RNA transcripts are a critical piece of the central dogma of biology yet are difficult to image due to their requirement for specific, fluorescent labeling and nanoscopic size. Bruker’s Vutara single-molecule localization microscope (SMLM) and fully integrated SRX software enables users to easily conduct smFISH imaging and analysis experiments.
Bruker offers the tools necessary for successful smFISH experiments including our:
Single molecule fluorescence in situ hybridization (smFISH) — also known as smRNA FISH or RNA FISH — is a cutting-edge technique for studying gene expression in single cells. This technique is similar to FISH in that it is used to visualize DNA, either specific genes or portions of genes, but differs in its unique ability to image and quantify individual RNA molecules. The applications of smFISH transcend life sciences disciplines and include, among others, genomics, cancer biology, neuroscience, and more.
If the research question requires investigation and visualization of RNA instead of DNA, smFISH is the most optimized technique. Fluorescence in-situ hybridization (FISH) operates under the same principles as smFISH — the hybridization of complementary fluorescent probes to a target sequence. However, FISH is optimized for targeting DNA sequences, such as entire genes or portions of genes.
Performing smFISH experiments with the Vutara not only allows for quantification of RNA, but also enables the visualization of the target RNA at subcellular resolution. The ability to image the RNA within the cell opens avenues to studying RNA distribution and localization in relation to other markers or proteins. The combination of the imaging and quantification with smFISH can also be used as a readout for prediction of protein content. The advantage of RNA imaging with smFISH with the Vutara is unique from RNA sequencing data, which only provides quantitative values of RNA abundance.
RNA imaging with smFISH enables quantitative and qualitative gene expression experiments within single cells. With smFISH, researchers can measure cellular mRNA and perform spatial localization and quantification in locations such as the cytoplasm and nucleus. Single RNA molecules can be traced over time for temporal analysis.
With smFISH, RNA targets are imaged via the hybridization and of many short, fluorescently labeled oligonucleotide probes.
Fluorescence in situ hybridization (FISH) is a technique used for the visualization of DNA, either specific genes or portions of genes, within a cell. This technique works via the use of probes that hybridize to the desired genomic region of interest and are labeled with fluorescent dye that is visible with SMLM. FISH is a powerful tool to investigate genomics questions, as it supports chromatin tracing applications. Like FISH, single molecule fluorescence in situ hybridization (smFISH) is a technique optimized for studying gene expression in single cells but is distinct in its ability to localize and quantify individual RNA molecules. With smFISH, RNA targets are imaged via the application of many short, labeled oligonucleotide probes that fluoresce when hybridized to the target sequence.
The advantage of our smFISH module is that it provides both visualization and quantification of RNA within the sample — unlike other techniques like RNA sequencing that only provide quantitative RNA abundance values.
The advantage of using Vutara’s smFISH module is that it provides both visualization and quantification of RNA within the sample — unlike other techniques like RNA sequencing that only provide quantitative RNA abundance values.
Advanced software is necessary to analyze smFISH data collected with SMLM. Bruker’s SRX software contains internal analysis tools, including those for segmentation and statistics, and is compatible with external open-source code. This software also features statistical analysis of distribution of smFISH probes in the cytosol and nucleus.
Bruker provides a variety of protocols for SMLM methods like smFISH. You can find them here.
An additional protocol for sample preparation and imaging with probes can be found here:
Trcek, T., Lionnet, T., Shroff, H. et al. mRNA quantification using single-molecule FISH in Drosophila embryos. Nat Protoc 12, 1326–1348 (2017). https://doi.org/10.1038/nprot.2017.030