Movies for minimal dataset. Smaczynska-de Rooij PLoS One 2019
Fluorescence microscopy: For co-localisation and live-cell imaging, cells expressing tagged proteins were visualized after growing to early log phase in synthetic medium with appropriate supplements.
Epifluorescence microscopy was performed using Olympus IX-81 inverted microscope with a DeltaVision RT Restoration Microscopy System (using a 100x/1.40 NA oil objective), Photometrics Coolsnap HQ camera with Imaging and Image capture performed using SoftWoRx™ image analysis and model-building application (Applied Precision Instruments, Seattle). Time-lapse live cell imaging of GFP-tagged Sla2 was performed with 1 sec time-lapse. All image data sets were deconvolved, using the SoftWoRx application.
Time-lapse live cell images of Rvs167-GFP was acquired using OMX DeltaVision V4 and a 60xUSPLAPO (numerical aperture, 1.42) objective with refractive index 0.1514 immersion oil (Cargille). Samples were illuminated using Insight Solid State Illuminator (10%), and images were taken simultaneously on separate scientific complementary metal oxide semiconductor (sCMOS) cameras (70 ms exposure). Seven 250 nm sections were acquired every 500 ms (181 time points). The stacks were then deconvolved and processed, using SoftWorx, to produce a movie composed of maximum intensity projections at each time point. The Rvs167-GFP lifetime was analyzed from those projections. Movies and kymographs were assembled using Fiji software. Images were exported as TIFF files and image size adjusted to 300 d.p.i. and assembled using Adobe Photoshop CS2.
