Ld plan neofluar 20x objective

Fluorescence microscopy was used to evaluate the membrane integrity of yeast cells that were exposed to compound 7b. For this purpose, C. albicans cell suspension (1 × 10⁷ cells/mL) that was prepared in sterile PBS was treated with MIC concentration of 7b and then incubated for two hours at 37 °C. Cells that were incubated in similar conditions without compound served as the negative control.
The suspensions were then centrifugated and the cell pellets were washed and resuspended in sterile PBS. PI solution was added to resuspended cells to achieve a final concentration of 1 μg/mL and the samples were incubated for another 30 minutes in the dark.
The cells were further analyzed with an inverted Zeiss Axio Observer Z1 microscope with a LD Plan Neofluar 20x objective (NA = 0.4, Zeiss). Concretely, the fluorescence images were collected using a Compact Light Source HXP 120 C mercury lamp, the light being reflected by a dichroic mirror while using an excitation filter BP 525/50. An AxioCam Icc digital camera was employed to capture the images, which were then processed while using the ZEN software.

The numerical aperture (NA) of the mini-microscope was calculated as approximately 0.32 at 10X magnification and 0.50 at 20X magnification. The lateral resolution of the mini-microscope was measured as approximately 1–2 μm (Supplementary Fig. 1). The point spread function (PSF) at 20X was also determined for the mini-microscope and the axial resolution was calculated as approximately 34 μm, based on the FWHM measurement (Supplementary Fig. 4). The distortion fields of the mini-microscope at 10X are shown in Supplementary Fig. 5, which were negligible in both directions. The benchtop microscope used for imaging was a Zeiss Axio Observer D1 microscope equipped with an AxioCam MRm Rev.3 camera (Carl Zeiss), an LD A-Plan 10X objective with a 0.25 NA, and an LD Plan-Neofluar 20X objective with a 0.4 NA. The confocal microscope was CSU-X1 (Yokogawa) equipped with a Zyla 5.5 camera (Andor) and a 20X objective with an NA of 1.15. A true background-free image of a stained mouse brain sample obtained from the confocal microscope in comparison with the benchtop microscope and mini-microscope is provided in Supplementary Fig. 5.

Images of axons in MAIDs were acquired on an Axiovert 200M microscope (Carl Zeiss) using LD Plan-Neofluar 20x objective and Volocity-controlled camera, filters, shutter, and stage. Images were taken with a 5% overlap to facilitate stitching (Perkin-Elmer; RRID:SCR_002668). Blank images were subtracted to correct for optical artifacts. The images were stitched automatically and ‘despeckled’, using a 3 × 3 median filter (ImageJ). To correct for large illumination artifacts, background was subtracted in ImageJ using the ‘Subtract background’ plug-in, with a 100 µm window and the sliding paraboloid algorithm.
Images of immunostained cells and neurons on coverslips (other than for neurite tracing) were acquired using an upright laser-scanning confocal microscope (LSM-510, Zeiss; RRID:SCR_014344) equipped with 40x or 100x oil-immersion objectives; 488, 543, and 633 nm lasers; and 505–530, 560–615, and >650 nm emission filters. Images for neurite tracing were acquired using Axio Observer.Z1 microscope (Zeiss) equipped with Hamamatsu ORCA-Flash 4 sCMOS camera, EC Plan-Neofluar 40x objective, Colibri 2 LED illumination and appropriate filters.