Multiplexed Error-Robust Fluorescence In Situ Hybridization, known as MERFISH, is a groundbreaking technique in biology. It allows scientists to visualize and count thousands of individual RNA molecules within cells and tissues. This method offers an unparalleled view into gene expression patterns, providing insights into how genes are turned on or off in specific locations. MERFISH represents a significant leap forward in understanding molecular activities that govern cellular processes and tissue organization, focusing on single-molecule detail.
The Core Principle of MERFISH
MERFISH builds upon single-molecule fluorescence in situ hybridization (smFISH) by vastly increasing the number of RNA types that can be simultaneously identified. It achieves this through a clever combination of “multiplexing” and “error-robustness.” Multiplexing identifies many different RNA molecules simultaneously within a single sample.
This is accomplished by assigning a unique binary barcode to each targeted RNA species. Instead of using a different color for each RNA, MERFISH reads out these barcodes through sequential imaging rounds. In each round, specific “readout probes” are introduced that bind to a portion of the barcode, and the fluorescent signal determines a “bit” in the barcode.
Error-robustness ensures accuracy despite potential measurement imperfections. Barcode sequences are carefully designed so that a few misread bits will not lead to an incorrect identification of an RNA molecule. This design allows for the detection and correction of errors, making the measurements highly reliable.
How MERFISH is Performed
The MERFISH protocol involves several distinct stages, from sample preparation to computational analysis. Tissue samples or cells are prepared, often by fixing, to preserve structure and make RNA targets accessible for probe binding. Specialized “encoding probes” are then introduced to bind to the RNA molecules of interest.
These probes carry unique barcode sequences. The core MERFISH process involves multiple rounds of hybridization and imaging. In each round, fluorescently labeled “readout probes” are added to bind to specific parts of the barcodes on the RNA molecules.
After each hybridization, the sample is imaged to detect signals. Probes are then chemically removed or “bleached” to prepare for the next round. This cyclical process is repeated approximately 8 to 20 times, depending on barcode complexity and the number of genes targeted. The precise position of each fluorescent spot is recorded in every imaging round.
Finally, collected images undergo computational analysis. Software decodes the binary barcode for each RNA molecule by piecing together fluorescence across all imaging rounds at each spatial location. This identifies the specific RNA species and its precise coordinates within the cell or tissue. Computational analysis also includes image processing to correct for artifacts, RNA molecule detection, and quality control.
What MERFISH Reveals
MERFISH provides a detailed view of gene expression by mapping the location and quantity of thousands of RNA molecules. It offers a “spatial transcriptomic” perspective, showing not just active genes, but where their RNA products reside within cells and tissues. It can simultaneously measure the copy number and spatial distribution of hundreds to tens of thousands of RNA species in individual cells with high precision.
MERFISH offers exceptional resolution, often reaching single-molecule and single-cell precision. Researchers can identify and count individual RNA molecules, providing a granular understanding of gene expression patterns. Data reveals how RNA molecules are distributed within a cell’s cytoplasm or nucleus, and how cell types are spatially organized within tissues. This detail allows identification of RNA species enriched in specific subcellular compartments, providing insights into their function and regulation.
Impact and Applications
MERFISH has impacted various fields of scientific research by providing a detailed view of gene expression in its native spatial context. In neuroscience, it is advancing brain mapping by allowing scientists to chart the expression patterns of thousands of genes across large brain regions. This helps in understanding complex neural circuits, the diversity of cell types in the brain, and the molecular underpinnings of behavior and cognition. For instance, MERFISH can reveal how gene expression changes in specific cell types contribute to neurodegenerative diseases like Alzheimer’s and Parkinson’s.
In developmental biology, MERFISH helps researchers understand how cells differentiate and organize to form tissues and organs, by visualizing gene expression changes during development. It allows for detailed studies of gene expression changes in tissues affected by genetic disorders, offering insights into the underlying molecular mechanisms. This spatially resolved gene expression information aids in understanding how cells communicate and interact to drive developmental processes.
MERFISH also contributes to immunology and cancer research. It enables detailed profiling of tumor microenvironments, revealing cellular diversity within tumors. By mapping gene expression across entire tumor tissues, researchers can gain a deeper understanding of cellular composition, tumor biology, and responses to various treatments. This can help identify specific oncogenes or immune cell populations, potentially guiding personalized therapies.
The technique’s ability to analyze both fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissue samples, which are commonly found in clinical biobanks, broadens its applicability to translational research. This allows scientists to leverage large archives of patient samples for studies on disease progression and treatment outcomes. MERFISH is a powerful tool for uncovering the molecular landscape of cells and tissues in both healthy and diseased states, driving new discoveries in biology and medicine.