Fluorescence quenching of free and bound NADH in HeLa cells determined by hyperspectral imaging and unmixing of cell autofluorescence

Abstract

Carbonyl cyanide-p-trifluoro methoxyphenylhydrazone (FCCP) is a well-known mitochondrial uncoupling agent. We examined FCCP-induced fluorescence quenching of reduced nicotinamide adenine dinucleotide / nicotinamide adenine dinucleotide phosphate (NAD(P)H) in solution and in cultured HeLa cells in a wide range of FCCP concentrations from 50 to 1000µM. A non-invasive label-free method of hyperspectral imaging of cell autofluorescence combined with unsupervised unmixing was used to separately isolate the emissions of free and bound NAD(P)H from cell autofluorescence. Hyperspectral image analysis of FCCP-treated HeLa cells confirms that this agent selectively quenches fluorescence of free and bound NAD(P)H in a broad range of concentrations. This is confirmed by the measurements of average NAD/NADH and NADP/NADPH content in cells. FCCP quenching of free NAD(P)H in cells and in solution is found to be similar, but quenching of bound NAD(P)H in cells is attenuated compared to solution quenching possibly due to a contribution from the metabolic and/or antioxidant response in cells. Chemical quenching of NAD(P)H fluorescence by FCCP validates the results of unsupervised unmixing of cell autofluorescence. Hidalgo, "Ultraviolet excitation fluorescence spectroscopy: a noninvasive method for the measurement of redox changes in ischemic myocutaneous flaps," Plast. Reconstr. Surg. 96(3), 673-680 (1995). 31. J. Eng, R. M. Lynch, and R. S. Balaban, "Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes," Biophys. J. 55(4), 621-630 (1989). 32. A. S. Galkin, V. G. Grivennikova, and A. D. Vinogradov, "→H+/2e-stoichiometry in NADH-quinone reductase reactions catalyzed by bovine heart submitochondrial particles," FEBS Lett. 451(2), 157-161 (1999). 33. J. P. Brennan, R. G. Berry, M. Baghai, M. R. Duchen, and M. J. Shattock, "FCCP is cardioprotective at concentrations that cause mitochondrial oxidation without detectable depolarisation," Cardiovasc.

Statistically strong label-free quantitative identification of native fluorophores in a biological sample

“…In comparison, in conventional fluorescently labelled microscopy images of cells and tissues a typical range of SNR is 20–40 dB 20 . However, in this study, the autofluorescent images of retina cells are characterised by a far lower SNR of ~5 dB; similar SNR values were observed in previous investigations 2 , 3 , 21 .…”

NAD+ Repletion Rescues Female Fertility during Reproductive Aging

“…In this work we utilized a fluorescence microscope adapted for multispectral imaging by adding a number of custom-made fluorescent channels (Gosnell et al, 2016a(Gosnell et al, , 2016bHabibalahi et al, 2019;Rehman et al, 2017) to characterize native fluorescence of oocytes. Images were taken in the following channels listed in the table below.…”

Investigating challenges with scattering and inner filter effects in front‐face fluorescence by PARAFAC

“…Samples 1, 6, and 7 are the ones where, respectively, tryptophan, riboflavin, and NADH are the most predominant component two of the samples in the data set). In order to estimate this last component, which was already expected to be the most challenging one due to its low quantum yield, [58][59][60] a four-component PARAFAC model had to be fitted. In this model, the first two extracted components ( Figure 4E,F) were related to riboflavin, as all their scores were linearly correlated to the concentration of riboflavin ( Figure 4I).…”