Budding yeast strain KBY8065 (Mat a CEN15(1.8)-GFP[10kb] ade2-1, his3-11, trp1-1, ura3-1, leu2-3,112, can1-100, LacINLSGFP:HIS3, lacO::URA3, Spc29RFP:Hyg) was grown in liquid yeast extract peptone dextrose at 24°C. Cells were imaged in liquid yeast complete medium at 24°C. Time-lapse images were acquired on an Eclipse Ti wide-field inverted microscope (Nikon) with a 100× Apo TIRF 1.49 NA objective (Nikon) and a Clara CCD camera (Andor) using the Nikon NIS Elements imaging software. Time lapses were 10 min in duration with 30 s intervals. At each interval, a seven-step Z-stack of 400-nm step size was acquired in the GFP, RFP, and Trans channels.
Metaphase yeast cells (medium budded cells with two Spc29-RFP foci) were cropped by hand from the original time-lapse images. Both original and denoised time lapses were automatically tracked using a custom MATLAB program. The motion of the two sister lacO/LacI-GFP foci and the two sister Spc29-RFP foci were the motion of one focus relative to the other as in Chacón et al. (2014) (link), and the radius of confinement was calculated by a custom MATLAB program. For the images shown as illustrations, the heterogeneous background was subtracted with the rolling-ball method (10 pixel radius) in FIJI. Tracking results are from original and N2V-denoised images.
Metaphase yeast cells (medium budded cells with two Spc29-RFP foci) were cropped by hand from the original time-lapse images. Both original and denoised time lapses were automatically tracked using a custom MATLAB program. The motion of the two sister lacO/LacI-GFP foci and the two sister Spc29-RFP foci were the motion of one focus relative to the other as in Chacón et al. (2014) (link), and the radius of confinement was calculated by a custom MATLAB program. For the images shown as illustrations, the heterogeneous background was subtracted with the rolling-ball method (10 pixel radius) in FIJI. Tracking results are from original and N2V-denoised images.
