Abstract: An imaging system for capturing spatial images of biological tissue samples may include an imaging chamber configured to hold a biological tissue sample placed in the imaging system; a light source configured to illuminate the biological tissue sample to activate a plurality of fluorophores in the biological tissue sample; and a plurality of Time Delay and Integration (TDI) imagers configured to simultaneously scan the biological tissue sample, where the plurality of TDI imagers may be configured to separately receive light from different ones of the plurality of fluorophores.

Abstract: In acquisition of spatial transcriptomic information, a plurality of images representing a common field of view of a sample are obtained and registered. Each pixel of the registered images is decoded by identifying a code word from a plurality of code words in a code book that provides a best match to data values in the plurality of registered images for the pixel. For each code word identified as a best match and each pixel, whether a bit ratio for an image word for the pixel meets a threshold for the code word is determined. The image word is formed from the data values in the plurality of registered images for the pixel. For at least one pixel that is determined to meet the threshold, a gene associated with the code word is determined. Pixels for which the bit ratio does not meet the threshold are screened.

Abstract: A fluorescent in-situ hybridization imaging system includes a flow cell to contain a sample, a fluorescence microscope, and a control system. The fluorescence microscope includes a variable frequency excitation light source to illuminate the sample, a plurality of emission bandpass filters on a filter wheel, an actuator to rotate the filter wheel, and a camera positioned to receive fluorescently emitted light from the sample. The control system is configured to cause the variable frequency excitation light source to emit a light beam having a selected wavelength, cause the actuator to rotate the filter wheel to position a selected filter in a light path between the sample and the camera, obtain an image from the camera, and coordinate the variable frequency excitation light source and filter wheel such that the selected filter has an emission bandpass associated with emission by the fluorescent probes when excited by the selected wavelength.

Abstract: A system and method of performing deep cell body segmentation on a biological sample is provided. The method includes receiving a first and a second stained image. The first image is processed using a trained machine learned model that outputs locations of a plurality of cell nuclei in the first stained image. Seed points are then determined based on the locations of the plurality of cell nuclei. The second image is then processed using the seed points to determine a plurality of cell membranes using a watershed segmentation. The second image is then post-processed and an output image is produced. The output image is then analyzed and gene sequencing is performed.

Abstract: In a fluorescent in-situ hybridization imaging system performs, as nested loops, the following: (1) a valve sequentially couples a flow cell to a plurality of different reagent sources to expose the sample to a plurality of different reagents, (2) for each reagent of the plurality of different reagents, a motor sequentially positions the fluorescence microscope relative to sample at a plurality of different fields of view, (3) for each field of view of the plurality of different fields of view, a variable frequency excitation light source sequentially emits a plurality of different wavelengths, (4) for each wavelength of the plurality of different wavelengths, an actuator sequentially positions the fluorescence microscope relative to sample at a plurality of different vertical heights, and (5) for each vertical height of the plurality of different vertical heights, an image is obtained.

Abstract: A fluorescent in-situ hybridization imaging and analysis system includes a flow cell to contain a sample to be exposed to fluorescent probes in a reagent, a fluorescence microscope to obtain sequentially collect a plurality of images of the sample at a plurality of different combinations of imaging parameters, and a data processing system. The data processing system includes an online pre-processing system configured to sequentially receive the images from the fluorescence microscope as the images are collected and perform on-the-fly image pre-processing to remove experimental artifacts of the image and to provide RNA image spot sharpening, and an offline processing system configured to, after the plurality of images are collected, perform registration of images having a same field of view and to decode intensity values in the plurality of images to identify expressed genes.

Abstract: Exemplary embodiments provide methods, mediums, and systems for processing multiplexed image data from a fluorescence in-situ hybridization (FISH) experiment. According to exemplary embodiments, a convolutional neural network (CNN) may be applied to the image data to localize and identify hybridization spots in images corresponding to different sets of targeting probes. The CNN is configured in such a way that it is able to discriminate hybridization spots in situations that are difficult for conventional techniques. The CNN may be trained on a relatively small amount of data by exploiting the nature of the FISH codebook.

Abstract: Methods, systems, and apparatus, including computer programs encoded on a computer storage medium, for obtaining a microscope image that depicts a sample and a plurality of fiducial markers, identifying the plurality of fiducial markers in the image, and using the plurality of fiducial markers to register the image. Identifying the plurality of fiducial markers in the image includes comparing a spatial intensity distribution of a plurality of regions of the image to a reference distribution function.

Abstract: A method of generating a codebook includes obtaining a plurality of gene-identifying code words for the codebook. Each gene-identifying code word is represented by a sequence of N bits that correspond to a best match to a pixel data value identifying a gene. A plurality of negative control code words is generated, and each negative control code word is represented by a sequence of N bits. The negative control code words have an equal number of on-values. On-values of the plurality of negative control code words are evenly distributed across the N bits such that each ordinal position in the sequence of N bits has a same total number of on-bits from the plurality of negative control code words, and a Hamming distance between each negative control code word and each gene-identify code word is at least a distance threshold.

Abstract: A method of method of spatial transcriptomics includes receiving a plurality of images of a sample from an mFISH imaging system and generating a pixel word represented by a sequence of N intensity values. For each pixel, the pixel word is compared to a codebook and a closest matching code word of a plurality of code words is identified. Each code word is represented by a sequence of N bits. The plurality of code words include a plurality of gene-identifying code words and a plurality of negative control code words, the plurality of negative control code words have an equal number of on-values, and on-values of the plurality of negative control code words are evenly distributed across the N bits such that each ordinal position in the sequence of N bits has a same total number of on-bits from the plurality of negative control code words.

