smICA: an open source repository for mapping the concentration of fluorescently labeled molecules in living cells on the basis of confocal imaging combined with time-correlated single-photon counting

Abstract: Advanced microscopy techniques are essential for visualizing and tracking cellular components and molecules in biomedical research. However, conventional fluorescence microscopy methods often struggle with accurately measuring molecule concentrations in cells. To overcome these limitations, we introduce a novel approach that integrates laser scanning confocal microscopy with time-correlated single photon counting (TCSPC), supported by an open-source analysis tool called smICA (single-molecule Image to Concentration Analyzer). Our method, validated against traditional fluorescence correlation spectroscopy (FCS), offers enhanced accuracy in determining fluorescent molecule concentrations, particularly in cases where molecules are immobile or unevenly distributed. This is demonstrated using fluorescently labeled mRNA in living cells, highlighting the approach's effectiveness.