Fluorescence lifetime images of the endogenous nicotinamide adenine dinucleotide (NADH), which is well known as autofluorescence chromophores, were obtained for rat normal fibroblast cells (WFB) and H-ras oncogene-transfected WFB cancer cells (W31) and for human normal lung fibroblast (MRC-5) and human lung large carcinoma (HCI-H661). In both cases, the average lifetime of the NADH autofluorescence was shorter in cancer cells than in normal cells, indicating that the difference in metabolism between healthy and cancer cells might alter the level of coenzymes such as NADH. It is also shown that application of nanosecond pulsed electric field (nsPEF) induces apoptosis in these cell, resulting in the morphological changes and lengthening the autofluorescence lifetime of NADH. Furthermore, we found that nsPEF more efficiently affected cancer cells than normal cells in cell viability, suggesting the possibility of drug-free cancer therapy by nsPEF.
Intracellular pH of a single cell can be imaged using FLIM of enhanced green fluorescent protein (EGFP). The
correlation between the intracellular pH and the fluorescence lifetime of EGFP in HeLa cells is explained by considering
the pH-dependent acid-base equilibrium of the p-hydroxybenzylidene-imidazolidinone structure of the chromophore of
EGFP. The equilibrium between different forms of chromophore depends on pH of the medium. The equilibrium
constant between the neutral and anionic EGFP chromophores in HeLa cells is obtained by analyzing the fluorescence
lifetimes observed with different values of intracellular pH. The intracellular pH dependence has been also observed in
HeLa cells where enhanced yellow fluorescent protein (EYFP) is expressed. The pH dependence of the fluorescence
lifetime of EYFP may result from the pH dependence of the molecular structure of the protein bound ionic form of
EYFP or the conformational change of the EYFP chromophore induced by lowering pH. The fluorescence lifetimes
both of EGFP and of EYFP are not uniform in the cell. At each pH, for example, the fluorescence lifetime of EGFP
located near the outer cell membrane is shorter than those located inside cell, whereas the lifetime of EYFP located near
the outer cell membrane is longer than those located inside the cell. These differences are ascribed to the different
distribution of the electric field surrounding the fluorescent chromophore in the cells, implying that the chromophores of
EGFP and EYFP show the opposite electric field effects of the fluorescence lifetime to each other. The fact that the
fluorescence lifetime of BCECF in solution is different from the one observed at the same pH in intact cells of
Halobacterium salinarum has been also ascribed to the local field produced by membranes in vivo.
A dramatic change occurs in the cellular microenvironment during cell stress, but it has been difficult to follow these
changes in vivo. Here, fluorescence lifetime imaging (FLIM) microscopy has been used to examine stress-induced
changes in the microenvironment in a single cell. It is observed that the fluorescence lifetime of HeLa cells expressing
an enhanced green fluorescent protein (EGFP)-tudor fusion protein changes under stress. The change in the fluorescence
lifetime appears to be due to an alteration in the local electric field in the protein matrix surrounding the chromophore of
EGFP. In fact, the fluorescence lifetime of the GFP chromophore in a polyvinyl alcohol film is found to decrease in the
presence of an electric field, based on the measurements of the
field-induced change in the fluorescence decay profile.
The results indicate that the rate of the non-radiative process of the chromophore of GFP is enhanced by an applied
electric field. The FLIM method allows noninvasive determination of the status of the individual cells.
Electroabsorption (EA) or Stark effect spectroscopy is a widely used method for characterizing the dopant
molecules in polymer films and the films themselves. In this method the spectrum of a small (~0.1%) absorbance
change induced by a strong (~1 MV/cm) electric field is measured. Analysis of the spectra provides information
about the change in dipole moment and the change in polarizability accompanying an electronic transition. We
measured near-UV absorption and EA spectra of indole-doped poly (methy1 methacrylate) (PMMA) film prepared
by a spin coating method. In the normal-incidence EA spectrum a negative contribution, being proportional
to the absorption, was observed. In the case of magic angle between the electric vectors of the absorbed light
and the applied field this contribution disappeared. These observations have been explained by field-induced
orientation/alignment of the ensemble of polar indole molecules. Since the ground state dipole of indole is nearly
parallel with the transition dipole, the absorption rate of light, which is polarized perpendicularly to the applied
field, should be smaller. Our study demonstrated that the angular movement of indole-sized molecules in PMMA
at room temperature is hindered, but not stopped.
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