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Electron Microscopy Check List
Electron microscopy provides the ultimate high-resolution images. Coupled with immuno-EM, subcellular localization of proteins/sugars is excellent.
However, as with any powerful technique, there are trade-offs. Review the list below to determine if EM is right for your study.
- Implications of magnification: size & density of your target
- Popular fixation conditions
The amazing detail of TEM images can inspire you for your samples. Remember, transmission EM is NOT a survey technique, but a detail technique. If your sample is large or your ultrastructural feature is sparse, TEM may not be appropriate! TEM sections are 5-50nm thick and <3mm across. It would take a lot of TEM sections to just examine a (3mm)3 cube. Similarly, if your ultrastructural feature is sparse (e.g. 1:100 mutant:wildtype ciliary structure), a lot of TEM sections will be needed to find a few features.
The microtome only holds (3mm)3 cube of fixed/embedded tissue. If you start with a larger piece, you must cut it to the (3mm)3 cube before we can proceed for TEM.
If your ultrastructural feature isn't found after surveying 10 regions each on 2-3 sections, we need explicit approval from the PI to continue. Sectioning could become extemely expensive. Perhaps a new strategy is needed!
Unlike TEM, scanning EM, particularly Field-Emission SEM, is ideal for surveying specimens. It can scan almost centimeters during survey, and then zoom in to view microvilli in a few micrometers, all on the same specimen. Remember that SEM only scans the surface features of prepared specimens, but you can survey for rare microscopic events. A great example is rare bacterial infection of gastointestinal epithelial.
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Aldehyde fixatives, such as formaldehyde and glutaraldehyde are preferred for electron microscopy. They are excellent for preserving proteinaceous ultrastructure. In contrast, although popular and successful for fluorescence microscopy, solvent fixatives, such as methanol, acetone, or ethanol, cannot be used for electron microscopy. Because these fixatives coagulate proteins (sub-wavelength) while extracting some lipids, they do a poor job of preserving ultrastructure.
For lipids, osmium tetroxide both stains and stabilizes their ultrastructure. However, lipids and lipid constituents have an extra 'race-condition' resulting from a fundamental antagonism: standard fixation requires permeabilization, or partial destruction, of membrane integrity so that fixatives can penetrate. It's critical whether stabilization or destruction wins the race! Standard protocols may need a lot of customization for different tissues/cells just to balance this fundamental antagonism.
High-pressure freezing can side-step the fundamental antagonism of standard fixation. By flash-freezing samples at 20atm in liquid N2, ice-crystal formation is suppressed. After freezing, fixatives, particularly osmium tetroxide, work fine on the frozen specimen! Vitreous ice holds everything in place while the fixatives crosslink and stabilize. For tricky specimens, try high-pressure freezing with automated freeze-substitution.
Please consult with us, both before and during attempts to develop fixation conditions. If you do it yourself, we can provide you with the recipes, protocols, and some appropriate quality (EM-grade) reagents to get you started. If you want us to do it, we will tweak the recipes and protocols as part of our normal service. We have many years of experience chemically fixing a wide variety of samples, and we can help you navigate the trade-offs for each decision (but there's no substitute for empirical proof!). Make us part of your research effort; just ask us!
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