We use 2P FLIM-FRET Microscopy System to collect FLIM and FRET Images. Any fluorescence imaging requires careful selection of excitation power at the specimen plane in order to reduce photobleaching on one hand, but on the other hand obtain reasonable photon counts in the acquired data to obtain a good statistical fit. We found that 30 seconds is the ideal data collection time for our CFP expressed cells to obtain a lifetime of 2.4-2.6 ns. The same data acquisition time was used for all FLIM and FRET images. We used unfused cells to obtain multiphoton excitation intensity spectra for CFP, YFP, GFP, BFP and dsRED within the ti:sapphire laser tuning range from 700-1000 nm. We selected the peak excitation wavelength from these spectra to excite fused and unfused cells of the above-mentioned GFPs for FLIM images and the lifetime numbers were measured using the lifetime data processing software (SPCImage; Becker & Hickl) at every pixel.
This FLIM imaging methodology acquired lifetime images of CFP-C/EBPD154 (donor) in the absence and presence of the acceptor YFP-C/EBPD154. Mouse pituitary GHFT1-5 cells expressing CFP-C/EBPD154 alone (i.e. donor alone) were identified using an arc lamp light source. As described above, the 820 nm laser line was used as an excitation wavelength to illuminate the cell and the SPC-730 board was used to acquire lifetime images using 480/30 nm emission filters. The FLIM PMT was synchronized (see explanation in the Instrumentation Section) to the excitation laser pulse. The accumulation time was 30 sec to obtain reasonable photon counts. The acquired image was processed using the SPCImage software to obtain a single exponential decay FRET-FLIM image (tD).
We next imaged cells expressing the double-label combination of CFP-C/EBPD154 and YFP-C/EBPD154 (i.e. donor in the presence of the acceptor). The lifetime images were acquired using the same excitation (820 nm laser line) and emission filter (480/30 nm) used for donor alone lifetime image acquisition (www.chromatech.com). The double exponential fluorescence lifetimes were processed and calculated. Both sets of images (tD and tDA1, tDA2 were processed for lifetime FRET images. All images were acquired at room temperature (74oF). We did not observe any detectable autofluorescence signals using the unlabeled cells in the same media used for FRET-FLIM imaging.
See the image we collected using our FRET-FLIM system and processed using SPCImage Software.