Publikationsdatenbank

Vector-analyzed fluorescence lifetime microscopy approach: new insights into cell metabolism and calcium signaling, in vitro and in vivo (2021)

Art

Hochschulschrift

Autor

Leben, Ruth (WE 2)

Quelle

Berlin, 2021 — XIV, 162 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/33111

Kontakt

Institut für Veterinär-Physiologie

Oertzenweg 19 b
14163 Berlin
+49 30 838 62600
physiologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

The ability to dynamically observe functional processes at the (sub-) cellular level holds a high potential to answer open questions in medical biology, e.g. immunology. The ubiquitous co-enzymes nicotinamide adenine dinucleotide (NADH) and its phosphorized variant (NADPH) play a major role in metabolic processes, for instance in the production of the energy carrier ATP, or in the generation of reactive oxygen species (ROS) for cellular defense against invading pathogens. Since these two co-enzymes are autofluorescent, they provide an endogenous sensor to study those basic mechanisms label-free in vitro and in vivo using multi-photon microscopy. When NADH/NADPH bind to another enzyme to catalyze one of these processes, the molecular structure of the assembled enzyme compartment changes and thus (in contrast to their emission wavelengths) their fluorescence lifetime, meaning the time the molecule remains in the excited state before it emits light. However, the evaluation and interpretation of NAD(P)H fluorescence lifetime images (FLIM) in real biological environments are challenging. Here, the method of phasor analysis has been adopted and applied to stimulated/non-stimulated ROS-producing lymphocytes. This way it was possible to directly image the temporal dynamics of NADPH oxidase activation and its requirement for triggering NETosis in phagocyting neutrophils. In order to meet the growing demand for a systematic interpretation of NAD(P)H-FLIM measurements, especially to reflect the complex distribution and activity of NAD(P)H-dependent enzymes, the phasor approach was extended by the analysis of the phase vectors involved. For this purpose, a priori knowledge about the specific fluorescence lifetimes of the most abundant enzymes in tissue was created by measuring NADH or NADPH with the respective enzyme in solution. Applied to 3T3-L1 cell line or the data of phagocyting neutrophils, this method reveals insights into the enzyme composition and its enzymatic activity, which was shown to be even more complex than the pure FLIM images suggest. In addition to NAD(P)H-FLIM, the phasor approach and the vector analysis application to extract further information from the FLIM images was applied to Förster resonance energy transfer (FRET). The CD19+ lymphocytes of the reporter mouse line YellowCaB, carry the genetically encoded calcium-sensitive FRET-construct TN-XXL. In the presence of calcium ions, a second messenger of signal transduction, this construct is folded and the donor quenched. The fluorescence lifetime of the donor is shortened proportionally to the cytosolic Ca2+ concentration. Using vector analysis in the phase domain, maps of the absolute calcium ion concentration of the different B-cell populations in the germinal center of the lymph node were generated and their cell-to-cell interaction visualized. Together, both NAD(P)H-FLIM and donor-FRET-FLIM, hold the power to map mechanisms in cellular metabolism, defense, and intercellular communication, providing new insights into their interaction.