Why does SeekSpace perform ssDNA staining?

Answer:
SeekSpace performs ssDNA nuclear staining to support accurate tissue visualization, image registration, and spatial reconstruction during downstream analysis.

Fluorescent nuclear staining enables clear visualization of tissue boundaries, nuclear distribution patterns, cell-dense regions and local tissue architecture.

Compared with unstained tissue images, ssDNA-stained images provide substantially improved structural information for identifying tissue regions and estimating cellular spatial organization.

An important advantage of ssDNA staining is that it introduces minimal tissue dissociation or segmentation bias in frozen tissue sections, while also having minimal impact on downstream mRNA capture efficiency. This makes ssDNA staining highly compatible with spatial transcriptomic workflows.

Role of ssDNA staining in the SeekSpace workflow

In the SeekSpace workflow, ssDNA-stained images are used together with spatial barcode distribution data to support image-guided spatial reconstruction. During downstream analysis, the stained tissue image is aligned with the spatial barcode coordinate matrix so that:

• Spatial barcode signals can be accurately mapped back onto tissue structure 
• Cell localization confidence can be improved 
• Tissue-covered regions can be distinguished from background regions 
• Abnormal or poorly aligned regions can be identified during QC

The ssDNA image therefore serves as an important spatial reference layer for downstream tissue registration and cell-centered spatial positioning.

Image registration and spatial alignment

In downstream analysis, ssDNA-stained images can be aligned with adjacent H&E images and spatial barcode distribution maps to improve tissue interpretation and spatial localization consistency.

Because ssDNA staining preserves clear nuclear distribution patterns and tissue architecture, it provides a stable structural reference for image registration across different tissue sections and imaging modalities.

For cases where adjacent-section alignment is required, image registration may be performed using third-party image processing software such as GNU Image Manipulation Program (GIMP). Manual adjustment of image scaling, translation, and rotation can help improve correspondence between:

• Tissue morphology 
• Nuclear distribution patterns 
• Spatial barcode coordinates 
• Gene expression regions 

This registration process helps improve interpretation of tissue regions and increases confidence in downstream spatial reconstruction analysis.

Detailed image registration tutorials are available in the SeekSpace documentation.