Attofluor live cell chamber

HeLa cells were transiently co-transfected with LOVTRAP plasmids 20 – 28 hours before imaging. Cells used for live cell imaging were seeded on coverslips coated with 10 mg/ml fibronectin in Ham’s F-12K medium free of Phenol Red and containing 0.5% FBS. Coverslips were mounted in an Attofluor live cell chamber (Invitrogen) and placed on a heated microscope stage (Warner). Images were acquired using an Olympus IX81-ZDC microscope equipped with a CoolSNAP HQ2 14-bit camera (Photometrics). Bandpass and neutral density filters (Chroma) were switched using motorized filter wheels (Ludl Electronic Products) controlled by Metamorph software version 7.6.4. YFP and mCherry images were acquired using a 100 W Hg arc lamp with a 1% ND filter and a 510–520 nm or 565–595 nm band-pass filter respectively, for 500 ms. For photoactivation, a 5% ND filter and a 426–446 nm band-pass filter were used. Unless otherwise indicated, activation was carried out using a pulse protocol alternating 5 seconds light with 5 seconds dark. This minimizes photodamage while maintaining greater than 80% activity for wild type LOV2. Images were processed post acquisition via shading correction, background subtraction, and binary masking using Metamorph software (Molecular Devices).