Manual fluorescence microscopy was performed with an AxioVision fluorescence microscope (Zeiss) equipped with the 100/1.4 oil-immersion objective lens (Plan-Apochromat) and the HBO103W/2 mercury vapor short-arc lamp. The GFP fluorescence was observed with an excitation maximum of 488 nm and an emission maximum of 520 nm, and the RFP fluorescence with an excitation maximum of 595 nm and an emission maximum of 615 nm. Images were taken with the Hamamatsu Orca-ER digital camera using Velocity software (Improvision). tdTomato cannot be visualized by using these settings and can only be viewed with the Evotech high-throughput microscope.
The resumption of growth after quiescence was induced by adding fresh YPD, and the protein dynamics were immediately monitored by fluorescence microscopy within a time frame of 10–30 min, as described previously (Weberruss et al., 2013 (link)).
The resumption of growth after quiescence was induced by adding fresh YPD, and the protein dynamics were immediately monitored by fluorescence microscopy within a time frame of 10–30 min, as described previously (Weberruss et al., 2013 (link)).