Yeast cells were grown to the mid-log phase and treated with rapamycin for indicated times. Cells were then concentrated by centrifugation and mounted on an agarose gel pad (SD-N containing 3.5% [wt/vol] agarose; Young et al., 2011 (link); Graef et al., 2013 (link)). Fluorescence microscopy was performed at room temperature using an inverted fluorescence microscope (IX83; Olympus) equipped with ×150 objective lens (UAPON 150XOTIRF; Olympus). A 488-nm blue laser (50 mW; Coherent) and 588-nm yellow laser (50 mW; Coherent) were used for the excitation of mNeonGreen and mCherry, respectively. Fluorescence was filtered with a dichroic mirror reflecting 405-, 488-, and 588-nm wavelengths (Olympus), separated into two channels using the DV2 multichannel imaging system (Photometrics) equipped with a Di02-R594-25x36 dichroic mirror (Semrock), and then passed through a TRF59001-EM ET bandpass filter (Chroma) for the mNeonGreen channel and FF01-624/40-25 bandpass filter (Semrock) for the mCherry channel. Images were acquired using an electron-multiplying CCD camera (ImagEM C9100-13; Hamamatsu Photonics K.K.) and MetaMorph software (Molecular Devices) and processed using Fiji (Image J; Schneider et al., 2012 (link)).