Simultaneous Two- and Three-Photon Deep Imaging of Autofluorescence in Bacterial Communities

Area of Science:

• Microscopy and Imaging
• Bacteriology
• Biophotonics

Background:

• Intrinsic fluorescence in bacteria offers label-free characterization and metabolic monitoring.
• Multiphoton microscopy enhances imaging by reducing scattering and photobleaching compared to single-photon methods.

Purpose of the Study:

• To demonstrate in vivo multicolor multiphoton microscopy of Streptomyces bacterial communities.
• To utilize endogenous fluorophores and an ultrafast Yb-fiber laser for advanced imaging.

Main Methods:

• Employing an ultrafast Yb-fiber laser amplifier for multiphoton excitation.
• Utilizing simultaneous two-photon and three-photon excitation at 1040 nm for multicolor imaging.
• Imaging Streptomyces bacterial communities in vivo.

Main Results:

• Achieved in vivo multicolor imaging of bacterial communities using a single laser source.
• Demonstrated deep-tissue imaging (over 800 micrometers) with three-photon excitation.
• Successfully excited blue, green, yellow, orange, and red emitting fluorophores.

Conclusions:

• Multiphoton microscopy, particularly with three-photon excitation, enables deep and multicolor imaging of bacterial communities.
• This label-free approach is valuable for studying bacterial functions and transport mechanisms.
• The Yb-fiber laser system provides a versatile platform for advanced bacterial imaging.