Detection and Quantification of Single Molecules of mRNA in Individual Cells

<div><p>(A) Schematic diagram depicting the mRNA detection method. Multiple fluorescent probes bind to each mRNA molecule, yielding a bright, localized signal.</p> <p>(B) Merged image of a three-dimensional stack of images from a CHO cell expressing the 7x-tetO gene depicted in (A), where each mRNA is hybridized to FISH probes that bind to the multimeric probe-binding sequence in its 3′-UTR (probe P1-TMR binding to the M1 multimer). Each spot corresponds to a single mRNA molecule.</p> <p>(C) Identification of the spots in the three-dimensional image stack in (C). Each particle found by the image-analysis algorithm is colored differently, showing that the algorithm is accurate and that individual molecules are uniquely identified. The scale bars are 5 μm long.</p>