For fluorescence measurements a long pass filter transmitting light above 515 nm was used. Fluorescence spectra of single
beta Interleukin 2 (44-56) were recorded with a polychromator and an image intensifying detection unit (IMD 4562, Hamamatsu Photonics, Ichino-Cho, Japan) [16] fixed on top of the microscope. For measuring fluorescence decay kinetics and lifetime images (FLIM) of single cells a time gated image intensifying CCD camera system (Picostar HR 12; LaVision, G?ttingen, Germany) with a time resolution of 200 ps was used, as depicted in Fig. 1 and described in detail elsewhere [17]. Fluorescence decay kinetics of individual
cells were recorded by shifting the time gate of 200 ps over an axis of 20 ns, whereas lifetime images were calculated according to the formula τ = Δt/ln (IA/IB) from two fluorescence images measured within time gates of 1 ns width immediately after the laser pulse (intensity IA) as well as Δt = 2 ns later (intensity IB). Since the contribution of mRFP in the emission spectra was very low, all fluorescence images and decay kinetics were related to EGFP.
