Single-molecule Fish
Single-molecule Fish - Related Videos
06:26
Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH
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Cited by 4 •
2020
Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.
06:59
Nanomanipulation of Single RNA Molecules by Optical Tweezers
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Cited by 13 •
2014
Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5’ and 3’ ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.
12:13
Single-Molecule Imaging of Nuclear Transport
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2010
Single molecule microscopy approch provided novel insights into nuclear transport.
08:18
Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes
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Cited by 2 •
2019
To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.
11:27
Studying DNA Looping by Single-Molecule FRET
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Cited by 13 •
2014
This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.
FAQs
Multiple probes bind different sequences along the same RNA molecule, so their combined fluorescence produces a discrete signal that microscopy can detect. This arrangement helps distinguish target transcripts from isolated nonspecific interactions and supports counting individual RNA molecules. The resulting spots provide a molecular readout that can be related to expression patterns in each cell.
Because the RNA remains in its cellular location, measurements can show whether transcripts are distributed throughout the cell or concentrated in particular regions. Position therefore complements abundance: two cells may contain similar transcript numbers but display different localization patterns. This spatial context helps connect gene expression with cellular organization and biological function.
Counting transcripts separately in individual cells exposes variation that an averaged measurement could conceal. A sample may contain cells with different RNA abundances or distinct expression patterns, allowing researchers to examine transcriptional heterogeneity directly. These differences can be evaluated alongside transcript position, making the technique useful for understanding how seemingly similar cells respond differently.
The method links each fluorescent spot to a counted or localized RNA signal within an individual fixed cell. Researchers can therefore examine transcript number, distribution, and variation on a cell-by-cell basis rather than relying only on a population average. This resolution is especially valuable when expression patterns differ among cells within the same biological sample.
A typical workflow preserves cells by fixation, allows fluorescently labeled oligonucleotide probes to hybridize with target RNA sequences, and uses microscopy to detect the resulting fluorescent spots. Image analysis can then count signals and determine their cellular positions. These stages connect molecular recognition with quantitative and spatial measurements in the original cell context.
Researchers can apply smFISH when they need to examine RNA expression, localization, or variability within individual cells. The approach supports investigations in developmental biology, cell signaling, disease mechanisms, and responses to environmental or pharmacological conditions. Its value lies in connecting transcript-level measurements with the specific cells and spatial patterns in which those measurements occur.