Key points are not available for this paper at this time.
SignificanceTo enable non-destructive longitudinal assessment of drug agents in intact tumor tissue without the use of disruptive probes, we have designed a label-free method to quantify the health of individual tumor cells in excised tumor tissue using multiphoton fluorescence lifetime imaging microscopy (MP-FLIM).AimUsing murine tumor fragments which preserve the native tumor microenvironment, we seek to demonstrate signals generated by the intrinsically fluorescent metabolic co-factors nicotinamide adenine dinucleotide phosphate NAD(P)H and flavin adenine dinucleotide (FAD) correlate with irreversible cascades leading to cell death.ApproachWe use MP-FLIM of NAD(P)H and FAD on tissues and confirm viability using standard apoptosis and live/dead (Caspase 3/7 and propidium iodide, respectively) assays.ResultsThrough a statistical approach, reproducible shifts in FLIM data, determined through phasor analysis, are shown to correlate with loss of cell viability. With this, we demonstrate that cell death achieved through either apoptosis/necrosis or necroptosis can be discriminated. In addition, specific responses to common chemotherapeutic treatment inducing cell death were detected.ConclusionsThese data demonstrate that MP-FLIM can detect and quantify cell viability without the use of potentially toxic dyes, thus enabling longitudinal multi-day studies assessing the effects of therapeutic agents on tumor fragments.
Smith et al. (Fri,) studied this question.