merfish readout probes  (Bio-Synthesis Inc)

Journal: eLife

Article Title: Three-dimensional single-cell transcriptome imaging of thick tissues

doi: 10.7554/eLife.90029

Figure Lengend Snippet: Images of nuclei (DAPI), total polyA mRNA, and two MERFISH bits were obtained using epifluorescence (Epi) and spinning-disk confocal microscopy respectively. Both epifluorescence and confocal images were taken with 1 s exposure time. The MERFISH bit-1 and bit-2 images were high-pass filtered to remove cellular background.

Article Snippet: MERFISH readout probes , conjugated to either Cy5, Cy3B, or Alexa488 dye molecules through a disulfide linkage, were purchased from Bio-Synthesis, Inc.

Techniques: Confocal Microscopy

Journal: eLife

Article Title: Three-dimensional single-cell transcriptome imaging of thick tissues

doi: 10.7554/eLife.90029

Figure Lengend Snippet: ( a ) A single-bit high-pass-filtered MERFISH confocal image of 242 genes in a brain tissue section taken with an exposure time of 0.1 s (left) and a magnified view of a single cell marked by the white box in the left image (right). ( b ) The correlation between the copy number of individual genes detected per field of view (FOV) using 0.1 s exposure time and those obtained using 1 s exposure time. The median ratio of the copy number and the Pearson correlation coefficient r are shown. The copy number per gene detected using 0.1 s exposure time is 24% of that detected using 1 s exposure time. ( c ) The same image as in ( a ) but after enhancement of signal-to-noise ratio (SNR) by a DL algorithm. ( d ) The same as ( b ) but after DL was used to enhance the SNR of the 0.1 s images. The copy number per gene detected with 0.1 s exposure time after DL-based enhancement is 89% of that detected using 1 s exposure time. Figure 1—source data 1. This source data file contains source data for .

Article Snippet: MERFISH readout probes , conjugated to either Cy5, Cy3B, or Alexa488 dye molecules through a disulfide linkage, were purchased from Bio-Synthesis, Inc.

Techniques:

Journal: eLife

Article Title: Three-dimensional single-cell transcriptome imaging of thick tissues

doi: 10.7554/eLife.90029

Figure Lengend Snippet: ( a ) Number of RNA molecules detected per field of view (FOV) at a single z-plane at the tissue depths of 10 µm and 90 µm in the first bit of the 242-gene MERFISH measurements in a 100-µm-thick section of the mouse cortex. ( b ) Logarithmic distribution of integrated photon counts of individual RNA molecules at the tissue depths of 10 µm and 90 µm identified in ( a ). In each boxplot, the midline represents the median value, the box represents the interquartile range (IQR), the lower whisker represents the smaller of the minimum data point or 1.5× IQR below the 25th percentile, and the upper whisker represents the greater of the maximum data point or 1.5× IQR above the 75th percentile. Molecule number (n, from left to right): 7565, 7914. ( c ) Number of RNA molecules detected per FOV at a single z-plane at tissue depths of 10 µm and 190 µm of in the first bit of the 156-gene MERFISH measurements in a 200-µm-thick section of the mouse hypothalamus. ( d ) Logarithmic distribution of integrated photon counts of individual RNA molecules at the tissue depths of 10 µm and 190 µm identified in ( c ). In each boxplot, the midline represents the median value, the box represents the IQR, the lower whisker represents the smaller of the minimum data point or 1.5× IQR below the 25th percentile, and the upper whisker represents the greater of the maximum data point or 1.5× IQR above the 75th percentile. Molecule number (n, from left to right): 9611, 7238. Figure 1—figure supplement 2—source data 1. This source data file contains source data for .

Article Snippet: MERFISH readout probes , conjugated to either Cy5, Cy3B, or Alexa488 dye molecules through a disulfide linkage, were purchased from Bio-Synthesis, Inc.