Abstract: Methods, systems, and apparatus, including computer programs encoded on a computer storage medium, for obtaining a fluorescence microscope image that depicts a sample and a plurality of fiducial markers, identifying the plurality of fiducial markers in the image, and using the plurality of fiducial markers to register the image. The sample and the plurality of fiducial markers have a common fluorescence color, and identifying the plurality of fiducial markers in the image includes comparing a spatial intensity distribution of a plurality of fluorescent regions of the image to a reference distribution function.

Abstract: A readout probe for use in fluorescent in-situ hybridization imaging has a targeting portion to bind to a first hybridization sequence in an encoding probe that targets an analyte in a sample, a first fluorophore to emit light at a wavelength range, and a first cleaving region between the first fluorophore and the targeting portion.

Abstract: A method of fluorescent in-situ hybridization imaging includes exposing a sample to a first plurality of first readout probes and a second plurality of second readout probes, obtaining a first image of the sample with first readout probes and the second readout probes bound to a first analyte and a second analyte, respectively, so as to emit light at a first wavelength range, treating the sample so as to modify the second readout probes, and obtaining a second image of the sample with the first readout probes bound to the first analyte so as to emit light at the first wavelength range and the second analyte substantially not emitting light at the first wavelength range.

Abstract: A computer program product includes instructions to cause one or more computers to obtain first and second images in the same color channel in the same hybridization step from a fluorescent in-situ hybridization imaging system, and to register the first image and the second image. One or more pixels having a first intensity exceeding a first threshold in the first image and having a second intensity exceeding a second threshold in the second image are identified and classified as activated in a first readout call, and one or more pixels having the first intensity exceeding the first threshold in the first image and the second intensity below the second threshold in the second image are identified and classified as activated in a second readout call.

Abstract: Methods, systems, and apparatus, including computer programs encoded on a computer storage medium, for obtaining a fluorescence microscope image that depicts a sample and a plurality of fiducial markers, identifying the plurality of fiducial markers in the image, and using the plurality of fiducial markers to register the image. The sample and the plurality of fiducial markers have a common fluorescence color, and identifying the plurality of fiducial markers in the image includes comparing a spatial intensity distribution of a plurality of fluorescent regions of the image to a reference distribution function.

Abstract: In acquisition of spatial transcriptomic information, a plurality of images representing a common field of view of a sample are obtained and registered. Each pixel of the registered images is decoded by identifying a code word from a plurality of code words in a code book that provides a best match to data values in the plurality of registered images for the pixel. For each code word identified as a best match and each pixel, whether a bit ratio for an image word for the pixel meets a threshold for the code word is determined. The image word is formed from the data values in the plurality of registered images for the pixel. For at least one pixel that is determined to meet the threshold, a gene associated with the code word is determined. Pixels for which the bit ratio does not meet the threshold are screened.

Abstract: For acquisition of spatial transcriptomic information, one or more filters are applied to deconvolution is performed on each image of a plurality of images to generate a plurality of filtered images, and registration of the plurality of filtered images is performed to generate a plurality of registered and filtered images. For each pixel of at least two pixels of the plurality of registered and filtered images, the pixel is decoded by identifying a code word from a plurality of code words in a code book that provides a best match to data values in the plurality of registered and filtered images for the pixel. For each pixel of at least two pixels, a gene is associated with the code word is determined and an indication that the gene is expressed at the pixel is stored.

Abstract: In a fluorescent in-situ hybridization imaging system performs, as nested loops, the following: (1) a valve sequentially couples a flow cell to a plurality of different reagent sources to expose the sample to a plurality of different reagents, (2) for each reagent of the plurality of different reagents, a motor sequentially positions the fluorescence microscope relative to sample at a plurality of different fields of view, (3) for each field of view of the plurality of different fields of view, a variable frequency excitation light source sequentially emits a plurality of different wavelengths, (4) for each wavelength of the plurality of different wavelengths, an actuator sequentially positions the fluorescence microscope relative to sample at a plurality of different vertical heights, and (5) for each vertical height of the plurality of different vertical heights, an image is obtained.

Abstract: A fluorescent in-situ hybridization imaging and analysis system includes a flow cell to contain a sample to be exposed to fluorescent probes in a reagent, a fluorescence microscope to obtain sequentially collect a plurality of images of the sample at a plurality of different combinations of imaging parameters, and a data processing system. The data processing system includes an online pre-processing system configured to sequentially receive the images from the fluorescence microscope as the images are collected and perform on-the-fly image pre-processing to remove experimental artifacts of the image and to provide RNA image spot sharpening, and an offline processing system configured to, after the plurality of images are collected, perform registration of images having a same field of view and to decode intensity values in the plurality of images to identify expressed genes.

Abstract: Methods, systems, and apparatus, including computer programs encoded on a computer storage medium, for obtaining a fluorescence microscope image that depicts a sample and a plurality of fiducial markers, identifying the plurality of fiducial markers in the image, and using the plurality of fiducial markers to register the image. The sample and the plurality of fiducial markers have a common fluorescence color, and identifying the plurality of fiducial markers in the image includes comparing a spatial intensity distribution of a plurality of fluorescent regions of the image to a reference distribution function.