Three-photon tissue imaging using moxifloxacin.

Area of Science:

• Biomedical Optics
• Cellular Imaging
• Fluorescence Microscopy

Background:

• Moxifloxacin is a clinically used antibiotic with potential as a cell-labeling agent for two-photon (2P) imaging.
• While 2P imaging with moxifloxacin offers enhanced fluorescence for visualizing cells in tissues, its imaging depth is limited by shorter excitation wavelengths (<800 nm).

Purpose of the Study:

• To investigate the feasibility of three-photon (3P) excitation of moxifloxacin at longer wavelengths to increase imaging depth.
• To develop and evaluate moxifloxacin-based 3P imaging for deeper tissue visualization in preclinical models.

Main Methods:

• Characterized moxifloxacin fluorescence under 3P excitation at wavelengths >1000 nm.
• Acquired excitation and emission spectra of moxifloxacin.
• Performed ex vivo 3P and 2P imaging of mouse bladder and small intestine tissues.
• Constructed a 3P imaging system using a high-energy Yb fiber laser (1030 nm) for in vivo imaging.

Main Results:

• Moxifloxacin exhibited fluorescence under 3P excitation (>1000 nm).
• 3P imaging provided better image contrast than 2P imaging at equivalent depths in ex vivo tissues.
• Initial 3P imaging with a Ti:sapphire laser was limited by excitation power, restricting speed and depth.
• A Yb fiber laser system enabled successful in vivo deep tissue 3P imaging of mouse small intestine.

Conclusions:

• Three-photon (3P) excitation of moxifloxacin is feasible and enhances imaging capabilities.
• Moxifloxacin-based 3P imaging offers improved contrast and greater imaging depth compared to 2P imaging.
• The developed high-energy Yb fiber laser system facilitates in vivo deep tissue imaging, suggesting potential for clinical applications.