The authors describe a method for imaging individual mRNA molecules in fixed cells using 48 or more short, singly labeled oligonucleotide probes. This approach allows each mRNA molecule to be identified as a computationally identifiable fluorescent spot via fluorescence microscopy. The method demonstrates simultaneous detection of three mRNA species in single cells and successful application in yeast, nematodes, fruit fly wing discs, mammalian cell lines, and neurons. The technique overcomes issues with heavy labeling, such as probe purification and self-quenching, by using a large number of singly labeled probes. The method is sensitive, specific, and quantitative, providing accurate integer counts of mRNA copy numbers and revealing intracellular locations of mRNAs. The authors also validate the method's effectiveness in detecting multiple mRNAs simultaneously and in capturing spatial information about mRNA localization, making it useful for studying development and gene expression patterns.The authors describe a method for imaging individual mRNA molecules in fixed cells using 48 or more short, singly labeled oligonucleotide probes. This approach allows each mRNA molecule to be identified as a computationally identifiable fluorescent spot via fluorescence microscopy. The method demonstrates simultaneous detection of three mRNA species in single cells and successful application in yeast, nematodes, fruit fly wing discs, mammalian cell lines, and neurons. The technique overcomes issues with heavy labeling, such as probe purification and self-quenching, by using a large number of singly labeled probes. The method is sensitive, specific, and quantitative, providing accurate integer counts of mRNA copy numbers and revealing intracellular locations of mRNAs. The authors also validate the method's effectiveness in detecting multiple mRNAs simultaneously and in capturing spatial information about mRNA localization, making it useful for studying development and gene expression patterns.