Soft imaging viewer

Fifty microliter of nanoparticles dropped in the parafilm and copper grid were kept above the samples and waited for 1 min. Then, the copper grid was taken out and dropped in the 2% phosphotungstic acid, and waited for 30 s. Afterwards, copper grid was taken out and dried with help of tissue paper, and then placed under the TEM microscope (Morgagni 268, Olympus). Pictures were taken at various magnifications (× 13,000), and accelerating voltage 70 kV and data were analyzed using Olympus soft imaging viewer.

Carbon-coated copper grids, 200 mesh (Science Services, Munich, Germany) were treated with oxygen plasma (Zepto, Diener electronic GmbH, Ebhausen, Germany). After this, 3 µL of precipitation-purified rAAV sample [25 (link)] was applied to the grid and incubated for 2 min. Excess liquid was drained off, the grid was dried at room temperature and washed with three drops of distilled water. Negative staining was performed using 3 µL 2% (v/v) uranyl acetate replacement stain (Science Services) for 30 s. Excess liquid was drained off and grids were dried before channeling the sample into the microscope. rAAVs were visualized with a CM100 (PW6021) instrument (Philips, Hamburg, Germany) with an acceleration voltage of 80 kV. Images were analyzed using the Soft Imaging Viewer (Olympus, Münster, Germany) and ImageJ [50 (link)].