The system consists of a Nikon TE300 epifluorescent microscope with a 100W Hg Arc Lamp. A Plan Fluor 60x NA 1.4 oil IR objective lens is used for 2p-FRET and 2p-FRET-FLIM image acquisition. TE300 is coupled to the Biorad Radiance2100 confocal/multiphoton system (www.cellscience.bio-rad.com). A 10W Verdi pumped, tunable (model 900 Mira, www.coherentinc. com) modelocked ultrafast (78 MHz) pulsed (<150 femtosecond) laser was coupled to the laser port of a Radiance2100 . This laser is equipped with x-wave optics for easy tunable range of the entire wavelength (700 to 1,000 nm). The system is equipped with laser spectrum analyzer (Model E201; www.istcorp.com) to monitor the excitation wavelength and power meter to measure the laser power at the specimen plane (Model SSIM-VIS & IR; www.coherentinc.com). The Radiance system is equipped with an external detector and four internal detectors for fluorescence imaging. The transmission detector is used for transmission imaging and also for Second Harmonic Generation (SHG) Imaging. LaserSharp2000 software is used to acquire both 2p-FRET and FLIM images using the internal or direct (external) detectors.
The Becker & Hickl GmbH company photon-counting module (TCSPSC, SPC-150) is widely used to acquire FLIM images for various biological applications (Bacskai et al., 2003; Eliceiri et al., 2003; König and Riemann, 2003). This board is installed in the Radiance2100 computer and the X and Y scan synchronizing pulses, together with a pixel clock signal from Radiance2100 control unit, are used to synchronize data collection in the SPC-150 board. This allows pixel-by-pixel registration of the accumulated photons with the laser scanning. Laser pulses are detected by a high-speed PIN photodiode and used by the SPC-150 board to determine the detection time of a photon (anode pulse from the PMT) relative to the laser pulses. This measurement system requires that the timer (a time-to-amplitude converter (TAC)) be activated only upon receipt of a detected photon rather than at every laser pulse. The SPC-150 system starts timing at the receipt of a detected photon and measures the time interval until the next laser pulse. A fluorescence decay histogram of photon emission times relative to the laser excitation pulse is generated from the distribution of inter-pulse intervals at each pixel of the image. The detector is a fast photomultiplier tube, with a FWHM response time of about 150ps (PMH-100, Becker & Hickl). This detector is fast enough to resolve lifetimes due to environmental changes in a biological system or protein-protein interactions. The data analysis software (SPCImage, Becker & Hickl) allows multi-exponential curve fitting of the acquired data on a pixel-by-pixel basis using a weighted least-squares numerical approach. The sum of all time bins is equivalent to the intensity image and this is displayed to an image, pseudo-colored according to the curve fit results. Therefore, each image can be easily displayed in a meaningful way to compare lifetimes within or between other images.
The FLIM PMT (model# PMH-100) was coupled in the middle of the arm connected to the direct detector. The coupler was removed in the arm and a flip mirror was inserted to direct the emission fluorescence signals from the specimen to the FLIM detector or to the direct detector. It's also possible to detect simultaneously both 2p-FRET and FLIM signal using this configuration. The six-position dual filter wheel is installed between the FLIM detector and the flip mirror in order to select appropriate emission filter depending on the fluorophore used for protein molecular imaging. In another filter wheel we used BG-36 glass filter which blocks the excitation IR laser light and also transmits the visible spectrum, about 70% at 500 nm (www.chromatech.com). The whole system, including the microscope, was covered with a black box in order to reduce the background counts to the lowest possible level.
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