Enhanced mRNA FISH with compact quantum dots.

ABSTRACT

Fluorescence in situ hybridization (FISH) is the primary technology used to image and count mRNA in single cells, but applications of the technique are limited by photophysical shortcomings of organic dyes. Inorganic quantum dots (QDs) can overcome these problems but years of development have not yielded viable QD-FISH probes. Here we report that macromolecular size thresholds limit mRNA labeling in cells, and that a new generation of compact QDs produces accurate mRNA counts. Compared with dyes, compact QD probes provide exceptional photostability and more robust transcript quantification due to enhanced brightness. New spectrally engineered QDs also allow quantification of multiple distinct mRNA transcripts at the single-molecule level in individual cells. We expect that QD-FISH will particularly benefit high-resolution gene expression studies in three dimensional biological specimens for which quantification and multiplexing are major challenges.