Fluorescence Lifetime Imaging Unravels C. trachomatis Metabolism and Its Crosstalk with the Host Cell

Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ2-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ2-NAD(P)H increase inside the chlamydial inclusion. τ2-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ2-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity

Experimental applications of TNF-reporter mice with far-red fluorescent label

This chapter provides protocols for in vitro and in vivo analysis of TNF-producing cells from a novel TNF reporter mouse. In these transgenic mice, genetic sequence encoding far-red reporter protein Katyushka (FRFPK) was placed under control of the same regulatory elements as TNF, thus providing the basis for detection, isolation, and visualization of TNF-producing cells

Fluorescence lifetime imaging for diagnostic and therapeutic intravital microscopy

Intravital imaging is now widely performed using wide-field microscopy, endoscopy, and state-of-the-art multiphoton microscopy for research and clinical assessment applications. Fluorescence lifetime imaging is increasingly being used as a complementary technology to greatly enhance the specificity and sensitivity in the analysis of the various fluorophores present within an intravital image. The fluorescence lifetime of a fluorophore. The fluorescence lifetime distribution for a fluorophore is an intrinsic property, arising from the emission of photons of light in the decaying to its original energy state after its molecules are excited by a specific wavelength of light and remain in an excited state for a range of times. This behavior for individual autofluorescent fluorophores, dyes, drugs, fluorescent proteins and antibodies is most frequently summarized in terms of their average fluorescence lifetime. Fluorescence lifetime differences are then used to identify and discriminate between molecules in various applications, including the assessment of drug distribution and metabolism, and in quantifying cell responses for toxicology. Fluorescence lifetime imaging microscopy (FLIM) and tomography involves the spatial representation of the fluorescent lifetimes of all molecules within image collected over a specified time period and resolution. Autofluorescence lifetime differences between normal and cancerous tissues have been used to define surgical margins during intraoperative surgery. Recent advances have enabled the rapid and robust collection of fluorescence lifetime information from tissues with high-resolution at video-rate speeds using endoscopic probes. Fluorescence lifetime imaging, combined with multi-spectral and anisotropic analysis, yields detailed redox state data from within a cell, arising from its metabolic state and enables intravital analysis of the transport and metabolism of fluorescent probes in cells. Intravital fluorescence lifetime imaging is becoming an indispensable diagnostic approach with broad therapeutic and clinical applications