Background Routine black box approaches quantify fluorescence intensity to profile the uptake of fluorophores, providing limited insight into microscopic events. Spatial intensity distribution analysis has previously been reported to quantify oligomerisation and number of particles from selected regions and profile intracellular distributions of labelled moieties. Methods In this study, the concentration and time-dependent behaviour of CellTrace™ calcein red-orange (AM) intracellular accumulation was examined in colorectal adenocarcinoma cell line and bovine aortic endothelial cells. Monolayers were subjected to fluorescence correlation spectroscopy, fluorescence intensity and SpIDA measurements to determine differences in the rate and extent of intracellular accumulation. Results Intracellular accumulation data derived from Spatial intensity distribution analysis were found to correlate with that of fluorescence correlation spectroscopy and fluorescence intensity profiles. The extent of intracellular accumulation was found to be time and concentration-dependent in both cell lines examined, with no significant differences in the rate of intracellular accumulation. Conclusions Spatial intensity distribution analysis applied at 'proof of concept' level is a rapid and user-friendly tool that can be applied to the quantification of intracellular concentration and kinetics of fluorophore uptake. General significance Confocal imaging as a routinely implemented tool for profiling fluorescently-labelled species is often under-exploited for yielding quantitative parameters. © 2014 Elsevier B.V. All rights reserved.
- Confocal microscopy
- Fluorescence correlation spectroscopy
- Spatial intensity distribution analysis