Freezing damages cell membranes and reduces the histological readability of biological specimens. This is a discussion of why, the consequences, and how to minimize or avoid the damage introduced when using freezing as the means to harden biological tissue in order to cut thin sections for histology.Pure water can exist in the solid state in three forms. Two forms are crystalline: one with a hexagonal lattice and the other with a cubic lattice. The third form capitalizes on the fact that water can be frozen so rapidly that it does not have time to form a crystal lattice and remains amorphous (vitreous form). Crystal formation is responsible for the expansion of water as it freezes (a property unique of water), which creates specimen preparation artifacts. Vitreous ice does not expand upon solidification. This makes it the only desirable form of ice appropriate for biological specimens.
Electron microscopy (EM) is an indispensable tool for the study of ultrastructures of biological specimens. Every electron microscopist would like to process biological specimens for either scanning electron microscopy (SEM) or transmission electron microscopy (TEM) in a way that the specimens viewed under the electron microscope resemble those seen in vivo or in vitro under the light microscope. This is, however, often easier said than done because biological tissue processing for EM requires careful attention of the investigator with regard to the numerous processing steps involved in specimen preparation, such as fixation, dehydration, infiltration, embedding, and sectioning.
AbstractCryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications.
A problem was presented to observe the packing densities of deposits of sub-micron corrosion product particles. The deposits were 5-100 mils thick and had formed on the inside surfaces of 3/8 inch diameter Zircaloy-2 heat exchanger tubes. The particles were iron oxides deposited from flowing water and consequently were only weakly bonded. Particular care was required during handling to preserve the original formations of the deposits. The specimen preparation method described below allowed direct observation of cross sections of the deposit layers by transmission electron microscopy.The specimens were short sections of the tubes (about 3 inches long) that were carefully cut from the systems. The insides of the tube sections were first coated with a thin layer of a fluid epoxy resin by dipping. This coating served to impregnate the deposit layer as well as to protect the layer if subsequent handling were required.
Microprobe analysis of biological tissue is now in the end phase of transition from instrumental and technique development to applications pertinent to questions of physiological relevance. The promise,implicit in early investigative efforts, is being fulfilled to an extent much greater than many had predicted. It would thus seem appropriate to briefly report studies exemplifying this, ∿. In general, the distributions of ions in tissue in a preselected physiological state produced by variations in the external environment is of importance in elucidating the mechanisms of exchange and regulation of these ions.
Calculations have been performed on the contrast obtainable, using the Scanning Transmission Electron Microscope, in the observation of thick specimens. Recent research indicates a revival of an earlier interest in the observation of thin specimens with the view of comparing the attainable contrast using both types of specimens.Potential for biological applications of scanning transmission electron microscopy has led to a proliferation of the literature concerning specimen preparation methods and the controversy over “to stain or not to stain” in combination with the use of the dark field operating mode and the same choice of technique using bright field mode of operation has not yet been resolved.
It has been an interest of our lab to develop a mammary epethelial cell culture system that faithfully duplicates the in vivo condition of the lactating gland. Since the introduction of collagen as a matrix on which cells are cultivated other E.C.M. type matrices have been made available and are used in many cell culture techniques. We have previously demonstrated that cells cultured on collagen and Matrigel do not differentiate as they do in vivo. It seems that these cultures often produce cells that show a disruption in the secretory process. The appearance of large ribosomal studded vesicles, that specifically label with antibody to casein, suggest an interruption of both protein maturation and secretion at the E.R. to golgi transition. In this report we have examined cultures on collagen and Matrigel at relative high and low seeding densities and compared them to cells from the in vivo condition.
A new specimen preparation technique for visualizing macromolecules by conventional transmission electron microscopy has been developed. In this technique the biopolymer-molecule is embedded in a thin monocrystalline gold foil. Such embedding can be performed in the following way: the biopolymer is deposited on an epitaxially-grown thin single-crystal gold film. The molecule is then occluded by further epitaxial growth. In such an epitaxial sandwich an occluded molecule is expected to behave as a crystal-lattice defect and give rise to contrast in the electron microscope.The resolution of the method should be limited only by the precision with which the epitaxially grown gold reflects the details of the molecular structure and, in favorable cases, can approach the lattice resolution limit.In order to estimate the strength of the contrast due to the void-effect arising from occlusion of the DNA-molecule in a gold crystal some calculations were performed.
Under a variety of electron microscope specimen preparation techniques different forms of chromatin appearance can be distinguished: beads-on-a-string, a 100 Å nucleofilament, a 250 Å fiber and a compact 300 to 500 Å fiber.Using a standardized specimen preparation technique we wanted to find out whether there is any relation between these different forms of chromatin or not. We show that with increasing ionic strength a chromatin fiber consisting of a row of nucleo- somes progressively folds up into a solenoid-like structure with a diameter of about 300 Å.For the preparation of chromatin for electron microscopy the avoidance of stretching artifacts during adsorption to the carbon supports is of utmost importance. The samples are fixed with 0.1% glutaraldehyde at 4°C for at least 12 hrs. The material was usually examined between 24 and 48 hrs after the onset of fixation.
3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy