Abstract text (incl. figure legends and references)

Conventional room-temperature preparation of biological samples for electron microscopy (EM) is often time-consuming and involves treatment of the sample with different fixation agents to stabilize the structure and enable its later observation in a vacuum under a high-energy electron beam. The fixation agents in general are extremely hazardous chemicals, some of which are subject to strict regulations, with uranium salts being the most complicated due to radioactivity. Some alternatives to uranium were proposed, but their use is mostly limited to post-staining of sections or negative staining. Here we propose to expand the range of their use by substituting uranyl acetate (UA) in the production of the platinum replicas from cell cortices. Our sample preparation procedure is based on previously published methods (1, 2). Cells, cultured on the glass coverslips, washed off culture medium with Ca²⁺-free Ringer solution, additionally "glued" using PLL 0.1 mg/ml, swollen in hypotonic buffer (1/3 of normal PHEM concentration), and subsequently unroofed by sonication in PHEM buffer of normal concentration. Cell cortices were immediately fixed in a mixture of glutaraldehyde and tannic acid in PHEM. After extensive wash with deionized water the samples were post-fixed with different metal salts in water, then dehydrated in ethanol series followed by hexamethyldisilazane, and air dried instead of critical-point drying to spare time. Platinum replicas were made by coating samples with 2 nm Pt/C; no baking out with carbon was used. Replicas were released with the help of HF acid and after extensive wash transferred to Formvar-coated 75 mesh TEM grids. Images were then acquired with a high-resolution SEM Hitachi S5000 or TEM Philips CM10 with bottom-mounted camera TemCam F-416 (in the latter case the images were digitally inverted to resemble SEM). The quality of all samples was compared to UA-treated sample taken as standard. Experiments revealed that UA could be substituted by gadolinium, hafnium, or iron chloride, as well as acetate salts of gadolinium, neodymium, samarium, or ammonium molybdate or osmium tetroxide without losing sample quality. Negligible sample thickness after unroofing allowed to shorten the time down to two hours from starting the preparation to image acquisition. With little modifications, it could be adapted for CLEM or gold-immuno EM by altering the primary fixation and introducing immunolabelling prior EM preparation procedure, and thus is a good alternative to existing routines.

1. Heuser, J. Traffic, 2000, 1, 545-52
2. Sochacki, K. A. & Taraska, J. W. 2017, 1663, 219-230