How smFISH works?

• multiple probes are designed to target adjacent segments of the mRNA transcript. Each probe is around 50 nucleotides long.

• these probes are labelled with fluorophores, which emit fluorescence when excited by a specific wavelength of light.

Visualising individual transcripts - When probes hybridise their target mRNA, the combined fluorescence makes the individual mRNA molecules visible as distinct puntca under a fluorescence microscope - The intensity of each punctum's fluorescence corresponds to the number of fluorophores, providing a quantitative measure of the transcript

50 nucleotides long

Early methods with electron microscopy - before the development of smFISH, transmission electron microscopy was used to visualise individual mRNA molecules in fibroblasts. - This involved labelling of the poly-A tail of the mRNA with a single large colloidal gold particle and labelling in-situ reverse transcribed cDNA with smaller gold particles