By Michael J. Dykstra, Laura E. Reuss
This e-book covers traditional mild microscopy, transmission electron microscopy, scanning electron microscopy, intermediate and excessive voltage transmission electron microscopy, electronic imaging and telemedicine, cryotechniques, fixation protocols, cytochemistry and immunocytochemistry, images and photomicroscopy. The textual content is geared up with a survey of every topic, and a strategies part, the place acceptable, with tried-and-true equipment that may produce publishable effects. the speculation at the back of a variety of technical techniques is equipped to assist the reader troubleshoot difficulties. whereas the recommendations sections usually are not intended to be encyclopaedic, they need to function a widely acceptable place to begin for a number of techniques to cytological examine. Biological Electron Microscopy is designed for an introductory one-semester path in organic electron microscopy and gives an creation to the entire significant technical methods for pattern practise and instrumentation usage to respond to cytological questions.
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Extra resources for Biological Electron Microscopy: Theory, Techniques, and Troubleshooting
Example text
Formaldehyde will toughen tissues, but they remain pliable (unlike osmium-fixed tissues, which become brittle). Cells can be easily pelleted through molten water agar without significant damage. e. Parameters Concentration. Most workers use 4% formaldehyde solutions, though some of the recent immunocytochemical procedures utilize as little as 2% formaldehyde made up from paraformaldehyde. Temperature. Room-temperature fixation is successful, though some workers believe that ice-bath temperatures provide better fixation.
Physiological activities of the cells are not stopped immediately, resulting in cells changing conformation in response to the fixative under some conditions. In addition, membranes become more permeable after primary glutaraldehyde fixation, and cellular compartments can subsequently leak. This will result in some hydrolytic enzymes escaping their normal cellular sequestration in lysosomes, which can cause some autolytic damage unless samples stored for prolonged periods are kept at temperatures considerably below the temperature at which the enzymes are most physiologically active (4°C is the temperature recommended for storing mammalian tissues that are normally at 36-38°C during life).
With larger sample sizes, some workers use higher concentrations of glutaraldehyde, since the solution becomes more dilute as it passes to the interior of a sample because the glutaraldehyde becomes bound and is no longer free in solution. Temperature. Historically, it has been recommended that glutaraldehyde fixation be done on ice or at 4°C. At the same time, it was established early on that microtubules become depolymerized when held at such temperatures. Glutaraldehyde fixation at room temperature seems to give adequate results without causing microtubule depolymerization.