Serial block-face scanning electron microscopy (SBF-SEM) makes it possible to obtain three-dimensional electron microscopy data over a large area of a biological sample in a semi-automated fashion. An image is taken of the block-face of a resin embedded sample and then a thin section is cut from the block using a diamond knife. Image collection is done using a backscattered electron detector in a scanning electron microscope. The process of imaging and sectioning is performed over and over, building up a three-dimensional data set. New protocols have been developed that deposit substantial amounts of metal into the sample in an attempt to overcome the problems of charging and signal generation. The problem with these is that the samples are fixed chemically at room temperature. Rapid freezing followed by freeze substitution gives superior preservation of morphology to processing chemically at room temperature. Quick freeze substitution protocols have now allowed processing of samples in less than 3 hours. New protocols will be presented that utilize the quick freeze substitution in combination with metals in an organic solvent such as acetone or methanol that produce samples that work well for SBF-SEM even when imaged in high vacuum mode. Chemicals such as lanthanum chloride or imidazole can be included into the freeze substitution media with the osmium tetroxide and these impart a high electron density to the sample. A double osmium method can be performed by using tannic acid or thiocarbohydrazide. Once warmed to room temperature other metal containing solutions such as uranyl acetate and lead acetate are used to give the sample more electron density. By utilizing the quick freeze substitution and rapid embedding methods the entire process can be completed in a day.