Table 1.

Technologies for single-molecule transcript imaging

Method	Single - molecule resolution	Live-cell imaging	Advantages	Disadvantages	References
Traditional FISH	no	no	Probes can be designed for a wide range of genes	Limited sensitivity and specificity; impossible to detect very short transcripts	15
Probes labeled with multiple fluorophores	yes	no	Generic approach suited for most transcripts; established protocols exist	High variability in the number of probes bound to target	21–26,39
Multiple probes labeled with single fluorophores	yes	no	Generic approach suited for most transcripts, low variability in the number of probes bound to target; commercially available labeled probes	Limited in the detection of very short transcripts	28,30,31 http://info.biosearchtech.com/singlemoleculefish
Rolling circle amplification of Padlock probes	yes	no	Can detect very short probes and discern between single nucleotide differences	Protocol requires reverse transcription and amplification steps	34
Branched DNA probes	yes	no	Commercially available labeled probes	Protocol requires amplification	38 www.panomics.com
Quantum dot-labeled probes	yes	yes	Bright and photostable; huge spectral range	Large physical size detrimental to target binding and can cause cell penetration problems	70–75
Sub-diffraction microscopy	yes	yes	Achieves a spatial resolution of 20 nanometers	Slow, expensive instrumentation	64–68
MS2-GFP	yes	yes	No need for external interventions (for example microinjection); yields spatial information	Requires generating transgenes, mRNA tend to form clumps	43,44,78
Molecular beacons	yes	yes	High specificity with no clumping; labeled probes are commercially available	Require micro-injecting probes	52–54