Abstract
Simultaneous quantification of multifarious cellular metabolites and the extracellular matrix in vivo has been long sought. Simultaneous label-free autofluorescence and multi-harmonic (SLAM) microscopy has achieved simultaneous four-channel nonlinear imaging to study tissue structure and metabolism. In this study, we implemented two laser systems and directly compared SLAM microscopy with conventional two-photon microscopy for in vivo imaging. We found that three-photon imaging of adenine dinucleotide (phosphate) (NAD(P)H) in SLAM microscopy using our tailored laser source provided better resolution, contrast, and background suppression than conventional two-photon imaging of NAD(P)H. We also integrated fluorescence lifetime imaging with SLAM microscopy, and enabled differentiation of free and bound NAD(P)H. We imaged murine skin in vivo and showed that changes in tissue structure, cell dynamics, and metabolism can be monitored simultaneously in real-time. We also discovered an increase in metabolism and protein-bound NAD(P)H in skin cells during the early stages of wound healing.
Highlights
In vivo imaging to visualize dynamic biological processes in their natural environment is a primary goal for biological applications and for clinical diagnosis and disease or treatment monitoring
Over the past three decades, nonlinear optical imaging technologies based on multiphoton-excitation fluorescence (MPEF) or multiphoton harmonic frequency conversions have been extensively developed into powerful platforms to image living cells or intact tissue [1,2,3,4,5,6]
Three-photon excitation is a higher-order nonlinear process that offers better spatial resolution [35,36]. 3PEF can dramatically reduce out-of-focus background noise, improving the signal-to-noise ratio (SNR) by orders of magnitude compared to 2PEF [2,36,37,38,39,40]
Summary
In vivo imaging to visualize dynamic biological processes in their natural environment is a primary goal for biological applications and for clinical diagnosis and disease or treatment monitoring. Over the past three decades, nonlinear optical imaging technologies based on multiphoton-excitation fluorescence (MPEF) or multiphoton harmonic frequency conversions have been extensively developed into powerful platforms to image living cells or intact tissue [1,2,3,4,5,6]. Endogenous fluorescent biomolecules such as flavins, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) coenzymes, and melanin allow one to monitor the metabolic changes of tissue through MPEF microscopy [7,8,9,10,11,12]. Multiphoton imaging tools have transitioned from bench-top to intravital platforms, especially for in vivo imaging of skin [5,10,20,21,22,23,24,25,26,27]
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