In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy

Jonathan A Palero,Hans C Gerritsen,Henricus J C M Sterenborg,Arjen N Bader,Angélique Van Der Ploeg Van Den Heuvel,Henriëtte S De Bruijn

doi:10.1364/boe.2.001030

Abstract

Nonlinear spectral imaging microscopy (NSIM) allows simultaneous morphological and spectroscopic investigation of intercellular events within living animals. In this study we used NSIM for in vivo time-lapse in-depth spectral imaging and monitoring of protein-bound and free reduced nicotinamide adenine dinucleotide (NADH) in mouse keratinocytes following total acute ischemia for 3.3 h at ~3 min time intervals. The high spectral resolution of NSIM images allows discrimination between the two-photon excited fluorescence emission of protein-bound and free NAD(P)H by applying linear spectral unmixing to the spectral image data. Results reveal the difference in the dynamic response between protein-bound and free NAD(P)H to ischemia-induced hypoxia/anoxia. Our results demonstrate the capability of nonlinear spectral imaging microscopy in unraveling dynamic cellular metabolic events within living animals for long periods of time.

Highlights

The autofluorescence of reduced nicotinamide adenine dinucleotides (NADH) can reveal the metabolic state of a cell
The mice were subjected to total acute ischemia during which the intrinsic fluorescence of the keratinocytes increased in intensity by an average of 71 ± 5% as depicted in Fig. 3, A and B
The results presented in this work demonstrate the dynamic effect of total acute ischemia on the autofluorescence spectra of in vivo mouse skin keratinocytes

Summary

Introduction

The autofluorescence of reduced nicotinamide adenine dinucleotides (NADH) can reveal the metabolic state of a cell. NADH, the principal electron donor in glycolytic and oxidative energy metabolism, has been used as a convenient noninvasive probe of cellular metabolic state [1] It exists in an oxidized (NAD+) and a reduced (NADH) form, only NADH is intrinsically fluorescent, whereas its oxidized product (NAD+) is not. Chance et al took advantage of this phenomenon and demonstrated that microfluorometry of NADH provides a means to probe the oxidation-reduction state of cells and tissues [1]. This pioneering work opened the door to more studies that utilized probing NADH fluorescence to reveal the metabolic activity within the cell. The emergence of nonlinear-excited fluorescence microscopy paved the way to a new era of cellular imaging in thick samples as well as living

Methods

Results

Conclusion