1972). In addition, Arabidopsis C59 wnt price thaliana is studied because it is widely used as one of the model organisms in plant sciences. Materials and methods Fluorescence lifetime imaging microscopy Multiphoton imaging was performed on a multiphoton dedicated Biorad Radiance 2100 MP system, coupled to a Nikon TE300 inverted microscope (Borst et al. 2003). A tunable Ti-Sapphire laser (Coherent Mira) was used as an excitation source which was pumped with a 5-Watt (Coherent) Verdi laser, resulting in excitation
pulses of ~200 fs at a repetition rate of 76 MHz. In the beam-conditioning unit (BCU), the excitation power was tuned by a pockell cell. The laser beam was collimated in the scanhead and focused by a Nikon 60x water immersion Apochromat objective lens (NA 1.2) into the sample. The fluorescence was detected by non-descanned direct detectors
(NDDs), which were coupled to the sideport of the microscope. Using this type of detection, Selleckchem BIBF 1120 the loss of fluorescence light was reduced, and 3–5 times more signal was obtained compared to internal detectors. Selleckchem VX-680 The emission light was split into two channels using a dichroic mirror filter wheel. FLIM measurements were performed by directing the fluorescence via a secondary dichroic (770DCXR, Chroma Technology Corp.) into a Hamamatsu R3809U photomultiplier, operated at 3.1 kV. Fluorescence was selected using a dichroic (FF 495—DiO2, Semrock) and 2x a bandpass filter (HQ700/75, Chroma Technology Corp). In the excitation branch, a longpass filter (RG 780 3 mm, Schott) was used for reduction of the excitation light. The multichannel-plate photomultiplier allows single photon detection at high time-resolution, with an IRF of 25 ps (van triclocarban Oort et al. 2008, 2009). The output of the detector was coupled to a Becker & Hickl single-photon-counting module (SPC 830) (Becker and Bergmann 2002). The signal
from the Hamamatsu triggers the START of the time ramping for the time-correlated single-photon-counting (TCSPC). The pulses from the Ti-Sapphire laser serve as the SYNC signal to stop the time ramping and allowing the timing of the arrival of the fluorescent photons. The time window (ADC) was set to 1,024 channels and typically fluorescence was recorded for 2 min at a photon count rate of approximately 20 kHz. The signal from the PMT is combined with the pixel clock and line predivider signals from the Biorad scanhead to create 2D lifetime images. Fluorescence decay curves were fitted to a sum of N exponentials Σaiexp(−τ/τ i ) (i runs from 1 to N), convoluted with the IRF (Digris et al. 1999, van Oort et al. 2008, 2009), which was determined from the decay of pinacyanol iodide in methanol. From these results, an average lifetime <τ> was also calculated, according to <τ> = Σa i τ i . The number of counts in the peak channels is ~100 in the fluorescence intensities images and traces.