Photostability of Fluorescent Protein Fusions

Photostability of fluorescent proteins in fusion constructs was measured on a wide-field fluorescence microscope (Axiovert 200 M; Carl Zeiss GmbH) equipped with a xenon arc lamp with monochromator (Cairn Research, Faversham, Kent, UK). Measurements were performed under continuous illumination for 900 s with 420 nm light (slit width 30 nm) to excite mTurquoise2. Supplemental Figure S3 shows the photobleaching results for the first 48 s continuous illumination (corresponding to the total illumination time during a FRET experiment with 200ms exposure time and 121 time frames) of the same experiments as shown in Fig. 5 and Supplemental Figure S2. The power was measured at the 20x objective (Zeiss LD-A-plan 20x Air/0,30 ph1 ∞) using a coherent power meter (FM Fieldmaster Power Energy Meter, 0210-761-99). Each 4 s, fluorescence intensity of FRET donor and acceptor was recorded with an exposure time of 200ms using a 40x objective (oil-immersion Plan-Neo- fluor 40×/1.30; Carl Zeiss GmbH). mTurquoise2 emission was detected with a BP470/30 filter, GFP/YFP emission was detected with a BP535/30 filter and OFP/RFP emission was detected with a BP620/60 filter^{49 (link)}. Image analysis was done in ImageJ. After subtraction of background signal, the mean fluorescence intensity of the cells was calculated for each time point.