Elyra microscope

Immunostaining was performed as described above, but slides were mounted using ProLong Gold mounting media instead of vectashield. Images were acquired using a Zeiss Elyra microscope.

Super-resolution microscopy was performed at the Harvard Center for Biological Imaging (HCBI) (Cambridge, MA, USA) using a Zeiss ELYRA microscope, which is capable of performing both dSTORM and SIM imaging. This microscope is equipped with 488 nm, 561 nm, and 638 nm laser lines, and a ×100 oil immersion objective lens (NA 1.4). For SIM imaging Aβ samples were dried onto pre-washed coverslips and covered in mounting medium (Vectashield H1000, Vector laboratories). For dSTORM imaging, samples were dried onto glass-bottom multi-well plates (Lab-Tek), and were then covered in photoswitching buffer solution immediately before imaging; this solution consisted of 100 mM mercaptoethylamine (MEA) in phosphate-buffered saline (PBS, pH 7.4), together with a glucose-enzyme oxygen scavenger (40 mg/ml glucose, 50 mg/ml glucose oxidase, 1 mg/ml catalase). The chamber was filled to the top, and was closed with a cap to minimize entrance of oxygen. Collected images were then processed using ZEN 2.3 software.

Imaging of pHrodo-labeled Mtb as described in Queval et al., 2017 (link). Mtb strains were grown to mid-log phase, then washed twice with an equal volume of PBS. Mtb was then resuspended in 100 mM NaHCO₃ with 0.5 M pHrodo dye (Invitrogen) and incubated at room temperature in the dark for 1 hr. The labeled cells were then washed three times with PBS, after which BMDM infections were performed as described above. At the indicated time post-infection, infected BMDM were washed with PBS and fixed in 4% paraformaldehyde. Nuclei were labeled with DAPI (1.25 mu-g/ml). Cells were then imaged on a Zeiss Elyra microscope with a 40× oil objective or a TissueFAXS confocal microscope with a 40× objective. Images were imported into CellProfiler (Carpenter et al., 2006 (link)) for analysis. Bacterial outlines were identified based on GFP signal; the outline was then expanded by five pixels, and pHrodo fluorescence intensity within the expanded outline was determined.

Cells grown on coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature. For immunostaining of plasma membrane PtdIns(4,5)P₂ and PtdIns(4)P, cells were fixed with 4% paraformaldehyde and 0.2% glutaraldehyde for 15 min at room temperature. Cells were washed three times with PBS containing 50 mM NH₄Cl. All subsequent steps were carried out on ice. Cells were blocked and permeabilized for 45 min in PBS containing 5% normal goat serum (NGS), 50 mM NH₄Cl, and 0.5% saponin. Primary antibodies were diluted in PBS containing 5% NGS and 0.1% saponin and applied to cells for 1 h. After three washes with PBS, cells were incubated with secondary antibody in PBS containing 5% NGS and 0.1% saponin for 45 min. Next, cells were washed with PBS for four times and post fixed in 2% paraformaldehyde in PBS for 10 min on ice and 5 min at room temperature, followed by three washes with PBS containing 50 mM NH₄Cl. Cells were mounted in ProLong^® Gold antifade reagent (LifeTechnology). Confocal images were acquired on an Olympus FV1200 laser-scanning microscope. Total internal reflection fluorescence (TIRF) and epifluorescence microscopy were carried out using a Zeiss Elyra microscope. The manufacturer’s software and FIJI software were used for data acquisition and analysis.

3D-SIM was performed on a Zeiss Elyra Microscope coupled to an optovar 1.6, ×63 objective, and a camera EM CCD Andor SIM. During z-stack acquisition, five rotations were applied. Deconvoluted structured illumination images were generated by the Zen software, and images were merged in ImageJ.

Widefield microscopy was carried out on an Olympus BX51 microscope. SIM was carried out on a Zeiss ELYRA microscope. The SIM image was calculated from five grating-angles and five phase shifts. A z-stack was acquired using 15 slices covering 1.65 μm axially. The raw images were processed into super-resolution images using the Zeiss ZEN (Black Edition) software with the default settings. To correct for chromatic shifts in the colour channels a z-stack was acquired of sub-diffraction multicolour fluorescent beads, using the same microscope set-up. The images were checked for any potential artefacts from SIM reconstruction as detailed in^{33 (link)}.