Characterization of endogenous fluorescence in nonsmall lung cancerous cells: A comparison with nonmalignant lung normal cells

Abstract

Monitoring fluorescence properties of endogenous fluorophores such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in normal and cancerous cells provide substantial information noninvasively on biochemical and biophysical aspects of metabolic dysfunction of cancerous cells. Time-resolved spectral profiles and fluorescence lifetime images of NADH and FAD were obtained in human lung nonsmall carcinomas (H661 and A549) and normal lung cells (MRC-5). Both fluorophores show the fast and slowly decaying emission components upon pulsed excitation, and fluorescence spectra of NADH and FAD show blue-and red-shifts, respectively, during their decay. All identified lifetime components of NADH and FAD were found to be shorter in cancerous cells than in normal cells, no matter how they were measured under different extra-cellular conditions (cells suspended in cuvette and cells attached on glass substrate), indicating that the changes in metabolism likely altered the subcellular milieu and potentially also affected the interaction of NADH and FAD with enzymes to which these cofactors were bound.The intensity ratio of NADH and FAD of cancerous cells was also shown to be larger than that of normal cells. K E Y W O R D S cancerous and nonmalignant lung cells, endogenous fluorophores, fluorescence lifetime microscopy, time-resolved emission spectra and decays Abbreviations: FAD, flavin adenine dinucleotide; FLIM, fluorescence lifetime microscopy; IRF, instrumental response function; NADH, nicotinamide adenine dinucleotide; ROS, reactive oxygen species; TCSPC, time-correlated single photon-counting.

Application of Nanosecond Pulsed Electric Field and Autofluorescence Lifetime Microscopy of FAD in Lung Cells

“…Representative decay profiles obtained as the sum of decay profiles at 21 × 21 pixels and histograms of lifetime distribution are also shown in these figures. As reported in our previous paper, 35 fluorescence decays of FAD of A549, H661, and MCR-5 cells attached on a glass substrate observed in the absence of applied electric field could be analyzed by assuming triexponential decay. In fact, the curve fitting for the observed decay profiles could be done in every case by using a triexponential function, as shown in the Supporting Information (SI) (Figures S1−S3), where the simulated decays are given, together with the residual distribution and the reduced chi-square, χ 2 .…”

“…Thus, the lung cancerous and normal cells were different in shape from each other, but the autofluorescence spectra of FAD, which were obtained in a suspension of cells, were nearly independent of the cell types (Figure ). Although there was no significant difference in the spectra, the AFL of FAD depended on the cell type . The difference of the lifetime between normal cells and cancerous cells probably originated from the difference in the intracellular environment surrounding fluorophores, which may alter the interaction of FAD cofactor with associated proteins, indicating that AFL imaging of FAD is a sensitive tool to detect such environmental difference. , Then, AFL imaging of FAD was carried out to examine the effects of application of nsPEF(50) on fluorescence characteristics of FAD in these lung cells in relation to field-induced change in intracellular function and mitochondrial function.…”

“…All the cells, i.e., A549, H661, and MRC-5, were cultured at 37 °C in a CO 2 humidified atmosphere (5%) following the provider’s protocols. The detailed processes were mentioned in our previous papers. ,, The autofluorescence spectra of FAD were measured in the suspension of A549, H661, and MRC-5 cells by a homemade time-resolved single photon counting system . Three-dimensional microelectrode chips with an interelectrode space of 200 ± 1 μm were constructed with the UV photolithographic technique on a microscopic quartz substrate and combined with an inverted fluorescence microscope (TE2000E, Nikon) .…”

“…Although fluorescence intensity gives information about the concentration of fluorophores, the lifetime of fluorescence is very sensitive to physical and chemical environments, and fluorescence lifetime coupled with spatial distribution provides valuable insight into the functionality of complex biological systems. − Therefore, lifetime imaging of autofluorescence arising from intracellular chromophores, i.e., endogenous fluorophores in cells and tissues, has received much attention because cellular environments are stable without exogenous probes and staining time is not necessary for diagnostic tests in medicine. ,,− It is considered that NADH autofluorescence is emitted not only from mitochondria but also from other intracellular compartments. On the other hand, the autofluorescence of FAD is mainly produced by mitochondrial flavins. , In recent decades, along with the characterization of photophysical and photochemical properties of FAD, studies have also focused on utilizing fluorescence imaging of FAD, e.g., for detecting intracellular pH changes and differentiating normal and cancerous cells. − FAD can be photoexcited with a longer wavelength than that of NADH, resulting in less phototoxic experiment than the NADH study. However, the image measurements of autofluorescence lifetime (AFL) of FAD seem to be still quite limited probably because of its weak intensity.…”

Solitary pulmonary nodule imaging approaches and the role of optical fibre-based technologies

“…They demonstrated that the fluorescence lifetime of NADH decreases in cancerous tissue. More recently, fluorescence lifetimes of NADH and FAD in human non-small cell lung carcinoma cells have been shown to be significantly shorter than in normal cells using FLIM [ 142 ]. The biochemical basis for these variations is unclear.…”