- MERFISH (Multiplexed Error-Robust Fluorescence In Situ Hybridization) is a powerful spatial transcriptomics technology that enables highly multiplexed imaging of RNA molecules within intact cells and tissues. Developed by Xiaowei Zhuang’s laboratory at Harvard University, this method can detect and localize thousands of RNA species simultaneously at single-molecule resolution.
- The technology works through a combinatorial labeling strategy where each RNA species is encoded by a unique binary barcode. These barcodes are detected through multiple rounds of hybridization and imaging. The system uses specially designed encoding schemes that enable error detection and correction, significantly improving the accuracy of RNA identification. This error-robust encoding is a key feature that distinguishes MERFISH from other spatial transcriptomics methods.
- The MERFISH workflow involves several critical steps. Initially, RNA molecules are labeled with encoding probes containing targeting sequences and readout sequences. Through sequential rounds of hybridization with fluorescently labeled readout probes, imaging, and probe removal, the binary code for each RNA is gradually revealed. Each round of imaging captures a specific bit in the binary code, and the complete sequence of bits uniquely identifies each RNA species.
- Data analysis in MERFISH requires sophisticated computational approaches. The system generates large image datasets that must be processed to identify individual RNA molecules, decode their identities, and map their spatial positions. Advanced algorithms handle image registration, spot detection, and barcode decoding. The error-correction capabilities built into the encoding scheme help distinguish true signals from technical artifacts, improving the reliability of RNA detection.
- Applications of MERFISH span various biological questions. In neuroscience, it helps map neural circuits by revealing gene expression patterns in different cell types. In developmental biology, it enables tracking of spatial gene expression changes during tissue formation. The technology is particularly valuable for understanding cellular organization in complex tissues, studying cell-cell interactions, and identifying rare cell populations.
- One of MERFISH’s key strengths is its high multiplexing capability combined with spatial resolution. The technology can detect thousands of RNA species in a single experiment while maintaining information about their subcellular localization. This comprehensive view of gene expression in spatial context provides insights into cellular organization and function that would be difficult to obtain through other methods.
- Technical considerations for MERFISH implementation include sample preparation, probe design, and imaging requirements. Tissue fixation and permeabilization must preserve RNA integrity while allowing probe access. The design of encoding probes requires careful consideration of specificity and hybridization efficiency. The imaging system must provide sufficient resolution and throughput for detecting single molecules across multiple rounds of hybridization.
- Recent developments have enhanced MERFISH’s capabilities. These include improvements in sample preparation protocols, increased multiplexing capacity, and enhanced analysis tools. Integration with protein detection methods allows simultaneous analysis of RNA and protein expression. Automated systems have been developed to increase throughput and reproducibility.
- Major applications include:
- Cell type identification in complex tissues
- Analysis of cellular organization and tissue architecture
- Study of gene regulatory networks in spatial context
- Investigation of RNA localization patterns
- Mapping of cell-cell interactions
- Analysis of developmental processes
- Disease-related changes in gene expression patterns
