Single‐cell redox states analyzed by fluorescence lifetime metrics and tryptophan FRET interaction with NAD(P)H

Abstract

Redox changes in live HeLa cervical cancer cells after doxorubicin treatment can either be analyzed by a novel fluorescence lifetime microscopy (FLIM)-based redox ratio NAD(P)H-a2%/FAD-a1%, called fluorescence lifetime redox ratio or one of its components (NAD(P)H-a2%), which is actually driving that ratio and offering a simpler and alternative metric and are both compared. Auto-fluorescent NAD(P)H, FAD lifetime is acquired by 2-photon excitation and Tryptophan by 3-photon, at 4 time points after treatment up to 60 min demonstrating early drug response to doxorubicin. Identical Fields-of-view (FoV) at each interval allows single-cell analysis, showing heterogeneous responses to treatment, largely based on their initial control redox state. Based on a discrete ROI selection method, mitochondrial OXPHOS and cytosolic glycolysis are discriminated. Furthermore, putative FRET interaction and energy transfer between tryptophan residue carrying enzymes and NAD(P)H correlate with NAD(P)H-a2%, as does the NADPH/NADH ratio, highlighting a multi-parametric assay to track metabolic changes in live specimens.

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

“…56 This could also be a major advantage, when this assay is finally transferred to clinical applications in the cancer or other fields. The FLIM assay-as we have shown 33 -can predict optimal drug choices and their effects in vitro in a matter of hours versus days or weeks in patients. This translational step depends on the availability of biopsies in the case of PCa or acute myeloid leukemia patient serum samples (or equivalent cell lines with diagnosed, known mutations), which is in the realm of possibilities.…”

“…The FLIM processing follows as previous published. 33 In short, photon reference images are normalized [Figs. 1(a)-1(b)] to compensate for varying intensities (FIJI, plugins → integral image filters → normalize local contrast): followed by zeroing the nucleus and creating single pixel region-of-interest (ROI) by an ImageJ/FIJI custom plugin.…”

“…25,26 First, intensity-based methods were expanded, [27][28][29][30][31] followed by fluorescence lifetime imaging assays to monitor REDOX changes as markers for changed metabolic states. 12,30,[32][33][34][35][36][37] These assays are of particular interest in measuring responses to treatment in various cancer pathologies, cancer being a metabolically heterogeneous pathology, being able to generate energy by oxidative phosphorylation (OXPHOS) and (often preferentially) by glycolysis. 9,[38][39][40][41] A third coenzyme, nicotinamide adenine dinucleotide (phosphate) [NAD(P)H], the phosphorylated form of NADH, cannot spectrally be differentiated from NADH, so this paper follows convention to describe the mixed lifetime/intensity signal as NAD(P)H. A heightened interest in expanding the application and simplification of this FLIM assay arises from its potential to test suitability and earliest response to drug treatment in cancer therapies.…”

“…We observed negligible SBT, tested first investigating single solutions of both coenzymes, followed by mixed solutions to mimic cellular conditions, and finally applied the assay to two prostate cancer (PCa) cell lines, this group has used in previous publications. [33][34][35] We compared the intensity spectrum and fluorescence lifetime results with other wavelengths and finally found the optimized excitation wavelength. The data are compared with the common standard assay of sequentially exciting cells with 740 nm followed by 890 nm at the new filter combination.…”

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

“…In these studies, tryptophan is the FRET donor and NAD(P)H is the FRET acceptor [i.e., NAD(P)H quenches tryptophan fluorescence]. These studies found that doxorubicin increases the abundance of FRET interactions between tryptophan and NAD(P)H. 303 New developments in super-resolution FLIM can localize molecular features within smaller structures and is growing in popularity along with other super-resolution techniques. 133,304…”