Two-photon microscopy assessment of the overall energy metabolism alteration of amoeba in hypertonic environment

In this study, a two-photon fluorescence microscopic imaging technique is reported for assessment the effect of dynamic hypertonic environment on the overall energy metabolism alteration and adaptation of soil-living amoeba Dictyostelium discoideum. For that purpose the fluorescence intensity of mitochondrial reduced nicotinamide adenine dinucleotide (NADH) was monitored and quantified in order to evaluate the corresponded metabolic state of monolayer cultured cells. The two-photon excitation of NADH with 720 nm near infrared irradiation produced blue fluorescence emission with maximum wavelength centered at 460 nm. The benefits of reported noninvasive microscopic technique are the significantly less cellular damage and avoiding the excitation of other biomolecules except of NADH. It enabled to acquire data for NADH levels of the observed cells on agar plate specimen and hypertonic nutrition media in a Petri dish. The method demonstrated also good sensitivity, reproducibility and the obtained results revealed that D. discoideum species form aggregation in hypertonic environment within several minutes with aim to survive. The formed aggregate had amorphous shape and it consisted from dozen amoeba cells, which kept their NADH amount in constant level for few hours. The reported imaging method might be applicable in various studies for characterization of metabolic events and assessment of the cell energy balance in hypertonic environment.