Techniques: Whisker Assay

Journal: eLife

Article Title: Three-dimensional single-cell transcriptome imaging of thick tissues

doi: 10.7554/eLife.90029

Figure Lengend Snippet: ( a ) Example high-pass-filtered bit-1 images of a 242-gene MERFISH measurement in a 100-µm-thick section of mouse cortex stained with different concentrations of encoding probes. The concentration values refer to the concentration of each individual encoding probe. ( b ) Distribution of integrated photon counts of individual RNA molecules identified at different encoding probe concentrations. In each boxplot, the midline represents the median value, the box represents the interquartile range (IQR), the lower whisker represents the smaller of the minimum data point or 1.5× IQR below the 25th percentile, and the upper whisker represents the greater of the maximum data point or 1.5× IQR above the 75th percentile. Molecule number (n, from left to right): 31606, 29190, 83644. The signals from individual RNA molecules increased with the encoding probe concentration and reached saturation at ~1.0 nM per probe. We thus used 1 nM encoding probe concentrations for staining thick-tissue samples. ( c ) A 100-µm-thick mouse brain slice was stained with the 242-gene MERFISH encoding probes, followed by sequential hybridization with readout probes corresponding to the first, second, third, and fourth bit of the barcodes, each bit using a different readout probe concentration. High-pass-filtered bit-1, bit-2, bit-3, and bit-4 MERFISH images (each with a different concentration of readout probes) are shown. ( d ) Distribution of integrated photon counts of individual RNA molecules identified at different readout probe concentrations. In each boxplot, the midline represents the median value, the box represents the IQR, the lower whisker represents the smaller of the minimum data point or 1.5× IQR below the 25th percentile, and the upper whisker represents the greater of the maximum data point or 1.5× IQR above the 75th percentile. Molecule number (n, from left to right): 1939, 3815, 3869, 5248. The signal increased with readout probe concentration, but the background also increased when the probe concentration reached beyond 5 nM. We thus used 5 nM readout probe concentration for thick-tissue imaging. In addition to the probe concentrations, we also optimized readout probe incubation time. ( e ) The number of RNA molecules per field of view per z-plane and the normalized intensity of individual molecules at different tissue depths. The encoding probe concentration was 1 nM per encoding probe, the readout probe concentration was 5 nM, and the readout probe incubation time was 25 min for these measurements. Figure 2—figure supplement 1—source data 1. This source data file contains source data for .

Article Snippet: MERFISH readout probes , conjugated to either Cy5, Cy3B, or Alexa488 dye molecules through a disulfide linkage, were purchased from Bio-Synthesis, Inc.

Techniques: Staining, Concentration Assay, Whisker Assay, Slice Preparation, Hybridization, Imaging, Incubation

Journal: eLife

Article Title: Three-dimensional single-cell transcriptome imaging of thick tissues

doi: 10.7554/eLife.90029

Figure Lengend Snippet: ( a ) Total copy number of decoded RNAs detected per field of view (FOV) per z-plane at different tissue depths in a 242-gene MERFISH measurement of the 100-µm-thick section. ( b ) Pearson correlation coefficients of RNA copy number of individual genes per FOV per z-plane detected at different tissue depths by MERFISH with the FPKM values measured by bulk RNA-seq. ( c ) Correlation of RNA copy number of individual genes per FOV per z-plane detected in the entire 100-µm-thick section by MERFISH with the FPKM values obtained by bulk RNA-seq. The Pearson correlation coefficient r is shown. ( d ) Example images of gel-embedded beads acquired in two rounds of imaging. Buffer exchanges were performed between imaging rounds, mimicking the MERFISH protocol. Because the gel expanded to a different extent in different rounds, the positions of beads changed from round to round in x, y, and z directions. Circles mark beads identified in both imaging rounds. Because the gel size changed, the x and y positions of the beads changed, and the brightness of these beads also changed due to the shift in their z positions. Arrows highlight beads detected in one of the imaging rounds, but not the other, due to the gel-size change, which move these beads out of focus. Figure 2—figure supplement 2—source data 1. This source data file contains source data for .

