Ace200

Samples of PLT-Exos-Ca²⁺ from volunteer 3 were used for this analysis. For each preparation to be stained, two microliters sample were adhered for 1 min onto glow-discharged Carbon coated grids (Agar S160-3 grids and Leica ACE200-glow discharge for 30 s at 10 mA). After removing excess liquid by blotting with Whatman filter paper, grids were negatively stained using 1% uranyl acetate for 1 min. Preparations were visualized in a JEOL JEM 1400 Plus transmission electron microscope operated at 100 kV, and images were acquired with an integrated Hamamatsu Flash sCMOS digital camera.
Measurements of the mean diameter of exosomes was performed on two TEM images (3.1 μm² each) from two different samples. First, using the vector drawing tool of Adobe Photoshop Adobe 22.5.0 (San Jose, CA, USA), the perimeters of all the exosomes identified in the micrographs were manually outlined. For very close or partially overlapping particles, perimeters were outlined in separate layers to avoid contact between contours. Subsequently, the outlines were filled in with the Adobe Photoshop paint bucket tool, and separate layers were saved individually and imported as a stack into Fiji-ImageJ 1.53f51 (NIH, Bethesda, MA, USA). The images were then converted to binary in Fiji-ImageJ, and the Feret’s diameters were measured using the Analyze Particles macro (NIH, Bethesda, MA, USA).

For scanning electron microscopy analysis, the SiOC scaffolds (without cells) were first coated with gold (Leica EM ACE200) and then analyzed using a JSM6010 PLUS-LA (JEOL) scanning electron microscope.
The scaffolds containing cells were first fixed (2.5% glutaraldehyde; 0.1 M sodium cacodylate buffer) for 1 hour at room temperature and stored at 4°C (in the same solution) until used. The samples were then washed (0.1 M sodium cacodylate buffer), post-fixed (1% osmium tetroxide; 0.1 M sodium cacodylate buffer) for 40 min, and washed again. The samples were dehydrated with a series of ethanol solutions with increasing concentrations (30%, 50%, 70%, 90%, and 100%), dried using a critical point (Leica EM CPD300), and coated with gold (Leica EM ACE200). The samples were analyzed using a JSM6010 PLUS-LA (JEOL) scanning electron microscope. For energy-dispersive X-ray spectroscopy (EDS), the post-fixation step with osmium tetroxide was omitted, and the samples were coated using carbon.

The specimens were preserved in 70–80% ethanol. Eight paratypes were mounted on slides with Euparal liquid. Drawings were made using a Zeiss Axioplan microscope with a camera lucida. Photographs of larvae were taken using a Leica Z16 APO macroscope and processed with Leica Application Suite™ Version 3.1.8 to obtain combined photographs with enlarged depth of field. Photographs were subsequently enhanced with Adobe Photoshop™ CS3.
Specimens used for SEM were dissected and dehydrated through a stepwise immersion in ethanol and then dried by critical point drying (Leica EM CPD300). The mounted material was coated with a 5 nm Au/Pd layer (Leica EM ACE200) and subsequently examined and photographed with a Zeiss EVO LS 15 scanning electron microscope. SEMs were subsequently enhanced with Adobe Photoshop™ CS3.

The scanning electron images for the material structure were obtained by coating freeze-dried chitosan scaffold with gold using a sputter coater (KYKY Technology Co. LTD., Beijing, China) and imaging with a scanning electron microscope (SEM) (Jeol Ltd., Akishima-Shi, Japan).
The structure of exosomes was observed by a scanning electron microscope with higher resolution. After exosomes were absorbed on the surface of the chitosan scaffold, using 4% paraformaldehyde (Tianjin Damao, Tianjin, China) in PBS to fix samples for 15 min and washing twice with PBS. Then dehydrating samples in a series of alcohol solutions (25%, 50%, 75%, 90%, 95%, 100% alcohol in PBS), followed by drying at ambient temperature for 30 min and coating with platinum using a sputter coater (Leica ACE200, Wetzlar, Germany) before imaging with a field emission scanning electron microscope (FESEM) (JSM-7800F, Akishima-Shi, Japan).

Negatively stained electron microscope (EM) grids were produced by applying 10 μL of particle preparations on to glow discharged (20 s at 10mA in an Ace 200, Leica) formvar/carbon film copper 400-mesh grids, (EM Resolutions Ltd., Sheffield, UK). After 5 min, excess sample was blotted away, and the grids were successively washed three times (each 15–20 s) by floating on water droplets and finally stained with 2% (w/v) uranyl acetate for 15 s. Excess solution was removed at each step by blotting with Whatman No. 1 filter paper, and the grids were air dried before examination with a Talos F200C transmission electron microscope (Thermo Fisher Scientific, Eindhoven, The Netherlands) operated at 200 kV, fitted with a OneView 4k × 4k CMOS (Gatan UK, Abingdon, Oxfordshire, UK) bottom-mounted camera (JIC Bioimaging Platform Facility). Automated data acquisition was setup using EPU, each image had a 1-s exposure with a sample dose of 40 e⁻/A², nominal magnification of 45,000× and a calculated pixel size of 3.5 Å. Single particle analysis of the resultant images was performed using cryoSPARC v. 2.15.0 [45 (link)].