The fluorescence lifetime of many fluorophores is highly dependent on their environment, making fluorescence lifetime imaging (FLIM) a sensitive tool for analyzing subcellular distributions of certain ions or gradients in viscosity or hydrophobicity. In this workshop, we will exemplary demonstrate the usage of a pH-sensitive dye to monitor changes in lysosomal pH (Tannert et al, submitted). Lysosomes are small acidic degradation compartments of eukaryotic cells. Their physiological pH is usually in the range of 4.5-5, depending on their subcellular localization and maturation status. Different (patho)physiological conditions, like autoimmune diseases, neurodegenerative disorder, or age-related complications can induce alterations in lysosomal pH. In most of these cases the lysosomal pH is increased. Lysosomal neutralization can also be induced by chemical stimulation, for instance using ammonium chloride. We will demonstrate a lysosomal staining procedure of HeLa cells and subsequent FLIM measurement. To be able to follow lysosomal pH changes over time, the measurement time for individual FLIM measurements in a time series has to be short. Therefore, the rapidFLIMHiRes module is applied. During a time series measurement, we will increase lysosomal pH by addition of 10 mM ammonium chloride after a certain baseline and monitor the alterations by detecting the lifetime of a pH sensitive lysosomal marker dye. Subsequently, FLIM data analysis of this time series will be explained in detail. Thereby we will give various tips for data analysis of standard FLIM or rapidFLIM acquisition conditions using the SymPhoTime 64 software package.
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Presenter:
Astrid Tannert, Scientific coordinator of Jena Biophotonics and Imaging Laboratory (JBIL) at Leibnitz IPHT, (Germany)
Fabian Jolmes, Sales and Application Specialist at PicoQunat GmbH (Germany)
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This video is recorded on 25.11.2021 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.
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21 окт 2024
