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Aspects of Cryofixation and Cryosectioning for the Observation of Bulk Biological Samples in the Hydrated State by Cryoelectron Microscopy

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Aspects of Cryofixation and Cryosectioning for the Observation of Bulk Biological Samples in the Hydrated State by Cryoelectron Microscopy Scanning Microscopy Volume 1996 Number 10 The Science of Biological Specimen Article 30 Preparation for Microscopy 12-17-1996 Aspects of Cryofixation and Cryosectioning for the Observation of Bulk Biological Samples in the Hydrated State by Cryoelectron Microscopy K. Richter University of Lausanne, Switzerland, [email protected] Follow this and additional works at: https://digitalcommons.usu.edu/microscopy Part of the Biology Commons Recommended Citation Richter, K. (1996) "Aspects of Cryofixation and Cryosectioning for the Observation of Bulk Biological Samples in the Hydrated State by Cryoelectron Microscopy," Scanning Microscopy: Vol. 1996 : No. 10 , Article 30. Available at: https://digitalcommons.usu.edu/microscopy/vol1996/iss10/30 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Scanning Microscopy by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. Scanning Microscopy Supplement 10, 1996 (pages 375-386) 0892-953X/96$5. 00 +. 25 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA ASPECTS OF CRYOFIXATION AND CRYOSECTIONING FOR THE OBSERVATION OF BULK BIOLOGICAL SAMPLES IN THE HYDRATED STATE BY CRYOELECTRON MICROSCOPY K. Richter• University of Lausanne, Lausanne, Switzerland (Received for publication September 17, 1996 and in revised form December 17, 1996) Abstract Introduction Cryoelectron microscopy allows the observation of Biological specimens are not well suited to electron hydrated samples at high spatial resolution, and it would microscopy at room temperature. One major restriction be of great interest in biology to apply this method to is the vacuum in the microscope column, which does not cells and tissues. However, because of technical prob­ allow to observe water at room temperature. For this lems, the cryo-observation of frozen hydrated ultrathin reason, biological specimens are usually observed as dry sections of bulk material has not become an established artifacts. Operating the microscope at temperatures method. The major limitations are due to the difficulty below 170 K, when water is stable in the vacuum, does of achieving the vitrification of such material, and the not improve the situation. When water crystallizes to structural deformation caused by ultrathin sectioning: 1. ice, the biological matrix separates as a dry network. The vitrification of cells in a physiological environment However, if the cooling is quick enough, water is requires high-pressure freezing. However, new results prevented from transforming into the crystalline solid, suggest that the pressure may alter the ultrastructure of and if this highly viscous vitreous state is reached, the the sample. 2. Cryosectioning compresses structures in fully hydrated ultrastructure of the biological samples the cutting direction about 40 % . This deformation does can be studied by cryoelectron microscopy (Dubochet not necessarily destroy the character of macromolecular and McDowall, 1981). assemblies, but since it depends on the properties of the Cryofixation is much quicker than chemical fixation, material, internal standards cannot be used to correct for ion-gradients are not washed out, the antigenicity of the deformation of all the structures in a cell. epitopes is not altered due to reactions with chemical fixatives, and no external mass is added as it is with the chemical crosslinkers. For these reasons, cryofixation is Key Words: Cryofixation, cryosectioning, cryoelectron also employed in other electron microscopy techniques microscopy, vitrification, plunge-freezing, high-pressure that end up with a dry specimen, e.g., freeze-substitu­ freezing. tion and freeze-drying. Since high-resolution structural preservation is sacrificed by dehydration, vitrification is not the necessary aim with these techniques: Cryo­ fixation is satisfactory as long as displacements of the organic matter due to ice-crystal growth are beyond the other resolution limits of the method. If, on the other hand, the structures are to be observed in the hydrated state, vitrification is a prerequisite. Cryoelectron microscopy of vitrified biological •Address for correspondence: samples is well established for objects small enough to K. Richter be observed directly without the need for ultrathin Biomedical Structure Analysis Dept. 0195 sectioning. These specimens are prepared as a thin layer Im Neuenheimer Feld 280 of aqueous suspension of about 80 nm in thickness and D-69120 Heidelberg 1 µm in diameter spanned throughout the holes of a Germany perforated carbon film. The thin layer is reproducibly