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Principles & ProtocolsPrinciples - Electron crystallographyJump to ProtocolIn order to visualize a sample in a transmission electron microscope the sample must (a) be thin enough such that a beam of electrons can penetrate it (<250nm, ideally <100nm), (b) be deposited onto an EM grid, which is a thin circular copper grid that is 3mm in diameter, and (c)withstand high vacuum and electron radiation within the microscope column. For tissue, samples are prepared by cutting thin sections (sectioning). For aqueous suspensions of macromolecules, including 2D crystals and ordered helical arrays, 1-5 microliters of the solution is pipetted onto the EM grid, which is then subjected to either negative staining,plunge freezing, or a combination of these sample preservation techniques, cryo-negative staining. Two dimensional crystals are commonly formed by membrane proteins, such as bacteriorhodopsin or aquaporins. These crystals ideally consist of a single layer of molecules that are held together by specific contacts, showing crystalling order. |