Spectral Imaging of Fluorescent Protein Interactions

tsA-201 cells were plated onto poly-d-lysine coated 35 mm glass bottom culture dishes (World Precision Instruments, Sarasota, FL)^{56 (link)}. Cells were transiently transfected with GFP-Cav3.1, GFP-Kv4.3, FMRP(1–297)-mKate or FMRP-mKate constructs to use as donor–acceptor fluorescent pairs^{56 (link)}. On the experimental day DMEM was replaced with imaging medium comprised of (mM): 148 NaCl, 3 KCl, 10 HEPES, 3 CaCl₂, 10 d-Glucose, 1 MgCl₂ (pH 7.3 with NaOH) at 25 °C. Cells were examined on a Nikon Eclipse C1Si spectral confocal laser-scanning microscope using a 40×/1.3NA oil immersion objective. Laser lines of 457 nm were used to excite GFP and 561 nm to excite mKate, with emission spectra recorded between 400 and 750 nm. Spectral images were linearly unmixed offline using ImageTrak software^{56 (link)}.