Single-molecule FISH (smFISH) is a powerful technique that uses multiple fluorophores to visualize specific RNA targets at the single-molecule level in cells . As detection of individual fluorophores by conventional fluorescence microscopy is difficult, in order to achieve single-molecule sensitivity, multiple fluorophore-tagged oligonucleotide probes are designed to target different regions of an RNA transcript , , . Hybridization of multiple probes to the same RNA molecule renders the target RNA sufficiently fluorescent upon excitation, allowing each target transcript to be imaged as a bright spot (Figure 1A, Table 1). Currently, smFISH is regarded as the gold standard approach to visualize intracellular distributions of single RNA transcripts in fixed cells and tissues . However, due to the required fixation steps, RNA dynamics data cannot be easily obtained using smFISH. Additionally, smFISH requires permeabilization to allow oligonucleotide probes to enter the cell and hybridize to the target RNAs and washing to remove unbound probes. Therefore, false-negative data can sometimes result from the loss of RNAs.Figure 1Commonly-used techniques for single-molecule RNA imagingA. smFISH labels an endogenous RNA molecule (blue line) in fixed cells using multiple oligonucleotide probes, with each probe designed to hybridize to a different region of the target RNA. B. The MS2 system requires engineering target RNA to harbor multiple MS2-binding sites (blue line) such that binding of GFP-MS2 fusion proteins (indicated in green and red, respectively) to the MS2-binding sites can cause the target RNA to appear as a bright fluorescent spot. C. MBs are stem-loop forming oligonucleotide probes that are labeled with a reporter (red circle) and a quencher (black circle). In the absence of MB target, the reporter is well-quenched. When hybridized to MB target, the fluorophore is separated from the quencher, resulting in restoration of fluorescence. When a target RNA is engineered to harbor multiple MB targets (blue line), hybridization of the MBs to MB targets can illuminate the engineered target RNA as a bright fluorescent spot. smFISH, single-molecule fluorescence in situ hybridization; MB, molecular beacon.Table 1Feature comparison of the currently-available approaches for single-molecule RNA imagingApproachSingle moleculeLive-cell imagingIn vivo stabilityImaging endogenous RNAsProbe size (kDa)Fluorescent selectionHigh signal-to-backgroundRefs.MBYesYesBackbone chemistry dependentYes∼10Organic fluorophoreYes, , MS2YesYesYESNo∼40Fluorescence proteinNo, , , , FISHYesNoN/AYes∼10Organic fluorophoreNo, , 