C4742 95 digital camera

For fluorescence microscopy, C. albicans cells were fixed in 50 mM thimerosal and stained for exposed β-glucan (1.5 µg/ml Fc-Dectin-1 plus anti-human IgG conjugated to Alexafluor 488; green) and exposed mannan (25 µg/ml Concanavalin A conjugated to Alexafluor 647; red). All samples were examined by phase differential interference contrast (DIC) and fluorescence microscopy using a Zeiss Axioplan 2 microscope. Images were recorded digitally using Openlab v 4.04 (Improvision, Coventry, UK) with a Hamamatsu C4742- 95 digital camera (Hamamatsu Photonics, Japan). Fluorescence was quantified using and processed using Openlab (Openlab v 4.04: Improvision, Coventry, UK).
C. albicans cell walls were examined by high-pressure freeze substitution transmission electron microscopy (TEM). C. albicans SC5314 cells were incubated for 6 h at 37 °C with extracts from the small intestine, cecum or large intestine (above). These fungal cells were then processed for TEM as described previously (Ene et al., 2012 (link)), cutting ultrathin (100 nm) sections. Samples were imaged with a Philips CM10 transmission microscope (FEI, United Kingdom) equipped with a Gatan Bioscan 792 camera, and the images recorded using a Digital Micrograph (Gatan, Abingdon Oxon, United Kingdom).

Transmission electron microscopy (Joel model JEM 10–10) was used to visualize cellwall thickening in a vancomycin-decreased susceptibility strain. Bacteria were exposure to sub-inhibitor concentrations of vancomycin and then analyzed during the exponential growth phase. As a control, ATCC 25923 and VISA Mu ATCC 700699 strains were also grown to the exponential phase. The bacteria were harvested, washed, and fixed with a glutaraldehyde/formalin (2.5%/10%) solution in 0.1 M phosphate-buffered saline (PBS), pH 6.0. After that, the bacteria were post-fixed with osmium tetroxide, contrasted with uranyl acetate, and dehydrated in graded concentrations of ethyl alcohol (20, 30, 40, 50, 60, 70, 80, 90, and 100%). Transverse thin sections from samples embedded in resin were mounted on grids, which was followed by treatment with lead citrate. The samples were stained with 1% phosphotungstic acid at pH 7.2 and visualized by transmission electron microscopy. Observation and acquisition was performed using a transmission electron microscope operated at 100 kV and equipped with a Hamamatsu C4742-95 digital camera. The images were processed at 100,000× and analyzed to determine the cellwall thickness from an average of 10 cells per bacterial strain [30 (link)].

For fluorescence microscopy, cells were fixed in 50 mM thimerosal (Sigma-Aldrich) and stained for β-glucan (1.5 µg/ml Fc-Dectin-1 plus anti-human IgG conjugated to Alexa Fluor 488; green), chitin (50 µg/ml wheat germ agglutinin conjugated to Alexa Fluor 350; blue), and mannan (25 µg/ml concanavalin A conjugated to Texas Red; red). All samples were examined by phase differential interference contrast (DIC) and fluorescence microscopy using a Zeiss Axioplan 2 microscope. Images were recorded digitally using the Openlab system (Openlab v 4.04: Improvision, Coventry, UK) with a Hamamatsu C4742- 95 digital camera (Hamamatsu Photonics, Hamamatsu, Japan).
High-pressure freeze substitution transmission electron microscopy on normoxic and hypoxic C. albicans cells was performed as described previously (103 (link), 104 (link)), cutting ultrathin sections of 100 nm in thickness. Samples were imaged with a Philips CM10 transmission microscope (FEI, United Kingdom) equipped with a Gatan Bioscan 792 camera, and the images were recorded using a Digital Micrograph (Gatan, Abingdon Oxon, United Kingdom). The thicknesses of the inner chitin-glucan and outer mannan layers of the cell wall were measured by averaging >30 measurements for each cell (n > 30 cells) using ImageJ.