In Situ Sequencing (ISS) is a technique that determines the genetic code of molecules while they remain fixed in their original cellular and tissue locations. Instead of extracting and homogenizing a biological sample, ISS performs the sequencing reaction directly on a thin slice of tissue or a group of cells. This method captures messenger RNA (mRNA) expression data at single-cell or subcellular resolution, providing both molecular information and precise spatial coordinates. The approach generates high-resolution genetic data without destroying the sample’s structural integrity, shifting the paradigm to spatially-aware molecular mapping.
The Need for Spatial Genomics
Traditional methods for analyzing gene expression, such as bulk RNA sequencing (RNA-seq), require scientists to break down a tissue sample. This averages genetic signals across millions of cells, masking subtle differences and obscuring cellular heterogeneity. Even advanced techniques like single-cell RNA-seq require cell dissociation, resulting in an irreversible loss of surrounding context.
The physical location of a cell within a tissue is linked to its function and state. For instance, a cancer cell’s interaction with immune cells differs based on whether it is at the invasive edge or deep within the tumor, and this difference is reflected in its gene expression. Losing this spatial information prevents accurate study of cell-to-cell communication or how the environment influences a cell’s molecular profile. Spatial genomics, including ISS, was developed to integrate the molecular “what” with the anatomical “where.”
How In Situ Sequencing Works
In Situ Sequencing combines molecular biology and high-resolution imaging, starting with sample preparation. The tissue is fixed to preserve morphology and treated to convert target mRNA into stable complementary DNA (cDNA) strands. Specialized DNA probes, known as padlock probes, are introduced to bind only to the specific cDNA targets of interest.
When a padlock probe binds to its target cDNA, the ends are chemically sealed by a ligase enzyme, forming a circular DNA molecule. This circular template undergoes Rolling Circle Amplification (RCA), which repeatedly copies the DNA to create a dense, localized cluster of identical sequences called a Rolling Circle Product (RCP). This amplification step is performed directly within the tissue, creating a bright, detectable signal at the location of the original mRNA molecule.
The cyclic sequencing reaction reads the genetic code one nucleotide at a time. In each cycle, a fluorescently labeled probe binds to the RCP, and the color corresponds to a specific nucleotide (A, T, C, or G). A high-powered microscope captures the image, recording the color and precise spatial coordinates of every RCP. The fluorescent label is then washed away, and the process repeats with a new probe to read the next nucleotide. By performing multiple cycles, researchers decode the full sequence of hundreds of target genes and precisely map their locations, transforming the tissue slice into a molecular coordinate map.
Mapping Cellular Organization in Tissues
The output of In Situ Sequencing is a molecular atlas of the tissue. This atlas allows researchers to visualize the expression patterns of hundreds of genes simultaneously, providing a view of cellular organization. By correlating gene expression profiles with their exact positions, scientists can identify previously unrecognized cell types based on their unique molecular signatures and anatomical niches.
This capability is transformative for studying complex biological structures where cellular interactions are important, such as the brain and the tumor microenvironment. ISS has been used to map the distribution of neuronal subtypes in the brain, revealing how these cells organize into functional circuits. In cancer research, the technology enables the spatial mapping of immune cells, fibroblasts, and tumor cells, showing how their proximity influences tumor progression and therapeutic response. ISS also provides spatial context to molecular changes in neurodegenerative conditions, such as the accumulation of amyloid-beta plaques in Alzheimer’s disease.
Expanding Our Understanding of Biology
In Situ Sequencing bridges genomics and histology by linking molecular data directly to tissue morphology. This spatial context provides a deeper understanding of how pathological mechanisms unfold and how biological systems are organized at the molecular level. Visualizing molecular events within an intact biological specimen allows scientists to pinpoint the cellular origins of disease and development.
This molecular mapping impacts drug discovery by helping to localize and validate new therapeutic targets with greater precision. For personalized medicine, ISS offers the prospect of tailoring treatments based not just on the presence of a mutation, but on the spatially-resolved molecular architecture of a patient’s disease, such as the distribution of drug-resistant cell clones within a tumor. The technique is helping to build comprehensive cell atlases, changing how researchers study health, disease, and development by providing an integrated view of the genome in its native habitat.