Article Snippet: MERFISH readout probes , conjugated to either Cy5, Cy3B, or Alexa488 dye molecules through a disulfide linkage, were purchased from Bio-Synthesis, Inc.

Techniques: RNA Sequencing Assay, Imaging

Journal: eLife

Article Title: Three-dimensional single-cell transcriptome imaging of thick tissues

doi: 10.7554/eLife.90029

Figure Lengend Snippet: ( a ) Quantification of gel expansion factor in various buffers used in the MERFISH protocol. The initial gel size was the same as the coverslip, and the expansion factor after buffer exchange was determined as the ratio between the gel size after buffer exchange and the coverslip size. ( b ) In each round of MERFISH imaging, the sample is incubated with the readout probes in a wash buffer (containing either 10% ethylene carbonate [EC] or 10% formamide) for a duration of 15 min. Subsequently, the sample was rinsed with the wash buffer (without readout probes) to remove any excessive readout probes, followed by a treatment with imaging buffer containing glucose-oxidase-based or protocatechuate 3,4-dioxygenase rPCO-based oxygen scavenger system to reduce photobleaching. After the imaging process, the sample is treated with tris(2-carboxyethyl) phosphine buffer to cleave off the fluorescent dye linked to the readout probe through a disulfide bond, and finally washed with a solution of 2× saline-sodium citrate (SSC). All buffers, including wash, imaging, and cleavage buffers, contained 2× SSC. Gel-expansion factor in these buffers used in the MERFISH protocol was quantified and shown here. Reagents marked by * were selected for final use in the 3D thick-tissue MERFISH experiment. The dashed line highlights the expansion factor in the 2× SSC buffer alone. ( c ) XZ projection images of fiducial beads embedded in a gel undergoing buffer exchange for the indicated time period. Wash buffer containing 15% EC in 2× SSC causes noticeable gel distortion, which was recovered after 15 min in 2× SSC buffer without EC. Figure 2—figure supplement 3—source data 1. This source data file contains source data for .

Article Snippet: MERFISH readout probes , conjugated to either Cy5, Cy3B, or Alexa488 dye molecules through a disulfide linkage, were purchased from Bio-Synthesis, Inc.

Techniques: Buffer Exchange, Imaging, Incubation, Saline

Journal: eLife

Article Title: Three-dimensional single-cell transcriptome imaging of thick tissues

doi: 10.7554/eLife.90029

Figure Lengend Snippet: ( a ) 3D images of DAPI and total polyA mRNA from a single field of view (FOV) in a 100-µm-thick mouse brain tissue slice (top), alongside a single z-plane at tissue depth of 50 µm marked by the yellow box in the top image (bottom). ( b ) Maximum-projection images of 10 consecutive 1 µm z-planes of individual MERFISH bits of the region marked in yellow box in the bottom panel of ( a ). ( c ) RNA molecules identified in the same region as in ( b ) with RNA molecules color coded by their genetic identities. ( d ) The RNA copy number for individual genes per unit area (100 2 µm 2 ) per z-plane detected in the 100 µm MERFISH measurements of mouse cortex versus the FPKM from bulk RNA-seq. The Pearson correlation coefficient r is shown. ( e ) The Pearson correlation between RNA copy number for individual genes per z-plane at different tissue depths detected by MERFISH and the FPKM values of individual genes from bulk RNA-seq. ( f ) Number of detected RNA molecules per FOV at different tissue depths. ( g ) The RNA copy number for individual genes per unit area (100 2 µm 2 ) per z-plane detected in the 100 µm MERFISH measurements of mouse brain sections in this work versus that detected by 10-µm-thick-tissue MERFISH measurements using an epifluorescence setup . The Pearson correlation coefficient r is shown. ( h ) The RNA copy number of individual genes per cell detected in the 100-µm-thick-tissue section versus that in individual 10-µm-thick z-ranges of the same sample. The 100-µm-thick section was evenly divided into ten 10 µm z-ranges to determine the latter. Data in this figure were generated from a 100-μm-thick section of the cortical region collected from an adult mouse. Figure 2—source data 1. This source data file contains source data for .

