Simultaneous Two-Photon Fluorescence Microscopy of NADH and FAD Using Pixel-to-Pixel Wavelength-Switching

Abstract

Two-photon fluorescence (TPF) microscopy of intrinsic fluorophores provides physiological and pathological information from biological tissues. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are two endogenous fluorescent coenzymes existing on the intracellular scale. Autofluorescence images of NADH and FAD have been applied to noninvasively record changes during metabolism, according to their distributions and concentrations. However, the widely used sequential (non-simultaneous) excitation scheme results in artifacts caused by sample motion or laser power fluctuation. The single-wavelength illumination scheme suffers from low excitation efficiency and spectral bleed-through. In this paper, we demonstrate a new imaging system simultaneously capturing autofluorescence images from NADH and FAD, with high excitation efficiency and negligible spectral bleed-through. Two temporally multiplexed and spatially overlapped excitation beams were achieved with fast-switching light paths based on an electro-optic modulator. The switching beams were centered at 750 and 860 nm, enabling independent excitations of NADH and FAD. Autofluorescence images of NADH and FAD were acquired at the wavelength ranges of 415–455 nm and 500–550 nm, respectively. The electro-optic modulator was synchronized with the pixel clock from the microscope, achieving pixel-to-pixel wavelength-switching. The capability of the system was demonstrated by performing TPF imaging of freshly excised mouse colon tissues. The microenvironment of the colon wall was depicted by the distributions of colonocytes, goblet cells, and crypts of Lieberkühn, and the relative concentrations of NADH and FAD were estimated. The experimental results show that the system can effectively perform simultaneous imaging of NADH and FAD, and is considered a promising tool for investigations into metabolism-associated processes and diseases.

Longitudinal tracking of acute kidney injury reveals injury propagation along the nephron

“…Using 2PM, S1 proximal tubule (PT-S1), S2 proximal tubules (PT-S2) as well as distal convoluted tubules, connecting tubules and collecting duct tubule segments are accessible [25,26], of which the latter three were collectively analyzed (DCT/CD) in this study. NADH and FADH (flavin adenine dinucleotide) play important roles for PT ATP production and are simultaneously excitable at 750 nm using 2PM [24,27]. Consistent with previous data [25], As expected, PT segments were more injured than DCT/CD segments.…”

“…2 h after reperfusion, in vivo 2PM of partial IRI kidneys, allowed discrimination of IR, Not-IR, and Mid regions based on distinct spatial distribution of necrotic cell death as detected by nuclear propidium iodide (PI)-staining. PI-positive tubule cells further revealed markedly reduced blue autofluorescence (λEx = 750 nm, λEm = 435-485 nm), indicative of NADH (nicotinamide adenine dinucleotide) depletion [24] (fig 1E). In IR regions, epithelial necrotic cell death averaged 21.78 ± 1.32% PI-positive nuclei per segment´s total nuclei (mean ± SEM, n = 263 segments from 6 mice), of which 76.04% of all analyzed tubule segments demonstrated epithelial necrotic cell death of varying severity (fig 1F , G).…”

A computational two‐photon fluorescence approach for revealing label‐free the 3D image of viruses and bacteria

“…Because of its key role in the conversion of energy, the fluorescence of reduced nicotinamide‐adenine dinucleotide (NADH) and oxidized FAD can be used as metabolic biomarker of cell. Moreover, various optical studies described in the literature present the two‐photon excitation and emission spectra of fluorescence of NADH and FAD and three‐photon excitation of tryptophan or nucleic acids [23–28]. All of these studies display a two‐photon excitation range of NADH and FAD located between 700 and 850 nm—as we have measured—and an emission range, centered at 450 nm covering the range between 400 and 600 nm.…”

“…Moreover, various optical studies described in the literature present the two-photon excitation and emission spectra of fluorescence of NADH and FAD and three-photon excitation of tryptophan or nucleic acids. [23][24][25][26][27][28] All of these studies display a twophoton excitation range of NADH and FAD located between 700 and 850 nmas we have measuredand an emission range, centered at 450 nm covering the range between 400 and 600 nm. This spectral range corresponds to the emission range we have detected.…”

Light-sheet autofluorescence lifetime imaging with a single-photon avalanche diode array

“…For example, NAD(P)H FLIM can probe the temporal and spatial regulation of macrophage metabolism during tissue damage and repair in live zebrafish 11 . NAD(P)H FLIM typically relies on two-photon (2P) laser scanning microscopy, which provides intrinsic optical sectioning, improved penetration depth, and high spatial resolution with minimal phototoxicity 16 , 17 . However, autofluorescence FLIM traditionally suffers from long acquisition times and limited spatial views, partly due to the low fluorescence quantum yield (normalΦf for NAD(P)H is between 0.02 and 0.10 depending on protein binding) as well as lower molar absorption coefficient [ϵ for NAD(P)H is 6200 cm−1 M−1], which makes the autofluorescence signal hundreds of times dimmer than that of conventional fluorophores (e.g., the green fluorescent protein [GFP] has Φf=0.79 and ϵ=25500 cm−1 M−1) 18 …”