Workshop 6: Label-free FLIM - Studying the cellular metabolism by NADH autofluorescence FLIM

During this session, we explore the potentials of autofluorescence imaging to investigate the cellular metabolic state. Adenosine triphosphate (ATP), the energy molecule of the cells is the product of different energy production pathways - glycolysis and Krebs cycle and oxidative phosphorylation. Two co-factors plays important role in the energy production pathways: NAD(P)H (reduced nicotinamide adenosine dinucleotide (phosphate)[H]) and FAD (oxidized Flavin adenine dinucleotide). The autofluorescence of both NAD(P)H and FAD exhibit two distinctive fluorescence lifetimes for bound and free forms. The changes between the fractions of the short and long lifetime of these co-factors indicate changes in the metabolic activity and pathways in the specimen. Instruments Leica SP8 FALCON This fluorescence microscope enables two-photon NAD(P)H and FAD autofluorescence FLIM. The system is equipped with FOUR HyD detectors in the non-descanned (NDD) configuration and FALCON module for sensitive and fast lifetime imaging. 00:00​ Intro 21:12​ Sample preparation 27:06 Instrumentation 42:00 Proper FLIM experiment 2:09:02 Metabolic data acquisition and analysis 2:52:06​ Biological interpretation and summary NOTE: the Q&A was open during the workshop and addressed between the chapters ================== Presenter: Ali Gheisari, Imaging specialist at CMCB light microscopy facility at Technischer Universität Dresden (Germany) Cornelia Wetzker, Imaging specialist at CMCB light microscopy facility at Technischer Universität Dresden (Germany) ================== This video is recorded on 26.11.2021 during the online FLIM symposium - Beginners Guide to Fluorescence Lifetime Imaging (FLIM) in Life Sciences takes place on November 23. to 27. 2020 via Zoom. ================== Website: https://webapp.biotec.tu-dresden.de/e...