Abstract
Time-correlated single-photon counting combined with multi-photon laser scanning microscopy has proven to be a versatile tool to perform fluorescence lifetime imaging in biological samples and, thus, shed light on cellular functions, both in vitro and in vivo. Here, by means of phasor-analyzed endogenous NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) fluorescence lifetime imaging, we visualize the shift in the cellular metabolism of healthy human neutrophil granulocytes during phagocytosis of Staphylococcus aureus pHrodo™ beads. We correlate this with the process of NETosis, i.e., trapping of pathogens by DNA networks. Hence, we are able to directly show the dynamics of NADPH oxidase activation and its requirement in triggering NETosis in contrast to other pathways of cell death and to decipher the dedicated spatio-temporal sequence between NADPH oxidase activation, nuclear membrane disintegration and DNA network formation. The endogenous FLIM approach presented here uniquely meets the increasing need in the field of immunology to monitor cellular metabolism as a basic mechanism of cellular and tissue functions.
Highlights
Since 1990, when Winfried Denk et al first introduced the concept of two-photon laser-scanning microscopy and showed the unique benefits of ultra-short pulsed, near-infrared excitation for imaging highly-scattering biological tissue [1], this laser-based technology tremendously expanded its field of application, especially in neurosciences [2,3,4] and immunology [5] and in other disciplines such as nephrology [6] or developmental biology
The ubiquitous coenzymes NADH and nicotinamide adenine dinucleotide phosphate (NADPH), hereafter NAD(P)H, are key players of the basic metabolism as well as of various other functions in cells. They are endogenous fluorescence probes, since they can be selectively detected by two-photon microscopy when they are excited at 760 nm and their fluorescence is detected at 460 nm (Figure 1b)
In the phasor plots in b and c “free” encodes free NAD(P)H, “enzym.” NAD(P)H bound to metabolic enzymes, “NOX” NADPH bound to NADPH oxidases and “#” oxidized lipids as defined by Datta et al Since the NADPH oxidase NADPH oxidase 2 (NOX2)—the main catalyzer of oxidative burst and highly expressed in Polymorphonuclear cells (PMNs)-plays a central role in the process of phagocytosis, we focus on the detection of its activation during phagocytosis of S. aureus coated beads by means of NAD(P)H-fluorescence lifetime imaging microscopy (FLIM)
Summary
Since 1990, when Winfried Denk et al first introduced the concept of two-photon laser-scanning microscopy and showed the unique benefits of ultra-short pulsed, near-infrared excitation for imaging highly-scattering biological tissue [1], this laser-based technology tremendously expanded its field of application, especially in neurosciences [2,3,4] and immunology [5] and in other disciplines such as nephrology [6] or developmental biology. The further development in multi-photon microscopy heads towards a better understanding of function in living cells, tissue or even organisms. In this sense, fluorescence lifetime imaging microscopy (FLIM) is a promising technology, which probes the immediate microenvironment of molecules, as the basis of cellular function [13,14,15,16,17,18,19]. The time-correlated single-photon counting (TCSPC), which requires pulsed excitation as delivered by multi-photon microcopy, proved to be a highly versatile (but rather slow, i.e., typically 1–10 s/frame) technology to comprehensively acquire the molecular complexity within biological samples. TCSPC directly measures the fluorescence decay of all contained fluorophores, its thorough analysis remains a challenge [24,25]
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