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

Journal Article · 20 January 2024 · Sensors

DOI: https://doi.org/10.3390/s24020667 · OSTI ID:2472413

Fernández, Alma ^[1]; Classen, Anton ^[2]; Josyula, Nityakalyani ^[2]; Florence, James T. ^[2]; Sokolov, Alexei V. ^[2]; Scully, Marlan O. ^[2]; Straight, Paul ^[2]; Verhoef, Aart J.  ^[2]

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1. Texas A & M Univ., College Station, TX (United States); OSTI
2. Texas A & M Univ., College Station, TX (United States)

The intrinsic fluorescence of bacterial samples has a proven potential for label-free bacterial characterization, monitoring bacterial metabolic functions, and as a mechanism for tracking the transport of relevant components through vesicles. The reduced scattering and axial confinement of the excitation offered by multiphoton imaging can be used to overcome some of the limitations of single-photon excitation (e.g., scattering and out-of-plane photobleaching) to the imaging of bacterial communities. In this work, we demonstrate in vivo multi-photon microscopy imaging of Streptomyces bacterial communities, based on the excitation of blue endogenous fluorophores, using an ultrafast Yb-fiber laser amplifier. Its parameters, such as the pulse energy, duration, wavelength, and repetition rate, enable in vivo multicolor imaging with a single source through the simultaneous two- and three-photon excitation of different fluorophores. Three-photon excitation at 1040 nm allows fluorophores with blue and green emission spectra to be addressed (and their corresponding ultraviolet and blue single-photon excitation wavelengths, respectively), and two-photon excitation at the same wavelength allows fluorophores with yellow, orange, or red emission spectra to be addressed (and their corresponding green, yellow, and orange single-photon excitation wavelengths). We demonstrate that three-photon excitation allows imaging over a depth range of more than 6 effective attenuation lengths to take place, corresponding to an 800 micrometer depth of imaging, in samples with a high density of fluorescent structures.

• Research Organization: Washington Univ., St. Louis, MO (United States)
• Sponsoring Organization: National Institute of General Medical Sciences (NIGMS); National Institutes of Health (NIH); National Science Foundation (NSF); Robert A. Welch Foundation; US Air Force Office of Scientific Research (AFOSR); USDOE Office of Science (SC), Biological and Environmental Research (BER)
• Grant/Contract Number: SC0023103
• OSTI ID: 2472413
• Journal Information: Sensors, Journal Name: Sensors Journal Issue: 2 Vol. 24; ISSN 1424-8220
• Publisher: MDPI AGCopyright Statement
• Country of Publication: United States
• Language: English