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: 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: Exemplary sample processing methods are described that include providing an initial sample to a sample processing system. The sample processing system includes a light-emitting-diode, a temperature control unit, and a fluid supply unit. The methods also include irradiating the initial sample with light emitted from the light-emitting-diode to produce an irradiated sample. The methods may still further include adjusting a temperature of the irradiated sample with the temperature control unit to between 0° C. and 60° C., and contacting the irradiated sample with a fluid from the fluid supply unit. The irradiated sample has a reduction in auto-fluorescence of greater than or about 50% compared to the initial sample. Exemplary sample processing systems are also described that include a light-emitting-diode, a temperature control unit, and a fluid supply unit.

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: 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: 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 calibration slide for calibrating a multiplexed fluorescence in-situ hybridization (FISH) system has a substrate having a surface and a first plurality of beads on the surface. Each bead of the first plurality of beads has at least one binding domain to be bound to a corresponding probe that has a tag and a targeting domain that binds to a binding domain.

Abstract: A method of calibrating a fluorescence in-situ hybridization (FISH) system includes contacting a calibration slide with a plurality of probes, obtaining one or more images of the calibration slide; and calibrating the FISH system based on the one or more images. The calibration slide has a surface with a first plurality of beads. Each bead of the first plurality of beads has one or more binding domains. Each probe has a tag and a targeting domain, and the targeting domain binds a binding domain from the at least one binding domain.

Abstract: This disclosure relates to methods for image registration in fluorescence assays for the detection and quantitation of analytes in a sample using functionalized alignment beads as fiducial markers to improve image registration.

Abstract: Functionalized alignment beads each have a plurality of binding sites, the plurality binding sites including a first subset and a second subset of binding sites. The first subset of binding sites include a first nucleotide sequence selected to bind to a complementary first nucleotide sequence of a first probe that has a fluorophore and that targets the first nucleotide sequence in a sample or in a first targeting probe that targets the sample. The second subset of the plurality of binding sites include a second nucleotide sequence selected to bind to a complementary second nucleotide sequence of a second probe that has a fluorophore and that targets the second nucleotide sequence in a sample or in a second targeting probe that targets the sample. The fluorophore of the first probe has a same emission wavelength and/or same excitation wavelength as the fluorophore of the second probe.

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 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: 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: 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.