chameleon ii 2 photon laser  (Coherent Corp)

Journal: Scientific Reports

Article Title: A fluorescence lifetime-based FLIM-timer for measuring the protein turnover of transcription factor Nrf2 in live cells

doi: 10.1038/s41598-025-14721-6

Figure Lengend Snippet: FLIM-based fluorescence timer for measuring Nrf2 turnover. ( a ) Nrf2 regulation by Keap1. At basal conditions (left), Nrf2 is targeted for proteosomal degradation by Cullin3–RING ubiquitin E3 ligase in a complex with its adaptor Keap1 that recruits Nrf2. Modifications of sensor cysteines in Keap1 by inducers ROS or electrophiles (right) inhibit Keap1-Cullin3-mediated degradation of Nrf2, stabilises Nrf2 and initiates oxidative stress response. ( b ) Design of a FLIM-timer protein tag, composed of the fast-maturing FRET donor and the slower maturing FRET acceptor, linked by a linker peptide. FRET between tag components shortens the fluorescence lifetime (tm) of the FRET donor and serves as readout for protein turnover/stability. ( c ) The pixels within cellular areas shown and quantified in Suppl Fig a-c were grouped by their GFP channel intensities into 37 equal intensity bins, and the Coefficient of Variation (CV) for mCherry/GFP ratio (left) or fluorescence lifetime (right) calculated for each bin was plotted against mean bin intensity. Blue dashed lines show average values of cellular area. ( d ) Fluorescence lifetimes sfGFP-mCherry FLIM-timer and the sfGFP control constructs in Dox-induced HeLa-FRT/TO cells is measured using InTune laser. Fluorescence lifetime (tm) determined in entire cellular areas using global binning as described are shown as dots, boxes outline the data range within lower and upper quartiles, thick line marks median and whiskers extend to the highest and lowest values excluding outliers. ( e ) Photon numbers (top panels) and fluorescence lifetime heat maps (bottom panels) of HeLa cells transiently transfected with sfGFP-mCherry-Nrf2 (FT-Nrf2), sfGFP-Nrf2 with or without co-transfection with free mCherry, or sfGFP-Nrf2-mCherry, as is indicated. The colours correspond to the tm values as shown underneath. Representative images from 7, 3, 7 or 10 FLIM datasets respectively, are shown. Quantification in Suppl. Fig b. ( f ) Strong negative correlation between ratiometric and FLIM-based measurements shown by Pearson Correlation Coefficient (r) and a slope of the linear fit of the data with the high goodness of fit (R ). Dots are globally measured values of fluorescence lifetime (tm) and GFP fluorescence intensities ratios measured in the same nuclear or cytoplasmic regions (N = 12) of HeLa cells transiently transfected with sfGFP-mCherry-Nrf2. Fluorescence lifetime acquired with 2-photon laser, followed by confocal imaging. ( g ) Confocal image of HeLa cells transfected with FT-Nrf2 displayed as an overlay of sfGFP (green) and mCherry (red) channels (left panel) or a fluorescence lifetime map obtained with 2-photon laser viewing FLIM (right). The false colours correspond to the tm values as shown underneath. See also Suppl Figs. and .

Article Snippet: Fluorescence lifetime was measured using SPC-150 Time-Correlated Single Photon Counter (TCSPC) module (Becker&Hickl) attached to LSM 710 microscope (Zeiss) equipped with either InTune pulsed laser with repetition rate 40 MHz and tunable wavelength (Zeiss) or 2-photon Chameleon laser with pulse repetition rate 80 MHz (Coherent) and HPM-100-40GaAsP detector that feeds to TCSPC module.

Techniques: Fluorescence, Ubiquitin Proteomics, Control, Construct, Transfection, Cotransfection, Imaging