Telephone Number: +49(6221)423263 vitrified by plunge-freezing of the grid into liquid ethane FAX Number: +49(6221)423459 at 110 K (Adrian et al., 1984). Cryoelectron micro­ E-mail: [email protected] graphs reveal the fully hydrated structure of embedded 375 K. Richter particles, e.g., viruses, and the image contrast corrected about 100 nm. In rare cases much thinner sections by the contrast-transfer function of the microscope can (down to 40 nm) could be studied. Sections were cut be interpreted as the native distribution of mass within with cryodiamond knives (Diatome, Biel, Switzerland) the sample. An extensive review about the benefits of of either 35 ° or 45 ° knife angle. The clearance angle cryoelectron microscopy is given by Dubochet et al. was set to 3 °. The cutting stroke was performed manual­ (1988). ly, as slow as possible. An ion-spray (Static Line, The smallest in-vivo system in biology is the cell. It Hauck, Biel, Switzerland) sometimes considerably would be of great interest to be able to look inside a cell improved the yield of flat sections. Its effect depends on by cryoelectron microscopy. Many questions concerning the distance from the knife edge and seems to depend on the organization of chromatin in the nucleus, the com­ the laboratory atmosphere. The proper distance (about partmentalization of the cytoplasmic gel or the extension 25 mm) must be found by trial and error. For most of of membranes by associated matrix-proteins are waiting the work discussed here this ion spray was not yet to be answered. As these structures depend upon hydra­ available. tion for their existence, they cannot be observed in a dry Slam-frozen and high-pressure frozen samples were specimen. However, two technical requirements hinder glued with a mixture of ethanol/propanol-2 (2:3) to the the employment of cryoelectron microscopy for hydrated cutting support. This cryoglue is sticky at 130 K and cells: (1) the vitrification of cells in their physiological becomes stiff below 115 K (Richter, 1994a). environment requires costly equipment and is difficult to Cryosections were manipulated with an eyelash to achieve, and (2) Ultrathin sectioning distorts the volume transfer them from the knife edge onto a carbon coated of the section. 600 mesh copper grid. The grids were held in place This article presents personal experiences with the nearby the knife edge by a pair of special tweezers cryofixation of cells and the interpretation of cutting which are part of the tool box of the cryomicrotome. artifacts in cryosections. The piston used to press the sections onto the grid was also part of this toolbox. Until observation in the Materials and Methods electron microscope, the loaded specimen grids were stored in small grid boxes (Gatan, Pleasanton, CA, USA) under liquid nitrogen. Plunge-freezing was performed with a KF80 (Rei­ All the way from the cryofixation device to the chert-Jung, Vienna, Austria) using ethane at 95 K as a electron microscope, the specimen should never be cryogen. In order to reduce the heat flow from the warmed up above 135 K. Transfers between cryo­ plunger to the cryogen, the plunge rod was modified. chambers were performed using small vessels to keep The end-fixation for the specimen pins was replaced by the samples covered by liquid nitrogen. In addition, care a yellow plastic disposable tip of the type normally used was taken that all tools coming in contact with the for laboratory micro-pipettes. The plunge depth was specimen were precooled. readjusted fixing a plastic tube of convenient length Cryo-observation took place in a CM12 electron underneath the original stop-face of the iron plunge rod. microscope (Philips, Eindhoven, The Netherlands) at 80 Parafilm was wrapped around this plastic tube to pro­ kV, using a Gatan 626 cold stage at 110 K. The micro­ hibit bouncing at the end of the plunge. As specimen scope was equipped with a double blade anticontamina­ carriers, small aluminum pins (purchased by a local tor (Marc Adrian, University of Lausanne, Switzerland). electronics store), 0.65 mm in diameter and only 33 mg Specimens stayed clean for at least 2 h observation time. in weight were stuck in the small orifice of the yellow Images were registered at 17000x primary magnification tip. on Kodak SO-163 film sheets (Eastman Kodak Co., For slam-freezing, the KF80 impact freezer was Rochester, NY), exposed to a speed of 2 and developed employed as described by the manual. High-pressure in D-19 (Kodak) full strength for 12 min at 20°C. For freezing was performed with a prototype freezer, kindly low-dose imaging, the programmable low-dose device of provided by M. Wohlwendt (Sennwald, Switzerland). the microscope was employed. The specimen area was Specimens were cut at 110 K with the Ultracut E exposed to 250 dnrn 2 for the registration of one micro­ ultramicrotome
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