*2.1. TEM Specimen Preparation*

The CNC material used is a National Research Council Canada-certified reference (NRCC), CNCD-1 [12,15–18]. The CNCs were extracted with sulfuric acid hydrolysis of softwood pulp, followed by neutralization and sodium exchange, purification and spray drying. The CNC aqueous solution was prepared by dispersing dry CNC powders in deionized water with a concentration of 0.02% (*w*/*v*). Vortex or sonication of the CNCs in aqueous solution was performed before preparing TEM specimens.

Continuous and perforated carbon-film-supported TEM grids were treated with glow discharge in air for 15 s at 15 mA current (PELCO easiGlow™, Ted Pella, Inc., Redding, CA, USA) before applying samples. CNC TEM specimens were prepared under four different conditions as described below. The first specimen (Sp1) was CNCs negatively stained with 2% uranyl acetate (UA) on TEM grids. Briefly, a 3 µL droplet of CNC aqueous solution was deposited on an ultra-thin continuous carbon-film TEM grid. Excess solution was blotted off with filter paper from the edge of the grid or the top of the droplet after 10 s incubation. The TEM grid was then tilted 30 to 45 degrees, rapidly washed with two running UA droplets and placed with a 2 µL droplet. After about 30 s of incubation, UA was blotted away with filter paper from the edge of the grids, and a thin layer of stain was left to air-dry on the TEM grids. The second specimen (Sp2) was pristine CNCs deposited on carbon TEM grids with the same preparation procedure as Sp1, except that the droplet incubation time was 60 s without UA staining. The third specimen (Sp3) was negatively stained CNCs on carbon TEM grids with gold nanoparticles as fiducial markers for electron tomography (ET). Colloid gold nanoparticles with a 5 nm diameter (0.1 mg/mL) were deposited on carbon-coated TEM grids and air-dried prior to CNC deposition. The same procedure for Sp1 was then followed to obtain negatively stained CNCs.

The fourth specimen (Sp4) was CNCs embedded in vitreous ice, and the cryo-TEM specimen was prepared using the plunge-freezing method [19]. Briefly, the plunge freezer Dewar/chamber (EMS-002 Rapid Immersion Freezer, Electron Microscopy Sciences, Hatfield, PA, USA) was precooled with liquid nitrogen for 30 min. Compressed ethane gas was liquefied in a Falcon tube surrounded with liquid nitrogen. The liquid ethane was then poured into a cryogen container immersed in a liquid nitrogen chamber with a temperature of −184 ◦C. The container was ready for use when the ethane was lightly frozen at the bottom and the walls of the container. One 4 µL droplet of CNC aqueous solution was placed on the perforated carbon side of a TEM grid facing up for 2 min. The tweezer securing the TEM grid was then mounted to the releasing anvil. Excess solution was blotted using filter paper from the back side of the TEM grid for 4 s, and the TEM grid was rapidly plunged into the liquid ethane. The frozen CNCs on the TEM grid were kept in liquid nitrogen and transferred for cryogenic-TEM (cryo-TEM) imaging.