Article Snippet: MERFISH readout probes , conjugated to either Cy5, Cy3B, or Alexa488 dye molecules through a disulfide linkage, were purchased from Bio-Synthesis, Inc.

Techniques: RNA Sequencing Assay, Generated

Journal: eLife

Article Title: Three-dimensional single-cell transcriptome imaging of thick tissues

doi: 10.7554/eLife.90029

Figure Lengend Snippet: ( a ) UMAP visualization of subclasses of cells identified in a 100-μm-thick section of the mouse cortex. Cells are color coded by subclass identities. IT: intratelencephalic projection neurons; ET: extratelencephalic projection neurons; CT: cortical-thalamic projection neurons; NP: near projection neurons; OPC: oligodendrocyte progenitor cells; Oligo: oligodendrocytes; Micro: microglia; Astro: astrocytes; VLMC: vascular leptomeningeal cells; Endo: endothelial cells; Peri: pericytes; SMC: smooth muscle cells; PVM: perivascular macrophages. ( b ) 3D spatial maps of the identified subclasses of excitatory neurons (left), inhibitory neurons (middle), and non-neuronal cells (right) within the 100 μm mouse cortex section. ( c ) UMAP visualization of major cell types identified in a 200-μm-thick section of the mouse anterior hypothalamus. Cells are color coded by cell type identities. ( d ) 3D spatial maps of the excitatory neuronal (left), inhibitory neuronal (middle), and non-neuronal (right) cell clusters identified in the 200-μm-thick mouse hypothalamus section. Cells are color coded by cell cluster identities in the top panels and two example excitatory neuronal (left), inhibitory neuronal (middle), and non-neuronal (right) cell clusters are shown in the bottom panels. ( e ) Boxplots showing the distributions of the nearest-neighbor distances from cells in individual inhibitory neuronal subclasses to cells in the same subclass (‘to self’) or other subclasses (‘to other’) in the mouse cortex obtained from the thick-tissue 3D MERFISH data. Cell numbers (n, from left to right): 44, 274, 125, 1161, 136, 797, 26, 330. *FDR <0.01 was determined with the Wilcoxon rank-sum one-sided test and adjusted to FDR by the Benjamini and Hochberg procedure. Only inhibitory neuronal clusters with at least 20 ‘self-self’ interacting pairs were examined and plotted. In each boxplot, the midline represents the median value, the box represents the interquartile range (IQR), the lower whisker represents the smaller of the minimum data point or 1.5× IQR below the 25th percentile, and the upper whisker represents the greater of the maximum data point or 1.5× IQR above the 75th percentile. ( f ) Distributions of the nearest-neighbor distances among all interneurons derived from the thick-tissue 3D MERFISH data of the mouse cortex. The distribution is fitted with a bimodal distribution (blue curve) with the two individual Gaussian peaks shown in red and green. ( g, h ) Same as ( e, f ) but for inhibitory neurons in the mouse anterior hypothalamus obtained from the thick-tissue 3D MERFISH data. Cell numbers in g (n, from left to right): 1404, 1222, 198, 567, 845, 1315, 866, 1263, 1149, 873, 455, 1517, 387, 1490, 409, 1321, 277, 1151, 773, 1871, 379, 830, 174, 1013, 598, 449, 811, 158, 194, 728, 103, 771, 119, 667, 217, 580, 98, 704, 1095, 1339, 186, 362, 82, 438, 41, 302. Data in this figure were generated from a 200-μm-thick section of the hypothalamic region collected from an adult mouse. Figure 3—source data 1. This source data file contains source data for .

Article Snippet: MERFISH readout probes , conjugated to either Cy5, Cy3B, or Alexa488 dye molecules through a disulfide linkage, were purchased from Bio-Synthesis, Inc.

Techniques: Whisker Assay, Derivative Assay, Generated