Ix81 wide field epifluorescence microscope

Yeast cells were grown to logarithmic phase on SC-Trp medium at 25°C. They were adhered to ConA coated coverslips. Cells were incubated for 10 min at room temperature on the ConA coated coverslip and then washed with SC-Trp medium. Cells were imaged on the coverslip in 40 µl of SC-Trp medium. All samples were imaged at room temperature using an Olympus IX81 wide-field epifluorescence microscope equipped with a 100×/1.45 objective. For single channel live cell imaging 488 nm laser light and images were acquired with 80–100 ms exposure time. Emission light was filtered using the GFP-3035C-OMF single-band filter set (Semrock, Rochester, NY). Fluorescence was detected using the Hamamatsu ImagEM EMCCD camera. For two color live cell imaging the samples were excited simultaneously with 488 nm and 561 nm laser light for 250 ms exposure. Excitation light was reflected with a OBS-U-M2TIR 488/561 (Semrock, Rochester, NY) dichroic mirror. Emission light was split and filtered with the DUAL-view (Optical Insights, LLC, Tucson, AZ) beam splitter. The beam splitter created two separated images, one for each channel, on the Hamamatsu ImagEM EMCCD camera sensor. Photobleaching was performed using a custom built setup with a 488 nm laser, focused on a ∼0.5 µm spot.
The wide-field epifluorescence microscope setup was controlled by Metamorph 7.5 (Molecular Devices, Sunnyvale, CA).

All in vitro experiments were performed at 23°C. Bead motility assays were carried out using a Olympus IX81 wide field epifluorescence microscope with LED illumination (pE4000, CollLED). Image acquisition was performed by a Hamamatsu c9100-13 EMCCD camera controlled by Micromanager 1.4 software^{70 (link)}. Multi-color images were taken through an UPlanS APO 40x or 60x oil objective.