Transmission Electron Microscopy (TEM) is the method used to obtain high-resolution images of plant and animal tissue, allowing morphometric analyses of the internal detail of sectioned cells or direct preps of macromolecules, localizing where substances reside within a cell, or immunologically marking specific antigens in a cell. There are two basic types of electron microscopy: transmission electron microscopy (TEM) yields high-resolution ultrastructural detail from a transmitted electron beam through the tissue; and scanning electron microscopy (SEM) yields high-resolution surface morphology from a secondary electron beam.
Different steps in the preparation of samples for TEM usually include tissue fixation, dehydration, embedding, sectioning, and staining, procedures that take several days to complete. Fixation is an important step that basically kills the cells and “locks” in place the cellular components, and may take place before or after the tissue is excised from the plant or animal subject. For most animal tissues, in vivo perfusion-fixation produces superior tissue morphology compared to immersion fixation; it removes blood from the tissues during a pre-fixation rinse and allows rapid fixation of cells and retention of tissue architecture by delivering the fixative directly while maintaining open vessels, open lumens, and interstitial spaces. When it is not convenient to perfuse the animal, collections of the tissue may be made by biopsy or excision. It is vitally important to work quickly and place the excised tissue immediately into the fixative, where it should be dissected into small (~1mm3 pieces) so it can more effectively absorb the fixative.
The fixation solution most often utilizes an aldehyde, preferably EM grade glutaraldehyde, as the fixation agent, but many EM fixative solutions contain a mixture of both paraformaldehyde and glutaraldehyde in order to utilize the ‘good’ features and reduce the ‘bad’ effects of both. It is vitally important to select the best fixative for the tissue you are preparing for electron microscopy, and there are numerous articles and books dedicated to fixation. Two published and commonly used fixatives for EM are Karnovsky’s fixative1 and Trump’s, also known as McDowell-Trump’s fixative2.
After fixation, the tissue is usually infiltrated with an electron-dense substance. Osmium tetroxide or ruthenium red are often used before dehydration, and uranyl acetate in 70% ethanol may be introduced during dehydration. We use a reduced-osmium formulation containing 2-Mercaptoethanol in order to avoid osmium artifacts in the finished product. Embedding the tissue involves the removal of moisture (dehydration) using increasing concentrations of ethanol or acetone and the subsequent infiltration with a formulated resin. There are many resin formulas currently in use for electron microscopy, but in this example, we will use an epoxy mixture known as EMBED 812.
- Karnovsky, MJ. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27:137-138A. 1965.
- EM McDowell, BF Trump. Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Pathol Lab Med. Aug;100(8):405-14. 1976.
- Tissue sample, fixed (submitted by PI in fixative or buffer)
- Small tissue forceps
- Sharp (double edge) razor blade
- Dental wax (Ted Pella, Inc., Redding, CA)
- Small labeled glass vials, one for each sample
- Embedding capsules, e.g. BEEM capsules (Ladd Research Industries, Williston, VT)
- 0.1M NaCacodylate buffer (Ladd Research)
- 2-ME buffer: 0.1M NaCacodylate with 0.13M sucrose and 0.01M 2-Mercaptoethanol
- 1% Osmium Tetroxide in 2-ME buffer (Ladd Research)
- Ethanols: 30%, 50%, 70%, 80%, 90%, 100%, Absolute
- Propylene Oxide
- 60oC oven
- EMBED 812 epoxy (Electron Microscopy Sciences, Fort Washington, PA, USA)
- EMBED 812 10mL
- DDSA 4.5mL
- NMA 6mL
- DMP-30 0.178mL/10mL
CAUTION: SAMPLES MUST NOT BE ALLOWED TO DRY OUT!
WHEN CHANGING SOLUTIONS, REMOVE AND REPLACE THEM IN ONE VIAL AT A TIME
LEAVE A SMALL AMOUNT IN THE BOTTOM OF THE VIAL
ADD NEXT SOLUTION AS QUICKLY AS POSSIBLE.
- Place the sample tissue on a piece of dental wax in a small droplet of buffer.
- Using a sharp razor blade, cut desired areas of the sample into 1mm3 pieces.
- Place 6-8 pieces of the 1mm3 tissue into small labeled glass vials containing buffer.
- Rinse with 0.1M NaCacodylate 10 min
- Rinse with 2-ME buffer, preferably on a shaker 3 x 20 min
- Decant buffer and barely cover tissue with 1% OsO4 in 2-ME buffer
- Wrap vials in foil and refrigerate, 4oC 60 min
- Rinse samples with quick exchanges of 2-ME buffer 3 times
- Rinse samples with 2-ME buffer until rinse does not turn brown 5 min each
- Rinse with 0.1M NaCacodylate buffer 10 min
- Dehydrate in each of following dilutions of ethanol 2 X 10 min
30%, 50%, 70% (may leave in 70% at 4oC if necessary) 80%, 90%, 100%
CAUTION: 100% EtOH AND PROPYLENE OXIDE ARE VERY VOLATILE AND WILL EVAPORATE ALMOST IMMEDIATELY: CHANGE THESE SOLUTIONS AS QUICKLY AS POSSIBLE.
- Absolute Ethanol 3 X 10 min
- 100% Propylene Oxide 2 X 10 min
- 1 part Embed formula (w/o DMP-30) : 1 part Propylene Oxide
(covered, on shaker) 1 hr
- 2 parts Embed formula (w/o DMP-30) : 1 part Propylene Oxide
(open, slight vacuum or covered, on shaker) Overnight
- 100% Embed formula (w/o DMP-30) (open, on shaker) 2 hr
- 100% Embed formula (with DMP-30) (open, on shaker) 3-4 hrs
- Place one piece of tissue in each labeled BEEM capsule and fill with epoxy + DMP-30
- Polymerize in 60oC oven 24 hrs