Processing unit 1004, which may be implemented as one or more integrated circuits (e.g., conventional microprocessors or microcontrollers), controls the operation of computer system 1000. Processing unit 1004 may include one or more processors. These processors may include single-core or multi-core processors. In certain embodiments, processing unit 1004 may be implemented as one or more independent processing units 1032 and / or 1034, each including a single or multi-core processor. In other embodiments, processing unit 1004 may be implemented as a quad-core processing unit formed by integrating two dual-core processors into a single chip.

[0049] In various embodiments, processing unit 1004 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 1004 and / or storage subsystem 1018. Through appropriate programming, processor 1004 may provide the various functions described above. Computer system 1000 may further include a processing acceleration unit 1006, which may include a digital signal processor (DSP), a special purpose processor, and / or the like.

[0050] The I / O subsystem 1008 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. User interface input devices may include motion sensing and / or gesture recognition devices such as a Microsoft Kinect® motion sensor that allows a user to control and interact with an input device, such as a Microsoft Xbox® 360 game controller, via a natural user interface using gestures and spoken commands. User interface input devices may also include eye gesture recognition devices such as a Google Glass® blink detector that detects a user's eye movements (e.g., "blinking" while taking a picture and / or selecting a menu) and translates the eye gestures as input to an input device (e.g., Google Glass®). Additionally, the user interface input devices may include a voice recognition sensing device that allows a user to interact with a voice recognition system (e.g., Siri® Navigator) via voice commands.

[0051] 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.

[0052] 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 1000 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.

[0053] Computer system 1000 may include a storage subsystem 1018 that contains software elements, which are currently shown as located in system memory 1010. System memory 1010 may store program instructions loadable onto and executable on processing unit 1004, as well as data generated during the execution of these programs.

[0054] Depending on the configuration and type of computer system 1000, system memory 1010 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 the processing unit 1004. In some implementations, system memory 1010 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 1000, such as during start-up, may typically be stored in ROM. By way of example, and not limitation, system memory 1010 further illustrates application programs 1012, program data 1014, and operating system 1016; application programs 1012 may include client applications, a web browser, a mid-tier application, a relational database management system (RDBMS), etc. By way of example, operating system 1016 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.

[0055] 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 1018. These software modules or instructions may be executed by the processing unit 1004. The storage subsystem 1018 may also provide a repository for storing data used in accordance with some embodiments.

[0056] Storage subsystem 1000 may further include a computer-readable storage medium reader 1020 that may be further connected to a computer-readable storage medium 1022. Together with, and optionally in combination with, system memory 1010, computer-readable storage medium 1022 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.

[0057] The computer-readable storage medium 1022 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.

[0058] By way of example, the computer-readable storage medium 1022 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. The computer-readable storage medium 1022 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 1022 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 1000.

[0059] The communications subsystem 1024 provides an interface with other computer systems and networks. The communications subsystem 1024 serves as an interface for receiving data from other systems and transmitting data from the computer system 1000 to other systems. For example, the communications subsystem 1024 may enable the computer system 1000 to connect to one or more devices via the Internet. In some embodiments, the communications subsystem 1024 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 1024 may provide a wired network connection (e.g., Ethernet) in addition to or instead of a wireless interface.

[0060] In some embodiments, the communications subsystem 1024 may also receive incoming communications in the form of structured and / or unstructured data feeds 1026, event streams 1028, event updates 1030, etc., on behalf of one or more users who may use the computer system 1000.

[0061] By way of example, the communications subsystem 1024 may be configured to receive data feeds 1026, 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.

[0062] Additionally, the communications subsystem 1024 may be further configured to receive data in the form of a continuous data stream, which may include an event stream 1028 of real-time events and / or event updates 1030, 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.

[0063] The communications subsystem 1024 may further be configured to output structured and / or unstructured data feeds 1026, event streams 1028, event updates 1030, etc. to one or more databases that may be in communication with one or more streaming data source computers coupled to the computer system 1000.

