Table 2. Techniques for multiplexed RNA imaging in living cells.

Technique	Multiplexing	Resolution	Principle	Advantages	Limitations	Ref.
Molecular beacons probes	Up to 4 RNAs	Can detect at single-molecule level	Hairpin DNA probes fluoresce upon hybridizing target RNA	Simple in design, real-time detection	Limited multiplexing, cell-to-cell delivery variations	62–64
Assembled DNA nanostructures	Up to 3 RNAs	Potentially at single-molecule level	Self-assembled DNA nanostructures with multiple probes	Enhanced stability and self-delivery capability	Limited multiplexing, relatively complex and large structures	68–73
Enzyme-free DNA circuits	<3 RNAs	Subcellular	Strand-displacement circuits amplify signal upon RNA binding	Amplified signal for sensitive RNA detection, potential logic analysis	Limited multiplexing, slow kinetics, risk in off-target activation	76–78
DNA probes + nanomaterials	Up to 4 RNAs	Can detect at single-molecule level	DNA-conjugated nanomaterials for enhanced fluorescence	Improved cellular delivery and stability, high sensitivity, multi-function	Limited multiplexing, potential cytotoxicity	64 and 87–90
Bacteriophage-derived RNA labelling tags	Up to 3 RNAs	Can detect at single-molecule level	Phage RNA tag with FP-fused RNA-binding coat protein	Genetically encoded system, real-time and long-term RNA tracking	Limited multiplexing, requires RNA tagging, background signal	91–96
CRISPR-dCas system	Up to 3 RNAs	Can detect at single-molecule level	Orthogonal FP-fused dCas variants or gRNAs with fluorogenic RNA tags	Target endogenous RNAs, genetically encodable	Limited multiplexing, background signal	101–111
Fluorogenic RNA aptamer probes	Up to 3 RNAs	Can detect at single-molecule level	Orthogonal spectrally distinct light-up RNA aptamer/dye pairs	Small tag, low background, genetically encodable, can target endogenous RNAs	Limited multiplexing, moderate brightness	118–129
SeqFRIES	Up to 4 RNAs	Potentially at single-molecule level	Orthogonal fluorogenic RNA aptamer/dye pairs with sequential imaging	Small tag, low background, genetically encodable, can target endogenous RNAs	Limited multiplexing, time needed for repeated cycles	128
RNA-stabilized protein tags	Up to 4 RNAs	Can detect at single-molecule level	RNA recruits FP via RNA-binding motifs for fluorescence	High signal-to-noise ratio, genetically encodable	Limited multiplexing, often requires RNA tagging	131–133