Fcf400 cu

The morphology of the BCNC was assessed by TEM imaging. Briefly, 5 µL of BCNC aqueous suspension at 0.01% (m/v) was applied on the grid (FCF400-Cu, Electron Microscopy Sciences (EMS), Hatfield, UK) and allowed to settle for 2 min at RT. The sample was then blotted off, and 5 µL of uranyl acetate (UA) (Sigma-Aldrich, St. Louis, MO, USA) for negative staining was directly applied. The excess solution was again blotted off and replaced by another 5 µL of UA. Each time, the UA was allowed to incubate for 30 s before it was removed. The sample was observed with a JEOL 2100 plus TEM device (JEOL, Tokyo, Japan), operated at 80 kV accelerating voltage. Several images were taken, considering areas far from each other and trying to represent the whole grid’s surface. The length (L) and width (W) of the BCNC were determined from at least 150 measurements by image analysis, using ImageJ software (Version 1.51j8, Bethesda, MD, USA) [42 ].

The TEM samples were
prepared on glow-charged (20 s oxygen plasma flash) Formvar carbon-coated
copper grids (FCF400-Cu, Electron Microscopy Sciences) according to
the protocol previously described by Castro et al.^{54 (link)} Three microliters of DNA origami solution
(c = 5.0 nM for DNA origami units, c = 5.4/5.7 nM for DNA origami dimers and c = 2.0–5.0
nM for 1D DNA origami arrays) was applied onto the carbon-coated side
of the grid and incubated for 3 min before excess sample solution
was blotted away with filter paper. After that, the sample was negatively
stained with 2% (w/v) aqueous uranyl formate solution containing 25
mM NaOH (added to increase the pH of the stain solution) in two subsequent
steps. First, the sample was immersed into a 5 μL droplet of
stain solution, after which the stain was immediately removed using
filter paper. Next, the sample was immersed into a 20 μL droplet
of stain solution for 45 s before the solution was blotted away with
a filter paper. The samples were left to dry under ambient conditions
for at least 15 min before imaging. All TEM images were obtained using
a FEI Tecnai 12 Bio-Twin electron microscope operated at an acceleration
voltage of 120 kV. The images were processed and analyzed (vertex
angle measurements) using ImageJ.

For negative stain EM, a 3 μL aliquot of the complex sample at a concentration of 0.01 μg μL⁻¹ was applied onto a glow-discharged carbon-coated grid (FCF400-CU, Electron Microscopy Sciences, EMS), blotted with a piece of filter paper (Whatman 1) and stained using 0.75% (w/v) uranyl formate (EMS) for 30 s. The images were collected on an FEI Talos L120C TEM at 120 kV coupled with a Gatan OneView camera. Each image was acquired in a low-dose mode at magnification of ×73,000 resulting in a pixel size of 2.0 Å, using a dose rate of ~24 e Å⁻². All single particle processing was performed using cryoSPARC^{63 (link)}.

AF-EVs were analyzed using a JEOL JEM-1230 electron microscope (JEOL USA, Inc, Peabody,MA, USA) in conjunction with formvar carbon-coated transmission electron microscopy grids (FCF400-Cu; Electron Microscopy Sciences,Hatfield, PA, USA). The copper carbon formvar grids were glow-dis-charged prior to loading an undiluted sample of EVs. The grids were then floated on 10mL of EV sample drop for 15 min, washed two times with water by floating on the drop of water for 30 s and then negatively stained with 2% uranyl acetate by floating on the drop of stain for 30 seconds. The grids were blot-dried with Whatman paper (Cytiva, Marlborough, MA, USA) and then imaged.