[Show abstract][Hide abstract] ABSTRACT: Fluorescence cross-correlation spectroscopy (FCCS) reveals information about the spatiotemporal coincidence of two spectrally well-defined fluorescent molecules in a small observation area at the level of single-molecule sensitivity. To simultaneously evaluate the activities of caspase-3 and caspase-9, we constructed a chimeral protein that consisted of tandemly fused enhanced cyan fluorescent protein (ECFP), monomeric red fluorescent protein (mCherry) and monomeric yellow fluorescent protein (Venus). In HeLa cell lysates, a combination of tumor necrosis factor-α (TNF-α)- and cycloheximide (CHX-)-induced apoptosis was monitored. In this, decreases of cross-correlation amplitudes were observed between ECFP and mCherry and between mCherry and Venus. Moreover, time-dependent monitoring of single cells revealed decreases in the cross-correlation amplitudes between ECFP and mCherry and between mCherry and Venus before morphologic changes were observed by laser scanning fluorescence microscopy (LSM). Thus, our method could predict the fate of the cell in the early apoptotic stage.
Biochemical and Biophysical Research Communications 01/2011; 404(2):706-10. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fluorescence lifetime images of HeLa cells expressing enhanced green fluorescent protein (EGFP) have been measured as apoptosis is induced by tumor necrosis factor-alpha (TNF-alpha) in combination with cycloheximide. The fluorescence lifetime of EGFP is found to decrease after the induction of apoptosis, indicating that the change in environment occurs around the chromophore of EGFP with the apoptosis process. The fluorescence lifetime imaging technique can be used to perform in vivo observation of cell death processes. Fluorescence lifetime measurements are useful to examine the induction of the apoptosis process, even when a morphological change of each cell cannot be observed because of a low spatial resolution.
Photochemical and Photobiological Sciences 07/2009; 8(6):763-7. · 2.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: pH dependence of the fluorescence decay profile of enhanced yellow fluorescent protein (EYFP) depends on the excitation wavelength. The correlation between the fluorescence lifetime and pH in solution is discussed in terms of the acid–base equilibrium of the chromophore of EYFP. Fluorescence lifetime images of EYFP in HeLa cells have also been measured at various values of intracellular pH. A remarkable pH dependence of the fluorescence lifetime image is observed in pH 4.5–6.0, indicating that pH in a single cell can be evaluated using the fluorescence lifetime image of EYFP especially in the acidic condition.
Journal of Photochemistry and Photobiology A: Chemistry. 235:65–71.