Under a super-resolution microscope, cells can become virtual microarrays with giga-pixel information density. We have demonstrated two barcoding methodologies, spectral and spatial, and shown that by using 7 of the current super-resolution fluorophore pairs, transcripts from 32 genes can be detected simultaneously in single yeast cells using spectral barcoding.
Super-resolution barcoding can be dramatically scaled up with the techniques presented in this paper. We found that the typical transcript size is about 100 nm ( and ,) and that transcripts are uniformly random distributed in yeast cells (Supplementary Fig.21
) at a mean density of 1.9±1.5 barcodes/μm2
=2463, s.d.). There are local regions that contain multiple mRNA barcodes within a diffraction-limited spot (Supplementary Fig.9
, Supplementary Note
) necessitating the use of super-resolution imaging. However, globally, super-resolution barcodes of the 32 profiled genes occupy less than 2% of the 2D super-resolution space available in a yeast cell, leaving a large amount of space open for further multiplexing. With one of the additional available emitters27
=792 genes can be spectrally coded.
Spectral Barcoding has a reconstruction fidelity dependent only on the hybridization of fluorophores, does not require linearization, has a low photon requirement and can be hybridized in a distributed pattern to increase robustness (). These advantages make it an excellent technique for barcoding mRNA and other molecules with unknown structures with the existing SRM dye palette. In comparison, spatial barcoding has a localized hybridization pattern, a lower reconstruction fidelity due to the much higher photon requirements and the tertiary structure of the barcoded molecule (). It has the advantage of scaling and may be more applicable to chromosome and splice isoform barcoding.
Scaling up beyond 1,000 genes will require the implementation of 3D SRM28
to improve the axial resolution by a factor of 20, and the synthesis of an additional far-IR fluorophore as an emitter (18
=18,532 genes can be spectrally coded with 1 additional emitter). At this higher barcode density, more sophisticated computational algorithms will also be necessary to identify barcodes in an intelligent and automatic fashion (Supplementary Note
Super resolution barcoding provides several advantages over existing transcriptional profiling techniques. First, direct imaging of the sample preserves the spatial information both within cells and amongst cells. With the application of light sheet microscopy29
, this technique can be extended into optically thick samples without photobleaching associated with z-sectioning in epi-fluorescence microscopy. This advantage makes it a powerful tool in studying signaling in heterogeneous systems such as microbial ecosystems, tissue and embryos, where interactions among different cellular populations play an essential role in cellular decisions. Second, because of the single-molecule and in situ
nature of the technique, the method is quantitative and avoids intrinsic bias in RNA extraction and conversion to cDNA. Lastly, many cells can be imaged simultaneously under a microscope quickly and throughput can be scaled up without considerable costs, compared to the high cost and long waiting time for sequencing single cells. After the initial cost of the probe set synthesis, the probe set can be hybridized many thousands of times to wild-type and mutant organisms. Super-resolution barcoding even at its current throughput can be a useful follow-up to existing high-throughput transcriptomics techniques by allowing genes of interest to be monitored with single cell resolution in spatially complex samples.
Lastly, the combinatorial labeling scheme can be applied to many types of molecules in situ
. It is a short leap to consider combinatorial labeling of chromosomes and proteins30
, for single cell proteomics and ChIP experiments. We hypothesize that for many types of biochemical techniques, such as microarrays, there is an equivalent in situ
single-cell experiment possible through super-resolution barcoding, removing the need for spatial separation traditionally performed by gels or dilution on a chip.