Correlativo luz microscopia eletrônica de varredura (CLEM) para rastreamento e Imaging proteína Viral associado estruturas nas células Cryo-imobilizado

This method helps to answer key questions in the biology field about how bio-proteins who are in cellular membranes to create environments that favor bio-replication and assembly.The two technical cornerstones that are combined here are first, the correlation between live cell imaging and electron microscopy, and second, high pressure freezing.Although this method has been designed and successfully utilized to tackle virus source interactions, it can be easily generalized to a range of applications in cell biology.Visual demonstration of this method is critical as there are many steps that require practical experience with success, especially Cryo-immobilization by high-pressure freezing and the subsequent steps.Begin by placing patterned sapphire discs with an alphanumeric pattern etched onto one of their surfaces into a 15 milliliter conical centrifugation tube, and washing the discs thoroughly with ethanol.Place the washed sapphire discs in a Petri dish lined with filter paper for drying, and use long, slim tweezers to transfer the dried sapphire discs onto glass slides.Using an inverted microscope at 4x magnification, confirm that the pattern of coordinates on every sapphire disc is readable.Then, sterilize the discs with an ultraviolet crosslinker for five minutes.In a biosafety cabinet, add half of the cell culture medium to a cell culture dish, and use long tweezers to carefully transfer the sapphire discs into the dish.Then, seed 1x10