Personal deltavision microscope system

Microscope imaging for sensitized emission experiments was done using a Personal DeltaVision microscope system (Applied Precision, Issaquah, USA), with Softworx 3.6.0 software. The microscope was equipped with an Olympus IX71 inverted microscope body, a 100x phase contrast objective (Olympus PlanApo 1.40 NA), a CoolSNAP HQ2 camera (Princeton Instruments), a 300W Xenon light source, and filters for imaging of CFP (ex. 430/24 nm; em. 472/30nm), GFP (ex. 470/40 nm; em. 525/50 nm), and mCherry (ex. 572/35 nm; em. 632/60 nm) from Chroma. For Cerulean and Venus the CFP/YFP/mCherry polychroic mirror was used (Chroma, 460–500, 525–575, 590–680 nm range) and for GFP, mCherry, mKate2, and tagRFP the GFP/mCherry polychroic mirror was used (Chroma, 400–470, 490–570, 580–630 and 640–730 nm range). Images were captured using 0.2s light exposure with 32% light transmission for every combination of filters. The FRET channel was set in the software by using the CFP or GFP excitation filter and the GFP or mCherry emission filter.