One appealing application of single transcript imaging is the validation of regulatory interactions. A traditional approach entails engineering mice in which a regulatory gene of interest is deleted, and searching for putative target genes with modified expression levels61
. Single-molecule FISH can enable detecting such targets in wild-type tissue by hybridizing to a sample probes for the regulatory gene and for a putative target gene. A measured positive correlation in the transcript abundance could imply either a direct regulation or alternatively regulation by a common upstream component. Single-molecule transcript imaging can also provide valuable information on the behavior of network motifs, modular circuit components such as feedback and feedforward loops which are highly abundant in transcriptional networks and often comprise only a few genes62
. The simultaneous quantitative in-situ
measurements of a handful of different transcripts can shed light on the behavior of these motifs within their tissue context. In a tumor single-molecule transcript imaging can highlight the role of transcriptional heterogeneity in tumor progression and the relation between spatial context and phenotypic states of cells, represented by their expression signatures.
Single-molecule transcript imaging techniques can be combined with high-throughput expression analysis in two complementary ways. One would be to start out with large gene expression screens that would suggest putative genes of interest, the detailed in-situ expression of which would then be described using single-molecule FISH. An alternative approach would be to start with an unbiased mapping of a tissue using a panel of single-molecule FISH probes to detect an interesting expression pattern in terms of spatial distribution within a tissue or an unusual co-expression pattern of a few genes in isolated cells. One could then enrich for such cells using FACS or laser capture and extend this core gene expression signature with high-throughput genome-wide expression measurements. This approach could provide a detailed description of rare cell populations residing within a tumor, such as putative cancer stem cells
A technical limitation of single-molecule transcript imaging is the inability to spatially resolve single molecules when they are closer than the diffraction limit, typically 200 nm. This could be a significant problem in highly expressed genes such as ribosomal components especially in smaller organisms such as yeast25
, and when mRNA molecules are physically localized in transport particles63
. Techniques that can address this limitation are sub-diffraction-limit microscopy methods such as STED64
, which enable resolving fluorescent molecules with nanometer resolution67
. While sub-diffraction microscopy outperforms other technologies in spatial resolution, enabling probing molecular structures in fixed and even live cells68,69
, scaling the technology to comprehensively measure gene expression in-situ
in many cells and in tissues is still a challenge due to long recording times, high intensity illumination, the prolonged use of which could potentially be harmful to the sample and expensive instrumentation.
Another exciting recent development is the use of probes coupled to quantum dots70–73
. The brightness of quantum dots makes them especially attractive for studying tissues, where cellular autofluorescense often masks the signal. Oligonucleotide probes labeled with quantum dots have been used to detect transcripts in cells and tissues70–72
, in paraffin embedded tissue74
and even in live cell imaging75
. Some limitations of using quantum dots, such as reduced permeability and steric hindrance difficulties when binding the targets, mainly caused by their relatively large size compared to conventional probes, as well as their tendency to turn on and off (‘blinking’)76
still limit the use of quantum-dot labeled probes for transcript imagingbut their attractive photophysical properties suggest a huge potential for single-molecule detection.
Finally, a challenge for the future is to combine transcript imaging approaches with quantitative measurements of other cellular constituents, namely proteins and DNA. While protocols tailored to simultaneously perform RNA FISH and immunofluorescense have been shown to be successful in some cases56,69
, their generic use is still limited by the variabilities inherent in immunofluorescense. Combination of RNA FISH with immunofluorescense or with GFP protein measurements will facilitate decoupling the relative contributions of transcriptional and translational regulation in cells and tissues, whereas combination with DNA FISH can address the expression variability of different clones within a tumor tissue. Such analysis can provide important insights into the combined regulation of protein expression in complex tissues.