MBs are antisense stem-loop forming oligonucleotide probes labeled with a fluorophore at one end and a quencher at the other end  (Figure 1C, Table 1). In the “closed” or “off” configuration, the complementary sequences flanking the loop domain anneal to form a stable stem, placing the quencher in close proximity with the reporter fluorophore, quenching its fluorescence. In the “open” or “on” configuration, target hybridization with the loop domain disrupts the stem, bringing the quencher away from the fluorophore to restore its fluorescence . With careful selection of fluorophore-quencher pair, MB fluorescence can increase 20–100 fold upon hybridization to target RNA . To date, MBs have been the most widely utilized tool for imaging endogenous RNA levels based on ensemble measurements , , , , , , , , , , , , , , , , , . To achieve single-molecule sensitivity, target RNA is engineered with tandem repeats of an MB target sequence, so that multiple MBs can hybridize to a target RNA, illuminating the RNA as a bright spot , , . Despite these advantages, one major limitation for the use of MBs is their biostability. The chemistry of the MB oligonucleotide backbone influences susceptibility to nuclease degradation or nonspecific protein binding, which could cause false-positive signals (FPSs) , .