For cryo‐TEM, 4 µL of the vesicle suspension was applied to a copper grid coated with a perforated lacy carbon 300 mesh (Ted Pella Inc) and blotted with filter paper to form a thin liquid film of solution. The blotted sample was immediately plunged into liquid ethane at its freezing point (−183°C) in an automatic plunger (Lieca EM GP). The vitrified specimens were transferred into liquid nitrogen for storage. Sample analysis was carried out under a FEI Tecnai 12 G2 TEM, at 120 kV with a Gatan cryo‐holder maintained at −180°C. Images were recorded with the Digital Micrograph software package, at low‐dose conditions, to minimize electron beam radiation damage. The measurements were performed at the Ilse Katz Institute for Nanoscale Science and Technology Ben‐Gurion University of the Negev, Beer Sheva, Israel.
Tecnai 12 g2 tem
Manufactured by Ametek
The Tecnai 12 G2 TEM is a transmission electron microscope (TEM) designed for high-resolution imaging and analysis of materials at the nanoscale. It is capable of operating at an accelerating voltage of 120 kV and provides a maximum magnification of up to 1,000,000x. The instrument is equipped with advanced features for electron optics and image acquisition, enabling detailed examination of the internal structure and composition of various samples.
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5 protocols using tecnai 12 g2 tem
1
Cryo-TEM Analysis of Vesicle Suspensions
2
Cryo-TEM Imaging of Nanoparticles
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Vitrified specimens were prepared according to a standard procedure [56 (link)]. Briefly, drops of an NP solution were applied to a copper grid coated with a perforated lacy carbon 300 mesh (Ted Pella Inc., Redding, CA, USA) and blotted with a filter paper to form a thin liquid film. The blotted samples were immediately plunged into liquid ethane at its freezing point ( ) using an automatic plunge freezer (EM GP, Leica Microsystems GmbH,Vienna, Austria) and transferred into liquid nitrogen for storage. The samples were analyzed using an FEI Tecnai 12 G2 TEM at with a Gatan cryo-holder, maintained at −180 °C. Images were recorded on a CCD camera (Gatan manufacturer, Pleasanton, CA, USA) at low-dose conditions.
3
Cryo-TEM Analysis of Extracellular Vesicles
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For cryo-TEM, 5 μL of the EV sample was applied to a copper grid coated with perforated lacy carbon 300 meshes (Ted Pella) and blotted with filter paper to obtain a thin liquid film of solution. The blotted sample was immediately plunged into liquid ethane at its freezing point (-183°C) with an automatic plunger (Lieca EM GP). The vitrified samples were stored in liquid nitrogen until analysis. Sample analysis was carried out with a FEI Tecnai 12G2 TEM at 120kV with a Gatan cryo-holder maintained at -180°C. Images were recorded digitally using the Digital Micrograph 3.6 software (Gatan, Munich, Germany). Crystal size of purified EVs was measured using ImageJ 1.53q software (U.S. National Institutes of Health, Bethesda, Maryland, http://imagej.nih.gov/ij/).
4
Cryo-TEM Analysis of Q-starch/PIP3 Complexes
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The morphology of the complexes, as well as a confirmation of their size, was evaluated by cryo-TEM. The Q-starch/PIP3 complexes were prepared to reach a final concentration of 250 nM PIP3 in Eppendorf tubes in a final volume of 1 mL. Specimens of Q-starch/cargo complexes were prepared on a copper grid coated with a perforated lacey carbon film, 300 mesh (Ted Pella Inc., Redding, CA, USA), under controlled temperature (cryogenic temperature). A 3 µL drop of the analyzed solution was applied to the grid and blotted with filter paper to form a thin liquid layer. The blotted samples were immediately and automatically plunged into liquid ethane at its freezing point (−183 °C) using Plunger (Leica EM GP, Wetzlar, Germany). The specimens were transferred into liquid nitrogen for storage. Samples were analyzed using FEI Tecnai 12 G2 TEM at 120 kV with a Gatan cryo-holder maintained at −180 °C. Images were recorded on a slow-scan cooled charge-coupled device camera (Gatan, Pleasanton, CA, USA) in low-dose conditions to minimize electron-beam radiation damage. The recording was carried out using the Digital Micrograph GMS3 software package.
5
Cryo-TEM Imaging of Hydrogel Microstructures
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Hydrogels of FD, 25 mol% FD-RGD and FD-RGD, each with overall 5% w/v (corresponding to total molar concentrations of 30, 28 and 22 mM, respectively) were prepared by dissolving in 100 μl NaOH 0.1 mM. Vitrified specimens were prepared on a copper grid coated with a perforated lacy carbon 300 mesh (Ted Pella Inc.). A typically 4 μl from a hydrogel sample was applied to the grid to form a thin film. The samples were immediately plunged into the liquid ethane at its freezing point (−183 °C, Lieca EM GP). The vitrified specimens were transferred into liquid nitrogen for storage. The samples were studied using a FEI Tecnai 12 G2 TEM, at 120 kV with a Gatan cryo-holder maintained at −180 °C, and images were recorded on a slow scan cooled charge-coupled device CCD camera. Images were recorded with the Digital Micrograph software package, at low dose conditions, to minimize electron beam radiation damage.
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