Hpm 100

NAD(P)H FLIM was performed on an upright LSM 510 microscope (Carl Zeiss) with a 1.0 NA ×40 water-dipping objective using a 650 nm short-pass dichroic and 460 ± 25 nm emission filter. Two-photon excitation was provided by a Chameleon (Coherent) Ti:sapphire laser tuned to 720 nm, with on-sample powers kept below 10 mW. Photon emission events were registered by an external detector (HPM-100, Becker & Hickl) attached to a commercial time-correlated single photon counting electronics module (SPC-830, Becker & Hickl) contained on a PCI board in a desktop computer. Scanning was performed continuously for 2 min with a pixel dwell time of 1.6 µs. Cell type (HC vs. SC) and z-position within the epithelium was determined prior to FLIM analysis using the mitochondrially targeted fluorescent dye TMRM. The dye was added to the recording medium, at a final concentration of 350 nM, 45 min before imaging. TMRM fluorescence was collected using a 610 ± 30 nm emission filter. Excitation was provided at the same wavelength as NAD(P)H to avoid possible chromatic aberration. The 585 ± 15 nm emission spectrum of TMRM ensured its fluorescence did not interfere with acquisition of the NAD(P)H FLIM images.

Intensity and FLIM images were obtained on an upright LSM710 (Carl Zeiss) microscope with a 40×/1.0 NA, water-dipping objective or 63×/1.40 Oil (Carl Zeiss). Acceptor intensity images were obtained with excitation at 512 nm using an Argon laser and registered on the Zeiss PMT detectors. Two-photon excitation was provided by Chameleon (Coherent) Ti:Sapphire laser tuned to 800 nm. FLIM emission events were recorded by an external detector (HPM-100, Becker & Hickl) attached to a commercial time-correlated single photon counting electronics module (Becker & Hickl) with a 480/40 (Chroma) emission filter. FLIM images were fitted using in SPCImage (Becker &Hickl). A single component incomplete multi-exponential model was used with a laser repetition time of 12.5 ns. Colour-coded lifetime maps and greyscale intensity images were exported from SPCImage. Acceptor intensity images were processed using FIJI. The histogram intensity weighted mean lifetimes for each image was generated by SPCImage 5.6; values were exported to OriginPro. The peak values were obtained by doing a Gaussian fit.

HEK-293T cells co-transfected with RFP-FGFR2 and Δ5-GFP or Δ10-GFP were cultured on glass coverslips. Serum-starved cells were fixed with 4% w/v paraformaldehyde for 20 min and mounted with mounting buffer. Confocal data were collected with an inverted confocal microscope system (Leica TCS SP2) and two internal photomultiplier tube (PMT) detectors (one displaying the GFP emission and the other one the RFP emission). FLIM data were collected with the same microscope system and a GaAsP hybrid detector (Becker & Hickl HPM-100). The samples were excited with a picosecond diode laser (Hamamatsu PLP-10 470) at 467 nm. The repetition rate of the laser was always 50 MHz, with pulse duration ~90 ps. A 63 × 1.2 NA water-immersion objective was used to acquire the images. A line scan speed of 400 Hz and an image size of 512 × 512 pixels were used for acquiring the confocal images. The FLIM images were acquired with an image size of 256 × 256 pixels. Images decays were analyzed, averaged, and fitted with a single exponential-decay model with SPCImage (Becker & Hickl). The GFP fluorescence lifetime histograms were created in Matlab.