Plan apo objective

The queen plasmid used in this study was purchased from NBRP Yeast Genetic Resource Center (NBRP/YGRC)^{12 (link)}. Queen-expressing budding yeast strains were generated by linearizing the constructs with PstI and then inserting them into the his3Δ1 locus of the YMY2012 and glucose-relevant mutant strains hxk2Δ, gpr1Δ, snf3Δrgt2Δ. Budding yeast cells were immobilized on a glass cover slip-coated with concanavalin A at 1 mg/ml before imaging. The immobilized cells were imaged by a Nikon Ti2 SDC microscope equipped with a Yokogawa CSU-W1 confocal spinning head, a Plan-Apo objective (100×1.45-NA), and a back-illuminated sCMOS camera (Prime95B; Teledyne Photometrics). Imaging lasers were provided by 405 nm/100 mW (Vortran), 488 nm/150 mW (Vortran), combined in a laser launch (iLaunch, GATACA Systems). The QUEEN ratio was calculated using Fiji software^{61 (link)} and was calculated as follows. First, both the QUEEN images were converted to signed 16-bit floating-pointed grayscale. The QUEEN signals were subjected to background subtraction using the mean intensity per pixel from the signals outside the cells. Then, the intensity of ex405 images was divided by ex480 images to calculate the QUEEN ratio at each pixel, as the intensity ratio to represent the cellular ATP concentration. The normalized queen ratio represents the value in the presence of ES divided to in the absence of ES.

Yeast strains were cultured overnight at 30 °C or 25 °C in the synthetic complete medium (SM) without tryptophan and reinoculated into fresh medium the following day to a final OD₆₀₀ = 0.15 for an additional 3 h of culture before imaging. Cells were immobilized onto concanavalin A (ConA, 1 mg/ml)-coated coverslips and imaged by spinning disc confocal (SDC) microscopy coupled with structured illumination microscopy (SDC-SIM). The imaging was performed by a Nikon Ti2 inverted microscope equipped with a Yokogawa CSU-W1 confocal spinning head, a Plan-Apo objective (100 × 1.45-NA), a back-illuminated sCMOS camera (Prime95B; Teledyne Photometrics), and a superresolution module (Live-SR; GATACA Systems). All image acquisition and processing were controlled by MetaMorph (Molecular Device) software. The images were acquired continuously at a 0.25-μm interval for a total range of 7.5 μm in the z-direction, using 200 ms exposure for actin cable and 100 ms for Spa2.