Three-photon tissue imaging using moxifloxacin

Seunghun Lee,Yong Woong Jun,Won Hyuk Jang,Bumju Kim,Jun Ho Lee,Taejun Wang,Ki Hean Kim,Myoung Joon Kim,Yeoreum Yoon

doi:10.1038/s41598-018-27371-8

Seunghun Lee, Yong Woong Jun + Show 7 more

Open Access

https://doi.org/10.1038/s41598-018-27371-8

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Abstract

Moxifloxacin is an antibiotic used in clinics and has recently been used as a clinically compatible cell-labeling agent for two-photon (2P) imaging. Although 2P imaging with moxifloxacin labeling visualized cells inside tissues using enhanced fluorescence, the imaging depth was quite limited because of the relatively short excitation wavelength (<800 nm) used. In this study, the feasibility of three-photon (3P) excitation of moxifloxacin using a longer excitation wavelength and moxifloxacin-based 3P imaging were tested to increase the imaging depth. Moxifloxacin fluorescence via 3P excitation was detected at a >1000 nm excitation wavelength. After obtaining the excitation and emission spectra of moxifloxacin, moxifloxacin-based 3P imaging was applied to ex vivo mouse bladder and ex vivo mouse small intestine tissues and compared with moxifloxacin-based 2P imaging by switching the excitation wavelength of a Ti:sapphire oscillator between near 1030 and 780 nm. Both moxifloxacin-based 2P and 3P imaging visualized cellular structures in the tissues via moxifloxacin labeling, but the image contrast was better with 3P imaging than with 2P imaging at the same imaging depths. The imaging speed and imaging depth of moxifloxacin-based 3P imaging using a Ti:sapphire oscillator were limited by insufficient excitation power. Therefore, we constructed a new system for moxifloxacin-based 3P imaging using a high-energy Yb fiber laser at 1030 nm and used it for in vivo deep tissue imaging of a mouse small intestine. Moxifloxacin-based 3P imaging could be useful for clinical applications with enhanced imaging depth.

Highlights

Two-photon microscopy (2PM) is a nonlinear fluorescence microscopy technique based on two-photon (2P) excitation of fluorophores1. 2PM has been used in various biological studies at the cellular level, including neurobiology[2], immunology[3], and cancer biology[4]
Nonlinear methods that use a longer excitation wavelength than that used in 2PM, such as three-photon microscopy (3PM)[11] and third-harmonic-generation microscopy (THGM)[12], have been developed to improve imaging depth. 3PM is a nonlinear fluorescence microscopy technique based on three-photon (3P) excitation of fluorophores
We developed a new system for moxifloxacin-based 3P imaging that uses a high-power fiber laser at 1030 nm and demonstrated its ability to perform in vivo tissue imaging

Summary

Introduction

Two-photon microscopy (2PM) is a nonlinear fluorescence microscopy technique based on two-photon (2P) excitation of fluorophores1. 2PM has been used in various biological studies at the cellular level, including neurobiology[2], immunology[3], and cancer biology[4]. It has a relatively deep imaging depth and low phototoxicity compared to those of other three-dimensional (3D) fluorescence microscopy techniques such as confocal microscopy. Moxifloxacin is an FDA-approved antibiotic used in the clinic to both treat and prevent bacterial infections It has intrinsic fluorescence[9] as well as good pharmacokinetic properties such as relatively high aqueous solubility and lipophilicity[10] for tissue penetration. Nonlinear methods that use a longer excitation wavelength than that used in 2PM, such as three-photon microscopy (3PM)[11] and third-harmonic-generation microscopy (THGM)[12], have been developed to improve imaging depth. We developed a new system for moxifloxacin-based 3P imaging that uses a high-power fiber laser at 1030 nm and demonstrated its ability to perform in vivo tissue imaging

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