Images

Types and format

SAW supports three styles of microscope images:

• nuclei-staining image,

• nuclei-staining + immunofluorescence Images,

• hematoxylin & eosin staining (H&E) image.

Nuclei-staining image

Nuclei-staining has no bias in labeling cells in a tissue slice, making it invaluable for determining tissue area and cell location. Stereo-seq experiments and bioinformatics analysis tools are compatible with two stains, ssDNA and DAPI.

STOmics R&D team has compared and tested various commercialized staining reagents and found that ssDNA staining has the least effect on the downstream mRNA capture rate. The blue-fluorescent DAPI nucleic acid stain is a commonly used nuclear counterstain with high specificity in staining nuclei and can be used alongside other fluorescent reagents. In addition, both ssDNA and DAPI staining allow visualization of tracklines.

ssDNA

Nuclei-staining + immunofluorescence images

Immunofluorescence (IF) is a widely used image-based technique to visualize the subcellular distribution of the proteome in cells. For instance, nuclei can be stained with DAPI, while T cells can be identified using CD3. Multiplex IF (mIF) can be tagged on the tissue section and scanned by the fluorescence microscope simultaneously. Stereo-seq experiments and bioinformatics analysis tools are compatible with DAPI and up to 6 user-defined IFs, enabling spatial discovery across a tissue sample.

DAPI

APAK3_IF

TESK2_IF

Hematoxylin & eosin staining (H&E) image

Hematoxylin and Eosin stain (H&E stain) is a widely used tissue stain that provides histologic information for medical diagnosis and is considered the gold standard. Hematoxylin mainly stains cell nuclei in purplish blue, and eosin colors cytoplasm and extracellular matrix in different shades of pink. By integrating spatially resolved sequencing-based feature data generated from Stereo-seq with the H&E staining image, the morphology of cells can be linked with spatially localized feature expression. The combined use of histology images and feature expression co-representation increases the amount of information that a tissue slice can provide.

H&E

Here is a summary of supported input image types and formats, for SAW and StereoMap :

Image Types	Image Format	Magnification Objectives	Stereo-seq Chip Size
Nuclei-staining image e.g. ssDNA, DAPI	8 or 16-bit grayscale single-page image	10X	Up to 2 cm x 3 cm
Nuclei-staining + immunofluorescence image e.g. DAPI + up to 6 IFs	8 or 16-bit grayscale single-page image	10X	Up to 1 cm x 1 cm
Hematoxlin & Eosin (H&E) staining image	24-bit color image	10X	Up to 1 cm x 1 cm

Tracklines

Stereo-seq chip surface has periodic tracklines (horizontal and vertical lines) to assist base calling and image registration. These tracklines are areas where the capturing probe was unloaded and will appear as narrow lines on the spatial feature expression density heatmap. Tissue staining and imaging standard operating procedures (SOPs) for the Stereo-seq technology have been designed and tested to minimize their impact on downstream mRNA capture rates and enhance the visibility of tracklines in microscopy images. Because the lines on the density heat map and the microscopy image are the embodiment of tracklines from the chip, these lines can be used as position markers for aligning images.

Here are examples of tracklines on the image and the density heatmap.

The images are adjusted to optimize visibility.

Tracklines on fluorescence image	Tracklines on color image	Tracklines on density heat map
Tracklines show as black lines.	Tracklines show as lighter white lines.	Tracklines show as black lines.

SAW embeds automated image processing algorithms to identify the boundaries of tissue and cells and detect tracklines on the Stereo-seq chip for aligning the image with the feature expression matrix. In cases where the tracklines cannot be detected or the tissue/cell boundaries are vague, you may need to outline or align manually.