Frozen Cell Crusher: Cryo-Press

The size of Nannochlorisbacillaris is about 5 microns. (DAPI staining, 345 nm exposure, magnification: 1,000 times)

After homogenizing, DNA (blue) appears. It usually takes 2 to 3 hours to homogenize the sample in the mortar. However, in Cryo-Press, sample homogenization is only 2 minutes (15 seconds x 8 times).

In the "homogenized" image, blue fluorescence indicates DNA. Separation of DNA from cells by successful homogenization

Department of Plant Life Systems Laboratory, University of Tokyo, Department of Frontier Science, Department of Integrated Biological Sciences

Traditional homogenization has a high risk of homogenization of pyrolysis of RNA, but this image shows that CRYO-PRESS extracts 28S and 18S rRNA.

Chicken sciatic nerve (Cryo-Press and conventional glass homogenizer) is homogenized in two ways, SDS is added and the protein is extracted

Both data show that the silo press is superior to conventional glass homogenizer. In addition, Cryo-Press helps save time. I used only one or two samples of air compressor output.

Even larger cells or even tissues can be prepared by thinning to CryoET by cryosectioning or focused ion beam (FIB) milling. For frozen sections, frozen cells or tissue pieces were cut into thin samples using a cryostat. In FIB milling, the immersed frozen sample is exposed to a focused ion beam (usually gallium). The focused ion beam accurately cuts the material from above and below the sample and leaves a sheet suitable for ECT imaging. The strong interaction between electrons and substances also leads to anisotropic resolution effect. When the specimen is tilted during imaging, the electron beam interacts with a relatively large cross-sectional area at a higher tilt angle. In fact, a tilt angle greater than about 60-70 ° does not produce too much information and is therefore not used. As a result, the information in the final tomographic image "loses the wedge", thereby lowering the resolution parallel to the electron beam.

There are several ways to prepare biological samples for electron microscopy. One technique is called cryogenic electron microscopy, or simply frozen EM where the sample is rapidly frozen and then observed under an electron microscope. Using this technique it is possible to create very detailed viral images, individual compartments within cells, and even individual proteins. In order to solve this problem and to support the spread of low temperature EM, Cianfrocco and Leschziner have developed an open "ready-made" system on Amazon's Elastic Cloud Computing infrastructure. This allows users to create software packages and clusters of up to about 480 computers for analyzing low temperature EM data.

In cold electron microscopy, the observed sample is usually frozen (frozen hydrated) for storage purposes. Here, it is possible to quickly insert a very thin slip of the sample into the liquid ethane bath and observe it in its natural state. To keep the sample stable, use a solvent such as water or salt solution. Here it is very important that the process of freezing the sample is very fast. This prevents cold water around the sample sample from forming cubic ice. In the case of ice formation, it easily absorbs the electron beam, which in turn blurs the sample. Therefore, it is necessary to quickly insert the sample into the coolant so that the water freezes only around the sample in order to obtain a clear image.