Plan neo fluor 40 1

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.

Cells were transfected with Tandem FP constructs containing a T2A linker, mTurquoise2 as reference and a fluorescent protein of interest. Cells expressing two separate FPs in equal amounts were imaged on a widefield fluorescence microscope (Axiovert 200 M; Carl Zeiss GmbH) equipped with a xenon arc lamp with monochromator (Cairn Research, Faversham, Kent, UK), using a 40x objective (oil-immersion Plan-Neo- fluor 40×/1.30; Carl Zeiss GmbH). Orange FPs were excited with 510 nm light and emission was detected with a BP572/25 filter. As reference, mTurquoise2 was excited with 420 nm light and emission was detected with a BP470/30 filter. Clover and mNeonGreen are excited with 500 nm light and emission was detected with a BP535/30 filter. To prevent cross excitation, reference mTurquoise2 was excited with 405 nm light and emission was detected with a BP470/30 filter. After subtraction of background signal, the mean fluorescence intensity of the cells was calculated. The fluorescence intensity of the protein of interest relative to the fluorescence intensity of the reference mTurquoise2 reveals the relative brightness of the protein of interest^{42 (link)}.

Transfected HeLa cells on glass coverslips were mounted in metal Attofluor cell chambers 18 hours after transfection. Live-cell FRET imaging was performed on a widefield microscope (Axiovert 200 M; Carl Zeiss GmbH), equipped with an oil-immersion objective (Plan-Neo- fluor 40×/1.30; Carl Zeiss GmbH) and a xenon arc lamp with mono-chromator (Cairn Research, Faversham, Kent, UK). Images were recorded with a cooled charged-coupled device camera (Coolsnap HQ, Roper Scientific, Tucson, AZ, USA). Samples were excited using 420 nm light (slit width 30 nm) and a 455DCLP dichroic mirror. CFP emission was directed to a BP470/30 filter, by rotating the filter wheel YFP emission was directed to a BP535/30 filter. RFP was excited with 570 nm (slit width 10) and using a 585CXR dichroic mirror. RFP emission light was directed to a BP620/60 filter. All acquisitions were background- and bleedthrough-corrected (55%).