Cryoelectron microscopy of refrozen cryosections

Cryoelectron microscopy makes it possible to record high-resolution detail from large and complex structures. However, its application to understanding cellular structure is limited by the requirement that samples should be no thicker than approximately 0.5-1 microm. Therefore it is important to develop the ability to section biological material so that it can be imaged in its native frozen state. Here we have adapted standard methods of preparing cryosections so that they can be imaged by cryoelectron microscopy. As used for immunolabeling, cryosections of chemically fixed, cryoprotected frozen rat cardiac muscle were thawed, applied to carbon-coated grids, and rinsed on a drop of buffer. The special step here is that the cryosections were then refrozen by being plunged into liquid ethane and imaged at approximately -180 degrees C in a 200-kV field-emission gun electron microscope. The unstained cryosections have good contrast, allowing the identification of optimum regions of the sample. Considerable fine detail is observed within the substructure of the sarcomere A-band and I-band. Fourier transform analysis of the micrographs shows that this method preserves high structural order, hence these sections are well-suited to 3D reconstruction. We conclude that this approach has considerable potential for obtaining intermediate- and high-resolution structural detail from bulk tissue.