Hc pl apo 100 8 na

16-Bit images were taken with a Leica DM6B-Z epifluorescence microscope equipped with a Leica-DFC9000GT camera using an HC PL APO ×100/8 NA 1.4 oil lens. Exposure was set to 500 ms for GFP (470_ex, 525_em) and 600 ms for TXR/mCherry (560_ex, 630_em) channels. Raw images were exported as TIF files and analyzed using an automated macro in Fiji (ImageJ 1.52n). First, lower and upper limits of the display range were set to 1,000–50,000 for GFP and 2000–65535 for mCherry, respectively. Total GFP puncta were quantified by down-sampling the GFP image to 8-bit and using the “Find maxima” command (prominence set to 12). Nonvacuolar GFP puncta were quantified by excluding Vph1-mCherry–stained vacuoles, which were segmented by down-sampling the mCherry image to 8-bit and applying automated thresholding (Huang method).

16-Bit images were taken with a Leica DM6B-Z epifluorescence microscope equipped with a Leica-DFC9000GT camera using an HC PL APO ×100/8 NA 1.4 oil lens. The GFP filter set (470_ex, 525_em) was used for detection of GFP, and the TXR filter set (560_ex, 630_em) was used for detection of RFP or mCherry. The software Leica Application Suite X 3.6.0.20104 was used to capture images with exposure times adjusted to avoid signal saturation and using the autofocus module applied at DIC filter conditions. Raw images were exported as TIF files and further processed using the open-source software Fiji (93 (link)). 16-Bit images were converted to 8-bit, and the same display range settings were applied to all images of a given set of samples.