Es80 epi fluorescence microscope

The effect of small molecules on the temperature-induced nuclear localization of Crz1 was determined as previously described [12 (link)]. Briefly, overnight cultures of strain XW253 containing Crz1p-mCherry and Nop1p-GFP in YPD at 30°C were diluted 1:50 into fresh YPD and mock treated (1% DMSO) or treated with ¼ the MIC of the test compound. The resulting cultures were incubated for an additional 4 hr at either 30°C or 37°C. Samples (1 mL) were harvested by centrifugation, washed with DPBS, re-suspended in DPBS, and imaged using a Nikon ES80 epi-fluorescence microscope equipped with a CoolSnap CCD camera. Images were collected using NIS-Elements Software and processed in PhotoShop. Each condition was imaged on multiple days from independent cultures. For quantitative analysis, at least 100 cells with clearly visible signals for both flurophores were analyzed for each replicate and the percentage of cells with colocalized Crz1p-mCherry and Nop1p-GFP were calculated. Data are presented as mean percentage with error bars representing standard deviation.

For colony morphology analysis, plates were incubated for the designated time and imaged using a Nikon ES80 epifluorescence microscope equipped with a CoolSnap charge-coupled-device (CCD) camera at ×40 magnification. Live cell fluorescence imaging was carried out with a multiphoton laser scanning microscope (SP8; Leica Microsystems). Overnight cultures in YPD media were collected and back diluted either into fresh YPD or in the hyphal inducing medium to achieve either yeast or hyphal growth, respectively. Cells were then harvested, washed twice in 1× phosphate-buffered saline (PBS) and resuspended in 1× PBS prior to imaging. The samples were treated with Hoechst 33342 and incubated for 15 min in the dark at room temperature followed by washing twice with PBS. For Hoechst 33342 dye, an excitation wavelength of 350 nm and emission wavelength of 461 nm were used, whereas to visualize the GFP tag, an excitation wavelength of 395 nm and emission wavelength of 509 nm were used, and sequential images were acquired using 25× water immersive lens objective with a 3.32× zoom factor. Images were further analyzed by using ImageJ software.