ORIGINAL RESEARCH article
Front. Cell. Infect. Microbiol.
Sec. Bacteria and Host
Volume 15 - 2025 | doi: 10.3389/fcimb.2025.1642009
This article is part of the Research TopicNavigating the Microbial Landscape: Integrating Mass Spectrometry Imaging and Other Multimodal Imaging Approaches for Spatially Resolved Microbial StudiesView all articles
Label-free metabolic imaging of Pseudomonas aeruginosa infection using two-photon fluorescence lifetime imaging microscopy
Provisionally accepted
- 1University of Maryland Baltimore Institute of Human Virology, Baltimore, United States
- 2University of Maryland Baltimore School of Dentistry, Baltimore, United States
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In this study we have applied high-spatial and temporal label-free imaging of individual live multidrug-resistant bacteria and bacteria-infected cells and animal tissue using two-photon fluorescence lifetime imaging microscopy (2p-FLIM). 2p-FLIM can identify and quantify fluorescence intensity and lifetimes among bacteria, infected cells, and tissues. We have implemented 2p-FLIM in combination with phasor plot analysis for quantifying molecular differences of NAD(P)H intensities and lifetimes for fast, sensitive, high-resolution, nondestructive imaging of live bacteria and bacteria-infected cells and tissues. We have further developed a coordinated workflow using 2p-FLIM for high-resolution temporal-spatial mapping of bacteria infected cells and tissues that can be performed for near real-time quantitation of NAD(P)H intensities and lifetimes for identifying changes in metabolism. 2p-FLIM may have broad applicability for characterizing microbial infection at the molecular, subcellular, cellular and tissue levels. The ability to quantitate and directly monitor changes in NAD(P)H metabolism in near real-time in bacteria cells and tissues during an infection, offers a potential mechanism for understanding microbial pathogenesis and evaluating therapeutic treatments that can be applied to multiple model systems. Overall, the application of this label-free imaging approach has the potential to address biomedical research needs and technical problems that occur broadly across multiple biological systems and diseases.
Keywords: NAD(P)H1, metabolism2, bacteria2, infected cells3, infected tissue4, oxidativephosphorylation5, two-photon fluorescence lifetime imaging microscopy6
Received: 05 Jun 2025; Accepted: 21 Aug 2025.
Copyright: © 2025 Snyder, Bergamin De Castro, Scott and Ray. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Krishanu Ray, University of Maryland Baltimore Institute of Human Virology, Baltimore, United States
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