Effect of Fixation and Mounting on Fluorescence Lifetime of Cellular Autofluorescence

Fluorescence lifetime measurements are often performed on live as well as fixed cells and tissues. Fixation and mounting processes are routinely used in cellular research or clinical diagnosis. In this paper, the effects of fixation and mounting on the fluorescence lifetime of cellular autofluorescence were studied by fluorescence lifetime imaging microscopy over time. Two endogenous fluorescent fluorophores, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), showed different results between live cells and fixed cells. The average lifetime of NADH in live HeLa cells was about 1.02 ns, while maintained about 1.57 ns during the fixation periods of 14 days. The average lifetimes of FAD in live and fixed HeLa cells within 11 days were similar around 1.75 ns but increased to 2.10 ns after 12 days. The free and bound states of the two kinds of fluorophores were further analyzed. It was found that the bound-FAD had two different groups, which was related to the cell division cycle. The effect of mounting medium on fluorescence lifetimes was also studied, indicating glycerol has a negative impact on the fluorescence lifetime compared with neutral balsam.