Microenvironment Analysis in Squid Axons Using Fluorescence Correlation Spectroscopy and Laser Scanning Microscopy

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We quantified the microenvironment based on the diffusion constants of fluorescent molecules, green fluorescent protein and fluorescence labeled peptide in living cells by using two fluorescence methods, laser scanning microscopy (LSM) for the macroscopic area, and fluorescence correlation spectroscopy (FCS) for the microscopic area. Fluorescence autocorrelation functions (FAFs) were analyzed with a three-component model. The averaged diffusion constants were 5.6×10^<-7> and 2.5×10^<-7> cm^2 /s in squid axons and HEK293 cell, respectively. Taking account of the molecular weight of the chromophore, we concluded that, unexpectedly, molecules move freely inside of the cell with only about 2-3 times slower than that in water. The observed high mobility can explain the rapid localization of the molecules involved in the signal-transduction process. We conclude that FCS is a noninvasive and sensitive method that can be used to examine a wide variety of molecular interactions within subcellular locations in living cells.
日本組織細胞化学会の論文