Especially for single-cell transcriptomics, fliFISH provides a more accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals. It takes ideas from stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM) to improve over smFISH. By using photoblinking patterns instead of fluorescence intensity, fliFISH provides more precise and better spatial resolution of RNA hybridization while requiring fewer oligonucleotide probes. fliFISH was applied in mouse pancreatic beta cells in culture and in islet tissue sections to quantitatively identify a radial gradient of single-cell gene expression values for insulin and its relevant transcription factor within normal islets.