Gatan digital micrograph software

Transmission electron microscopy was used to visualize the ultrastructure of plastids present in the mature fruit flesh. Fresh flesh (1mm cube) from the mesocarp region was fixed for 3 hours using 2.5% glutaraldehyde and freshly prepared 0.1M Sörensens phosphate buffer and washed using the 0.1M Sörensens phosphate buffer. After washing the tissue was post-fixed using 1% osmium tetroxide and 0.1M Sörensens phosphate buffer for an hour followed by a serial dehydration using increasing concentration of ethanol (30%, 50%, 70%, 90%, 100%). The fixed tissues were infiltrated with 100% epoxy resin. After overnight infiltration, fresh resin was used to embed the tissue in embedding molds and polymerized for two days at 60˚C. Ultrathin (70nm) sections were taken using Leica EM UC6 ultramicrotome (Leica Microsystems, Vienna, Austria) provided with DiATOME diamond knife. The thin sections were placed on charged 200-mesh copper grids and stained with uranyl acetate and lead citrate. Grids were imaged in FEI Tecnai 12 TEM (Thermo Fisher Scientific, Eindhoven, The Netherlands) using Gatan UltraScan 1000 camera. All the images were processed and analysed using Gatan Digital Micrograph software (Gatan Inc. Pleasanton, CA, USA).

Virus samples were deposited on Formvar carbon-coated grids (TED Pella, Redding CA, USA) and incubated for two minutes. After that, the excess solution was removed, and the samples were then stained for 20 s with a 2% water solution of phosphotungstic acid (PTA; Sigma–Aldrich), pH 7.0. Samples were viewed with a transmission electron microscope Jeol JEM-2100 (JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 200 kV, in parallel beam mode, with a defocus of 0.8 microns. Images were obtained and processed using a Gatan Orius SC200D (2 k × 2 k) detector and Gatan Digital Micrograph software (Gatan, Inc., Pleasanton, CA, USA).

Analysis of samples containing outer membrane vesicles by dynamic light scattering (DLS) was performed using commercial service at SOLVE analytical laboratory (Lund, Sweden). OMV containing samples were analysed using Zetasizer Nano ZS instrument (Malvern, UK) and Zetasizer Software 8.01 (Malvern, UK). Transmission electron microscopy (TEM) was done at BioVis core facility at Uppsala University (Sweden). A 5 µl drop of the sample was placed on a Formvar- and carbon coated 200-mesh copper grid. The excess solution was immediately removed by blotting with filter paper. The sample was then washed on three consecutive drops of MQ water followed by two drops of 2% uranyl acetate. Excess of uranyl acetate was removed by blotting on filter paper. Dried grids were examined by Tecnai™ G2 Spirit BioTwin transmission electron microscope (Thermo Fisher/FEI) at 80 kV with an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.).

Samples were fixed in 2.5% glutaraldehyde (Ted Pella) + 1% paraformaldehyde (Merck) in PIPES pH 7.4 and stored at 4°C until further processed. Samples were rinsed with 0.1 M PBS for 10 min prior to 1 h incubation in 1% osmium tetroxide (TAAB; Berks, UK) in 0.1 M PBS. After rinsing in 0.1 M PBS, samples were dehydrated using increasing concentrations of ethanol (50%, 70%, 95% and 99.9%) for 10 min each step, followed by 5 min incubation in propylene oxide (TAAB). The samples were then placed in a mixture of Epon Resin (Ted Pella) and propylene oxide (1:1) for 1 h, followed by 100% resin and left overnight. Subsequently, samples were embedded in capsules in newly prepared Epon Resin and left for 1–2 h and then polymerised at 60°C for 48 h. Ultrathin sections (60–70 nm) were cut in an EM UC7 Ultramicrotome (Leica) and placed on a grid. The sections were subsequently contrasted with 5% uranyl acetate and Reynold's lead citrate and visualised with Tecnai™ G2 Spirit BioTwin transmission electron microscope (Thermo Fisher/FEI) at 80 kV with an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.).

EV samples were mixed with an equal volume of 4% paraformaldehyde, added onto a formvar and carbon-coated 200-mesh grid (Oxford 11 Instruments) and incubated for 20 min. After incubation, the grid was dried and washed first 3 times with PBS, followed by 8 washes in MilliQ water. The samples were stained in a drop of uranyl oxalate, pH = 7, for 5 min, after which they were stained with a drop uranyl acetate 4% pH = 4 with 2% methylcellulose on ice for 10 min. The dried grids were imaged in a Tecnai G2 transmission electron microscope (TEM, FEI company) with an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.).

Characterization of Tau-F, Tau-O and BDTau-F was performed by transmission electron microscopy (TEM) using negative staining. Tau-F were diluted 1:10 in distilled H2O and placed on a formvar and carbon coated 200-mesh copper grid (Ted Pella). The sample was directly stained with 2% uranyl acetate. Dried grids were examined by TEM (FEI Tecnaii G2) operated at 80 kV with an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.).

The X-ray diffractometer (Rigaku D/Max-3c, Rigaku, Tokyo, Japan) with Cu K_α radiation (λ = 0.154 nm) is employed to analyze the phase composition of the nanofibers. The working voltage and current were set to be 40 kV and 15 mA, respectively. The scan range was set from 5° to 80° with the speed of 5° min⁻¹. A field-emission scanning electron microscope (FESEM, SU8020, Hitachi, Tokyo, Japan) and transmission electron microscope (JEM-2800, JEOL, Tokyo, Japan) were utilized to characterize the morphologies and structures of the nanofibers. The EDX mapping experiment was also conducted on the same TEM. All the TEM images were processed on Gatan^® DigitalMicrograph software (Gatan, Inc. Pleasanton, CA, USA).