posted on 2019-04-02, 13:09authored byKathryn Ayscough, Iwona Smaczynska-de Rooij
<div>Set of movies files to provide minimal dataset for Smaczynska-de Rooij 2019 PLoS One figure 6</div><div>Methodological information</div><div>
<p><b></b>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.</p>
<p>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. </p>
<p>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.<a></a> 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. </p>
</div>