By Michael J. Dykstra, Laura E. Reuss
This publication 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 recommendations part, the place applicable, with tried-and-true tools that would produce publishable effects. the speculation in the back of numerous technical ways is supplied to aid the reader troubleshoot difficulties. whereas the strategies sections will not be intended to be encyclopaedic, they need to function a widely acceptable start line for quite a few ways to cytological study. Biological Electron Microscopy is designed for an introductory one-semester direction in organic electron microscopy and offers an creation to the entire significant technical methods for pattern training and instrumentation usage to reply to cytological questions.
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Additional info for Biological Electron Microscopy: Theory, Techniques, and Troubleshooting
Ideal Qualities Ideal qualities of embedding media include uniformity of batches of resin components, good solubility in dehydration agents, and low viscosity as a monomer, along with minimal shrinkage, stability under the electron beam, minimal granularity, and good sectioning characteristics. Unfortunately, some of these goals are mutually exclusive. For example, resins with low viscosity tend to shrink significantly during polymerization (see Table 2 for a listing of resins and resin viscosities).
Typically, cells are placed in primary fixative and rinsed several times in buffer before being immersed in molten water agar (at 45-50°C) and immediately centrifuged to bring the sample into the tip of the centrifuge tube (see the detailed instructions in the section Chapter 1 Techniques). Another use of agar for specimen handling is the agar-peel technique for cells in situ. In this procedure, adherent cells in flasks or Petri dishes are rinsed free of growth medium with PBS to remove media proteins that otherwise would be fixed by aldehyde fixatives.
The presence of the stabilizer does not seem to adversely affect fixation. The shelf life of acrolein at room temperature is not long because of its propensity for auto-polymerization. Storage at 4°C is recommended. Diluted solutions will be less prone to polymerize. 4, or if there is turbidity in the concentrated solution from which the dilute solution is made, discard the solution. This fixative has a high vapor pressure and is extremely toxic, so always work carefully with it under a fume hood.
Biological Electron Microscopy: Theory, Techniques, and Troubleshooting by Michael J. Dykstra, Laura E. Reuss