Abstract
Objective: Despite wide usage of fluorescent dyes in light microscopy and related fields there is no reliable standard for characterization and comparison that would ensure high reproducibility (RP) across different labs and objective identification of the best dye. Typically, dyes (i.e. chromophores) are characterized by their excitation and emission peaks (Ex/Em) and working concentration, this is not enough for RP results especially for functional imaging dyes. The current study aims to develop rigorous dye characterization and application standard protocol. Methods: The pilot study was performed on fluorescent dyes with fast transmembrane voltage sensitivity that were applied to cardiac cells/tissues. Images were recorded by commercial fluorescent widefield and confocal microscopes, and custom built macroscope. The study exclusively focuses on all aspects of dye application: storage, delivery, labeling, imaging, and related analysis. Results: The influence of a large number of variables on image processing was measured. Long term stability and importance of maintaining physiological conditions and healthy cells was estimated as well. It was found that for many dyes the published Ex/Em spectra (especially functional) are measured at nonphysiological conditions and may be shifted by up to 100 nm. Conclusions: The most critical variables for RP are optimal dye storage, loading, maintenance of sample health, Ex/Em provision, bleaching minimization, background removal, max contrast, and signal to noise ration (S/N). Suboptimal conditions in any of the above steps can significantly affect the signal intensity (normalized). Although some of protocol imperfections can be easily compensated (e.g., increasing excitation intensity and detector exposure time) full compensation may be inefficient if not impossible. Shifted Ex/Em spectra missing application protocol data make dye comparison (especially originating from different labs/manufacturers) quite inaccurate.