Axiovert 100s microscope

FRET was carried out using a Zeiss LSM510 with 'META' spectral detector mounted on an Axiovert 100S microscope with a 63x Planapochromat, 1.4 NA oil-immersion objective (Zeiss). ECFP and EYFP (Karpova et al., 2003 (link)) were excited with 458 nm laser light, emitted fluorescence was collected in 8 images each separated by 10 nm between 467 nm and 638 nm in lambda scanning mode. Separation of ECFP and EYFP fluorescence spectra was carried out using the linear unmixing algorithms of the Zeiss LSM510 software (Zeiss), using reference spectra taken from cells expressing the ECFP or EYFP fusion proteins alone or untransfected cells. The fluorescence spectrum was separated into ECFP, EYFP and background signals. FRET was assayed by acceptor (EYFP) photo-bleaching. Bleaching was accomplished using 50 iterations of 514 nm laser light with no attenuation from the acousto-optical tuneable filter (AOTF).

For BiFC HEK293a cells were transfected with plasmids allowing expression of mCherry (control for transfection efficiency), vYNE-SPIRE1/2 (human SPIRE1/2 fused to the C-terminus of enhanced vYFP (accession CAO91538.1) N-terminus fragment (amino acids 1–173)) and vYCE-Rab27a wild type and mutants (Rab27a (rat)) fused to the C-terminus of enhanced vYFP C-terminus fragment (amino acids 155–239) using Fugene 6 transfection reagent (Promega E2691). Forty-eight hours later mCherry and YFP (BiFC) fluorescence was recorded from living cells in low power fields of view for each condition using the Zeiss Axiovert 100 S microscope, using the same exposure setting for each channel and each condition. Normalised BiFC signal was determined using ImageJ software. Briefly mCherry images were converted to binary and ROIs corresponding to transfected cells were saved to the regions manager. Mean vYFP for each ROI was extracted and normalised BiFC determined for each ROI by dividing the vYFP signal by the corresponding mCherry signal to normalise for differences in the level of transfection in each cell. Median BiFC signal for ROI was determined using Graphpad Prism^{7 (link)} software (Graphpad, La Jolla, USA).