Tecnai spirit biotwin microscope

The MIC of SFN and PEITC were added to bacterial cultures (OD₆₀₀ = 0.4) and incubated in 37 ◦C. Nontreated cultures were used as control. Next, at the time indicated, bacteria were pelleted by centrifugation (4000× g, 10 min) and resuspended in a fresh medium. 5 μL of the diluted (100 times) bacteria culture (before or after laser treatment) was dropped and dried on a copper grid for TEM observation. Electron microscopic analyses were performed using the Tecnai Spirit BioTWIN microscope (FEI Company, Eindhoven, Netherlands) at 120 kV.

Transmission electron microscopy (TEM) was used to determine changes in structure of bacterial cells treated with AgNPs (or AgNO₃), 3ChPL, and their combination. Inocula in late log phase (cultured for 6 h, at 37°C and 150 rpm) in CA-MHB medium (0.5 McF, 1.5 × 10⁸ CFU/mL) were treated with agents, or combinations of agents, at concentration corresponding to 5× MBC for 90 and 180 min (37°C, 150 rpm). Following the treatment, the bacterial cells were centrifuged (2,800 ×g, 10 min) and washed twice with phosphate-buffered saline. Bacterial pellets left in tubes were fixed with 2.5% glutaraldehyde (Polysciences, Warrington, PA, United States), and then with 1% osmium tetroxide (Polysciences, Warrington, PA, United States). After ethanol dehydration, bacteria were embedded in Epon 812 resin (Sigma-Aldrich). The ultramicrotome Leica UC7 was used to prepare ultrathin sections (55 nm). Lead citrate and uranyl acetate were added as contrasting agents. The entire study was performed at 120 kV using Tecnai Spirit BioTWIN microscope (FEI).

70S ribosomes were negatively stained with 0.2% uranyl acetate. Carbon coated grids were first glow-discharged to increase the surface hydrophilicity using a Harrick Plasma cleaner. 4 µL aliquots of 70S ribosomes (~10 nM) were placed on grids for about 1 min, and excessive liquid was absorbed by filter paper. After that 0.2% uranyl acetate was applied on the grid for about 1 min and absorbed using filter paper. The grids were air-dried and examined using an FEI Tecnai Spirit BioTwin microscope (FEI, Hillsboro, OR) (120 KV) at 49,000× magnification.

A Leica Ultracut UCT ultramicrotome was used to prepare the ultrathin sections (50 nm). Ultrathin sections were blocked in 1% BSA (Aurion, Wageningen, The Netherlands) in PBS buffer for 15 min and then incubated in primary antibodies in a 1:10 dilution overnight at 4° C. Next day ultrathin sections were incubated with secondary antibody goat anti rat conjugated with colloidal gold 10 nm (Sigma Aldrich, Poland) in a 1:50 dilution for 2 h, followed by washing in PBS buffer and distilled water. Negative controls were created by omitting the primary antibody step (Supplementary Material 1). Lead citrate (Microshop, Poland) and URANYLess (Microshop, Poland) were added as the contrasting agents. The cells were visualized using a Tecnai Spirit BioTWIN microscope (FEI) at 120 kV (Laboratory of Electron Microscopy, Faculty of Biology, University of Gdańsk).

Cells were fixed in 0.1 M sodium cacodylate (pH 7.4) buffer containing 2.5% glutaraldehyde, 3% paraformaldehyde and 5% sucrose, pelleted, and post fixed in 1% OsO₄ in veronal-acetate buffer. Next, they were stained en bloc overnight with 0.5% uranyl acetate in veronal-acetate buffer (pH6.0), dehydrated, and embedded in Embed-812 resin. Sections cut on a Leica EM UC7 ultra microtome with a Diatome diamond knife at a thickness setting of 50 nm were stained with 2% uranyl acetate, and lead citrate. The sections were then examined using a FEI Tecnai spirit BioTwin microscope (FEI) at 80 keV and photographed with an AMT XR16 CCD camera. About 30 EM cross-sections per cell line were imaged for morphometric analysis of the nuclear envelope. The NE membrane enclosed in each image was measured with Fiji^{64 (link)}, and the quantitative analysis of the number of nuclear blebs was performed using GraphPad (La Jolla, CA) Prism.

Samples were deposited on Formvar/carbon-coated 200-mesh copper grids (Electron Microscopy Sciences) for 1 min at room temperature, and then grids were washed and stained with 0.75% (w/v) uranyl formate (Electron Microscopy Sciences) solution in water. Prepared grids were imaged using a Tecnai Spirit BioTWIN microscope at 80 kV (LaB6 gun, 0.34 nm line resolution) equipped with a 4k × 4k Eagle CCD camera with a high-sensitivity scintillator from FEI.

Overall, 5 µl of TorsinA filaments or TorsinA–liposome mixtures were loaded on glow-discharged (EMS 100, Electron Microscopy Sciences) continuous carbon film grids (CF200-Cu, Electron Microscopy Sciences) immediately after diluting them. TorsinA filaments were diluted in the same buffer which they were dialyzed against (typically 20 mM HEPES/NaOH pH 8.0, X mM NaCl, 10 mM MgCl₂, 0.5 mM ATP). TorsinA–liposome mixtures were also diluted in dialysis buffer (20 mM HEPES/NaOH pH 8.0, 100 mM NaCl, 10 mM MgCl₂, 0.5 mM ATP), except lacking sucrose to prevent interference with negative staining. Samples containing only liposomes were prepared identically to TorsinA–liposome mixtures, and the final sucrose concentration after dilution was about 1% w/v. After 45 s of adsorption on grids, the samples were blotted and the specimen on the grid was immediately stained with 2% w/v uranyl acetate for 30 s. The specimen was blotted, stained once more, re-blotted, and air dried. Electron micrographs were recorded on an FEI Tecnai Spirit BioTwin microscope (FEI) operated at 80 keV and equipped with a tungsten filament and an AMT XR16 CCD detector.