Plan apochromat 63 1.40 oil objective

Conidia of sdeA::GFP cyp51A::mRFP strain were inoculated in glass-bottomed dishes (MatTek Corporation) containing 2 mL of MM and incubated for 8 h at 37°C. Filter sets 38HE and 63HE (Carl Zeiss) were used to detect GFP and mRFP, respectively. Germlings were analyzed under a 100× magnification oil immersion objective (NA 1.4). The nuclei were visualized by staining with Hoechst (20 μg/mL). Images were captured with an AxioCam MRm camera coupled to a Zeiss Observer D.1 microscope and processed using ZEN software. For the SdeA::GFP relocation assays in the presence of antifungals, confocal laser scanning microscopy was performed using a Carl Zeiss LSM 800 confocal microscope with a Plan Apochromat 63×/1.40 Oil objective. The detection parameters for SdeA::GFP experiments were fixed as follows: laser, 488 nm; 35% 1.89 AU/84 μm detection wavelength, 488 to 574 nm; and detection gain, 850 V. The z-stack increments were 0.3 μm. The images were analyzed using ZEN Blue 2.3.

PC12 cells were plated on 4-well glass-bottom cell-culture plates (Greiner Bio-One, Frickenhausen, Germany) at a concentration of 4 × 10⁴ cells/well. Forty-eight hours after seeding, the cells were incubated in the presence of either 100 nM ⁵⁴⁶Alexa-rGIIA(D49S) or 100 nM ⁵⁴⁶Alexa-rGIIA for 3 h. The cells were then stained for mitochondria using MitoTracker™ Green FM (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer instructions, washed with the complete culture medium and imaged using an inverted confocal laser scanning microscope (Axio Observer Z1 LSM 710, ZEISS, Oberkochen, Germany) with a Plan-Apochromat 63/1.40 oil objective. Fluorophores were excited sequentially using argon (488 nm) and helium–neon (543 nm) lasers and the emitted light was collected through SP 545 and LP 545 filters. The mean degree of the colocalization of the red and green signals of at least 7 images was calculated using ZEISS ZEN software. Colocalization was presented as the Manders’ coefficient [45 (link)], i.e., the ratio of the summed intensities of the pixels from the green channel, for which the intensity in the red channel is above the threshold, to the total intensity in the green channel. An image of the colocalization was displayed using the “Colocalization” tab in the ZEISS ZEN software.

Cells were fixed in 4% paraformaldehyde in PBS for 15 min and stained for the indicated proteins. Images were obtained with a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Germany) using a Plan-Apochromat × 63/1.40 oil objective. The pinhole size was set to 1 airy unit for all channels. Z-stack images were collected at optimal intervals as suggested by the ZEN software (Zeiss), and the three-dimensional images were produced using Volocity software (Perkin-Elmer, Waltham, MA, USA).

Cells were washed three times in QH₂O then suspended in QH₂O. Overall, 15 μl of cell suspension was dropped on 30 μl of 1.5% agar film on a glass slide and sealed with DPX mountant (Sigma−Aldrich) between the glass slide and a coverslip. Fluorescence images were taken with an inverted fluorescence microscope (AxioObserverA1m, Zeiss) equipped with a Hal 100 halogen lamp, a high intensity HBO 100 mercury lamp and an ORCA-ER camera (Hamamatsu). Excitation light was first filtered by a 470/40 nm bandpass filter, then reflected by a 495 nm dichroic beam splitter to the sample through an objective (Plan-Apochromat × 63/1.40 oil objective, Zeiss). Fluorescence emission was filtered by a 520/40 nm bandpass filter before detection by the camera. Each fluorescence image was taken with a 0.1 s exposure time and 50 electron multiplication gain.

Cells were fixed in 4% paraformaldehyde in PBS for 15 min and stained for the indicated proteins. Images were obtained with a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Germany) using a Plan‐Apochromat 63 × /1.40 oil objective. The pinhole size was set to 1 airy unit for all channels.