[0064] Computer system 1000 may be one of various types of computer systems, including a handheld portable device (e.g., an iPhone® cellular phone, an iPad® computing tablet, a PDA), a wearable device (e.g., a Google Glass® head-mounted display), a PC, a workstation, a mainframe, a kiosk, a server rack, or any other data processing system.

[0065] Because the nature of computers and networks is constantly changing, the description of the computer system 1000 shown in this figure is intended to be a particular 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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 spatial omic images of a biological tissue sample, comprising: an imaging chamber configured to immobilize a biological tissue sample placed within the imaging system; a time delay and integration (TDI) imager comprising at least one scan line; a light source configured to illuminate an area on the biological tissue sample being captured by the TDI imager; a controller configured to cause the TDI imager to scan the tissue sample using one or more TDI scans of the tissue sample; An imaging system comprising:

2. 10. The imaging system of claim 1, wherein the controller causes the TDI imager to scan the biological tissue without requiring mechanical repositioning, stabilization, or focusing between captured scan lines within a row.

3. The imaging system of claim 1 , wherein the light source provides light to the tissue sample through a fiber optic line that projects light onto a substantially circular illumination area on the tissue sample.

4. The imaging system of claim 1 , wherein the light source provides light to the biological tissue sample through an aperture having a rectangular shape.

5. The imaging system of claim 4 , wherein the aperture projects a rectangular illumination area onto a rectangular field of view of the TDI imager on the biological sample.

6. The imaging system of claim 1 , wherein the light source provides light to the biological tissue sample through an aperture having a shape that matches the shape of the field of view of the TDI imager.

7. The imaging system of claim 1 , wherein the light source delivers light to the biological tissue sample through a fiber optic line having an oval shape.

8. The imaging system of claim 1 , further comprising a fiber optic bundle including a plurality of fiber optic lines, each of the plurality of fiber optic lines configured to provide a different wavelength.

9. The imaging system of claim 8 , wherein each of the plurality of fiber optic lines has an oval or rectangular shape.

10. The imaging system of claim 8 , wherein each of the plurality of fiber optic lines is positioned to direct light to a different portion of the biological tissue sample.

11. 1. A method for capturing a spatial omic image of a biological tissue sample, comprising: Immobilizing 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; causing a time delay and integration (TDI) imager to scan the biological tissue sample illuminated by the light from the light source using one or more TDI scans of the biological tissue sample; A method comprising:

12. The method of claim 11 , further comprising assembling equally spaced lines from a plurality of rows of scan lines from the TDI imager.

13. 12. The method of claim 11, wherein the light from the light source comprises a plurality of different wavelengths that are simultaneously projected onto the biological tissue sample.

14. The method of claim 13 , further comprising filtering the different wavelengths using a plurality of filters before the TDI imager.

15. The method of claim 13 , further comprising filtering the different wavelengths using a beam splitter before the TDI imager.

16. 1. An imaging system for capturing spatial omic images of a biological tissue sample, comprising: an imaging chamber configured to immobilize a biological tissue sample placed within the imaging system; a time delay and integration (TDI) imager; a light source configured to illuminate an area on the biological tissue sample being captured by the TDI imager with a plurality of different wavelengths; a controller configured to cause the TDI imager to simultaneously capture a plurality of different images of the biological tissue sample, each of the plurality of different images corresponding to one of the plurality of different wavelengths; and An imaging system comprising:

17. 17. The imaging system of claim 16, wherein the TDI imager comprises a plurality of scan lines, each of the plurality of scan lines capturing one of the different plurality of images.

18. 17. The imaging system of claim 16, wherein the TDI imager comprises a plurality of individual TDI cameras, each of the plurality of individual TDI cameras aimed at a different location on the biological tissue sample to simultaneously capture the different images.

19. 17. The imaging system of claim 16, further comprising a filter wheel in front of the TDI imager, the filter wheel comprising a plurality of filters corresponding to the different wavelengths.

20. 17. The imaging system of claim 16, wherein the light source comprises a broad spectrum light source that combines the different wavelengths onto a single illumination area.

Images