Time- and spectrally resolved characteristics of flavin fluorescence in U87MG cancer cells in culture

Abstract

Early detection of cancer is crucial for the successful diagnostics of its presence and its subsequent treatment. To improve cancer detection, we tested the progressive multimodal optical imaging of U87MG cells in culture. A combination of steady-state spectroscopic methods with the time-resolved approach provides a new insight into the native metabolism when focused on endogenous tissue fluorescence. In this contribution, we evaluated the metabolic state of living U87MG cancer cells in culture by means of endogenous flavin fluorescence. Confocal microscopy and time-resolved fluorescence imaging were employed to gather spectrally and time-resolved images of the flavin fluorescence. We observed that flavin fluorescence in U87MG cells was predominantly localized outside the cell nucleus in mitochondria, while exhibiting a spectral maximum under 500 nm and fluorescence lifetimes under 1.4 ns, suggesting the presence of bound flavins. In some cells, flavin fluorescence was also detected inside the cell nuclei in the nucleoli, exhibiting longer fluorescence lifetimes and a red-shifted spectral maximum, pointing to the presence of free flavin. Extra-nuclear flavin fluorescence was diminished by 2-deoxyglucose, but failed to increase with 2,4-dinitrophenol, the uncoupler of oxidative phosphorylation, indicating that the cells use glycolysis, rather than oxidative phosphorylation for functioning. These gathered data are the first step toward monitoring the metabolic state of U87MG cancer cells.

An in vitro cell irradiation protocol for testing photopharmaceuticals and the effect of blue, green, and red light on human cancer cell lines

“… 27 , 34 , 42 , 45 , 46 , 50 – 53 However, the concentrations of flavins and porphyrins in cancer cells compared to normal cells is debatable with very few direct or significant comparisons available. 23 , 54 – 56 Additionally due to the variety of media compositions, determining the specific chromophore cocktail makes it difficult to discern which compound is responsible for the blue light-specific cell death. 52 , 53 …”

“…27,34,42,45,46,[50][51][52][53] However, the concentrations of flavins and porphyrins in cancer cells compared to normal cells is debatable with very few direct or significant comparisons available. 23,[54][55][56] Additionally due to the variety of media compositions, determining the specific chromophore cocktail makes it difficult to discern which compound is responsible for the blue light-specific cell death. 52,53 Finally, the variable genetic mutations found in cancer cells lead to significant differences in phenotypes, such as circumvention of programmed cell death, up-regulated proliferation, recovery via autophagy, and/or increased tolerance to oxidative stress.…”

Inside the Joint of Inflammatory Arthritis Patients: Handling and Processing of Synovial Tissue Biopsies for High Throughput Analysis

“…Two-photon FLIM (2P-FLIM), can be utilized in order to assess protein bound vs. free nicotinamide adenine nucleotide [NAD(P)H] and as a result provide an overview of the cell's metabolic state ( 85 , 86 ). In the aforementioned application, FLIM is utilized to capture the fluorescence signal of NAD(P)H, a natural fluorochrome with importantly, distinct fluorescence lifetimes dependent on whether it is protein bound and therefore utilized in oxidative phosphorylation (OxPhos) (fluorescent lifetime of ~2.5 ns when bound compared to 0.4 ns if in free state) ( 87 ). Due to the single cell resolution that FLIM offers, it has previously been utilized for the characterization of the metabolic state of rare populations of cells ( 23 , 24 ), herein, we have implemented FLIM in order to assess the metabolism of synovial tissue cell suspensions before or after treatment with OxPhos inhibitor carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) ( Figure 3B ) ( 88 , 89 ).…”

The review of the light parameters and mechanisms of Photobiomodulation on melanoma cells

“…However, the concentration of flavin and porphyrin in cancer cells is low compared with normal cells, and it is debatable whether it is a photoreceptor that causes blue light to inhibit cell viability 34 . In addition, due to the various components in the cell culture medium, it is difficult to determine which compound causes cell death induced by blue light 35 …”