Digital micrograph software gms 3

Cells were fixed by incubation for 24 h in 4% paraformaldehyde and 1% glutaraldehyde (Sigma, St-Louis, MO, USA) in 0.1 M phosphate buffer (pH 7.2). They were then washed in phosphate-buffered saline (PBS) and post-fixed by incubation for 1 h with 2% osmium tetroxide (Agar Scientific, Stansted, UK) in 0.15 M phosphate buffer. They were dehydrated in a graded series of ethanol solutions and propylene oxide, impregnated with a mixture of (1:1) propylene oxide/Epon resin (Sigma), and then embedded in pure Epon resin, which was allowed to polymerise for 48 h at 60 °C. The Epon blocks were precisely resized for the cutting of a ribbon of serial ultrathin sections (80 nm thick) of individual cells with a Leica Ultracut UCT ultramicrotome (Leica Microsysteme GmbH, Wien, Austria). These serial sections were then placed on TEM nickel one-slot grids (Agar Scientific, Ltd., Stansted, UK) coated with Formvar film. The resulting TEM grids were stained for 20 min with 2% uranyl acetate (Merck, Darmstadt, Germany) and 5% Reynolds lead citrate for observation with a Jeol 1011 (Jeol Ltd., Tokyo, Japan) electron microscope. Electron micrographs of the sections were recorded with a digital camera driven by Digital Micrograph software (GMS 3, Gatan, Pleasanton, CA, USA) for the same region of cells in each of the series of sections.

Electron microscopy: Digital Micrograph software (GMS 3; Gatan). Confocal microscopy: Fiji software (version 2.0.0), Zen (version 2.3, Zeiss) and Imaris 9.2.0 software (Oxford Instruments Group). WB: Image Lab Touch Software (version 2.0.0.27, Chemidoc Imaging System from Bio-Rad). Flow cytometry: FlowJo (version X; BD). Statistics and graphs: GraphPad Prism8 (8.4.3; Graphpad software, LLC).

Particles on the polycarbonate filters were resuspended in distilled water by sonication. A drop of suspension was deposited on ultrathin C or lacey C supported nickel TEM grids and allowed to dry. The grids were analyzed in a JEOL JEM-2100 analytical TEM, operated at 200 keV. This TEM is equipped with a 60 mm² window, JEOL EX-230 Silicon Drift Detector (SDD) that allows acquisition of EDS spectra with a spatial resolution of 3 nm in STEM mode, and in the range of 5–10’s of nanometers in parallel illumination TEM mode. Images were acquired at different magnifications to characterize morphology and size of particles. SAED patterns were acquired from representative particles and analyzed using Gatan Digital Micrograph software (GMS 3) to measure crystalline features. EDS spectra were collected from the same regions. Minerals were identified based on both EDS spectra and SAED patterns.