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

Jang Hyuk Lee; Jose J Rico-Jimenez; Chi Zhang; Aneesh Alex; Eric J Chaney; Ronit Barkalifa; Darold R Spillman Jr; Marina Marjanovic; Zane Arp; Steve R Hood; Stephen A Boppart

doi:10.1364/BOE.10.005431

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

Biomed Opt Express. 2019 Oct 1;10(10):5431-5444. doi: 10.1364/BOE.10.005431.

Authors

Jang Hyuk Lee^{1

2}, Jose J Rico-Jimenez^{1

2}, Chi Zhang¹, Aneesh Alex³, Eric J Chaney¹, Ronit Barkalifa¹, Darold R Spillman Jr¹, Marina Marjanovic¹, Zane Arp³, Steve R Hood⁴, Stephen A Boppart^{1

5

6

7}

Affiliations

¹ Center for Optical Molecular Imaging, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
² Co-first authors with equal contribution.
³ GlaxoSmithKline, Collegeville, PA 19426, USA.
⁴ GlaxoSmithKline, Stevenage, UK.
⁵ Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
⁶ Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
⁷ Carle Illinois College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61820, USA.

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.