Journal: Data in Brief
Article Title: Quantifying nuclear wide chromatin compaction by phasor analysis of histone Förster resonance energy transfer (FRET) in frequency domain fluorescence lifetime imaging microscopy (FLIM) data
doi: 10.1016/j.dib.2020.105401
Figure Lengend Snippet: Workflow behind a histone FLIM-FRET experiment performed on the Olympus confocal scanning microscope that is coupled to an ISS FastFLIM box for time resolved detection. (a) To find a U2OS cell nucleus co-expressing H2B-eGFP and H2B-mCH at a specific acceptor-donor ratio we first set up a light path within the Olympus FV3000 confocal laser scanning microscope that empoyed the internal continuous wave lasers and GaAsP PMTs to detect eGFP and mCherry signal (top panel), and then used the Olympus Fluoview software to acquire a two-channel intensity image of a selected nucleus co-expressing the histone FRET pair (bottom panel). (b) To acquire a FLIM image of histone FRET within the selected U2OS cell nucleus we modified the Olympus FV3000 light path set up to employ an external modulated 488 nm laser source (80MHz) and then directed only the donor fluorescence (eGFP signal) to the external ISS FastFLIM box for time resolved detection. (c) Prior to FLIM acquisition of histone FRET experiments we calibrated the ISS FastFLIM system and phasor space by measurement of fluorescein at pH 9 (top panel) and then in the ISS Vista Vision software applied the derived phase versus modulation corrections to all subsequent FLIM acquisitions of donor fluorescence (H2B-eGFP) performed on the same day (bottom right panel). The fluorescence lifetime of H2B-eGFP recoreded in each pixel of a FLIM image was then transformed into histone FRET by phasor analysis in the SimFCS software, which enabled chromatin compaction to be spatially mapped and quantified (bottom left panel).
Article Snippet: Workflow behind a histone FLIM-FRET experiment performed on the Olympus confocal scanning microscope that is coupled to an ISS FastFLIM box for time resolved detection. (a) To find a U2OS cell nucleus co-expressing H2B-eGFP and H2B-mCH at a specific acceptor-donor ratio we first set up a light path within the Olympus FV3000 confocal laser scanning microscope that empoyed the internal continuous wave lasers and GaAsP PMTs to detect eGFP and mCherry signal (top panel), and then used the Olympus Fluoview software to acquire a two-channel intensity image of a selected nucleus co-expressing the histone FRET pair (bottom panel). (b) To acquire a FLIM image of histone FRET within the selected U2OS cell nucleus we modified the Olympus FV3000 light path set up to employ an external modulated 488 nm laser source (80MHz) and then directed only the donor fluorescence (eGFP signal) to the external ISS FastFLIM box for time resolved detection. (c) Prior to FLIM acquisition of histone FRET experiments we calibrated the ISS FastFLIM system and phasor space by measurement of fluorescein at pH 9 (top panel) and then in the ISS Vista Vision software applied the derived phase versus modulation corrections to all subsequent FLIM acquisitions of donor fluorescence (H2B-eGFP) performed on the same day (bottom right panel).
Techniques: Microscopy, Expressing, Laser-Scanning Microscopy, Software, Modification, Fluorescence, Derivative Assay, Transformation Assay
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