Quantitative analysis of Radachlorin fluorescence lifetime images using phasor plot

Abstract

In this work we present investigation of Radachlorin fluorescence lifetime images of cells of the three lines: HeLa, A549 and 3T3. The collected time-resolved fluorescence signals, obtained from different intracellular structures demonstrated that Radachlorin fluorescence lifetime inside the cells may vary within a quite wide range from 3.5 to almost 6 nanoseconds. Thorough investigation of fluorescence signals of Radachlorin in organic and water solutions at various pH values allowed us to conclude that the observed variations of Radachlorin fluorescence lifetimes inside the cells are primarily due to different acidity of microenvironment in lysosomes and other intracellular compartments. The assumption was validated by cells treatment with bafilomycin leading to malfunction of lysosome proton pumps and generation of more homogeneous distributions of pH values inside the cells. Comparative analysis of FLIM images of control samples and those treated with bafilomycin demonstrated much lower variations of Radachlorin fluorescence lifetime in the latter case. The most prominent variations of Radachlorin fluorescence lifetimes were observed within the pH range from 4 to 8, which perfectly corresponds to the typical pH values in living cells. Besides variations of Radachlorin fluorescence lifetimes with the solution pH we observed some increase of this value with ethanol concentration in the alcohol-water solution. Relative quantum yield of Radachlorin fluorescence and singlet oxygen generation rate was also studied as a function of microenvironment acidity. The financial support from Russian Science Foundation under the grant # 21-72-10044 is gratefully acknowledged.

Speaker

Andrey Belashov
Ioffe Institute
Russia

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