Simultaneous label-free autofluorescence and multi-harmonic imaging reveals in vivo structural and metabolic changes in murine skin

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.

Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H

“…EVs were isolated from cell conditioned media using differential ultracentrifugation, embedded in a 0.2% (w/vol) agarose gel, and imaged using a previously described custom laser scanning multiphoton setup with FLIM capabilities 34 . The mean fluorescence lifetime, τ , of each EV was calculated using the phasor approach for FLIM analysis (Supplementary Fig.…”

“…EV and cell samples were imaged at room temperature (about 25 °C) using a previously described custom laser scanning multiphoton setup with FLIM capabilities 34 . The glass bottom imaging dishes allow for illumination and detection from below the sample.…”

“…The fluorescence lifetime of fluorophores depends on factors that affect the nanoscale potential energy distribution of electrons such as pH, temperature, and molecular interactions 29 , 31 – 33 . The fluorescence lifetime of NAD(P)H in cells and tissues has been widely studied 26 , 27 , 34 – 38 , and it has been found that when bound to a protein, NAD(P)H exhibits a longer fluorescence lifetime of approximately 1.5–6 ns, whereas free NAD(P)H exhibits a characteristically shorter fluorescence lifetime of approximately 0.3–0.8 ns 25 , 31 .…”

Recent advances in the use of stimulated Raman scattering in histopathology

“…Autofluorescence from the cell is typically from oxidative chain respiratory proteins NADH and FAD and high levels of NADH have been associated with glycolysis or regions of hypoxia. 37,38 Bower et al, have demonstrated that incorporation of fluorescence lifetime imaging microscopy (FLIM) is able to separate free and bound NADH in living samples, and developed a video rate FLIM system which could add information on metabolic activity to the images. 39 6.2 Imaging in the C-H region 6.2.1 DNA Imaging.…”

Single‐beam optogenetic multimodal χ(3)/χ(5) nonlinear microscopy and brain imaging

“…As significant recent milestones along this route, 3PEF imaging has been extended to neuronal activity studies deep in intact mouse brain, [12] wide-field three-photon optogenetic stimulation, [13] and brain structure and function detection through an intact skull, [14] as well as 3PEF lightsheet microscopy. [15] In most recent studies, [16,17] 3PEF has been successfully combined with THG to provide a powerful tool for a multimodal optical imaging of skin and diagnostics of its pathologies. Here, we extend this approach and demonstrate its potential for imaging and understanding a vast variety of biological systems and settings, ranging from HeLa to brain cells.…”