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Transmission electron microscopy
Acknowledgments
We thank Richard Davey (SA Pathology) for his assistance in preparing the illustrations for this chapter and Dr Alan Curry (Manchester Royal Infirmary) for his helpful advice on microsporidia.
Introduction
Transmission electron microscopy (TEM) is a significant tool in demonstrating the ultrastructure of cells and tissues both in normal and disease states. In particular, TEM can be crucial in the diagnosis of various renal pathologies, the recognition of subcellular structural defects or the deposition of extracellular material (e.g. in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, CADASIL) and in the typing of microsporidia. This chapter details the methods used to process and prepare tissue samples appropriately for examination in the transmission electron microscope (EM). The fundamental advantage of TEM over conventional light microscopy (LM) is that the EM has a resolution approximately 1000 times greater. With this increased resolving power, the EM is able to demonstrate the ultrastructure (substructure) of individual cells. Contemporary EMs with digital imaging systems are capable of resolving 0.2 nm (or less): using cryotechniques, this allows cell structures to be examined at the molecular level. However, in practical terms, biological tissues prepared using standard methods cannot be examined at such high resolution due to the limitations of chemical fixation and routine preparation techniques.
Tissue preparation for transmission electron microscopy
The basic preparation methods used in routine TEM are provided in this chapter. More detailed discussions of these, plus alternative and specialized procedures, can be found elsewhere ( ). A flow chart summarizing the steps required for preparing the basic range of diagnostic TEM specimens is given in Fig. 21.1 .
The fundamental principle underlying TEM is that electrons pass through the section to give an image of the specimen. The electron beam is only capable of penetrating a resin section effectively to a depth of approximately 100 nm, so to obtain a high-quality image and optimize the resolution of the instrument, it is necessary to section the tissue to a thickness of around 80 nm.
Sectioning at this level requires tissues to be embedded in a rigid material which can withstand both the vacuum in the microscope column and the heat generated as the electron beam passes through the section. The wax embedding media used in LM are not suitable for this purpose and tissues must be embedded in an epoxy or acrylic resin. Both hydrophilic and hydrophobic media are available but, for routine purposes, a hydrophobic epoxy resin such as Araldite, Epon or Spurr’s is preferred (see Chapter 8 ).
Specimen handling
In order to preserve the ultrastructure of a cell it is crucial that samples are fixed as soon as possible after the biopsy is taken. The most sensitive cellular indicators of autolytic/degenerative change are mitochondria and endoplasmic reticulum, both of which may show signs of swelling (a reflection of osmotic imbalance) only a few minutes after the cells are separated from a blood supply.
The standard approach is to immerse the specimen in fixative immediately on collection. Once in fixative, the specimen is cut into smaller samples using a scalpel or razor blade. At this point the tissue should be oriented and dissected to optimize exposure of the critical diagnostic features during sectioning and screening. Dissection must also facilitate the penetration of fixatives and processing reagents ( ). The final tissue blocks may be in the form of thin sheets or small cubes (approximately 1 mm 3 ), although the risk of sampling error increases as the sample size decreases. In general, the volume of fixative should be at least 10 times the volume of the tissue. It is also vital to ensure that the tissue remains completely submerged in the fixative. One should be aware that small pieces may adhere to the inside of the lid of the biopsy container, and therefore these will be poorly fixed even if they have been exposed to fixative vapor. Gentle agitation of the vial on a mechanical rotator helps to overcome this problem and improves fixation.
The importance of using small samples cannot be overemphasized. The use of cold fixative (4°C) helps to minimize postmortem changes but fixation may be hindered as a consequence. In addition, the penetration rate of most TEM fixatives is quite slow, increasing the risk of artifact formation. It should also be noted that fixatives and processing reagents penetrate different tissues at different rates. Some tissues, e.g. liver, fix poorly and needle biopsies may need to be cut longitudinally to ensure adequate fixation. If a delay in fixation is unavoidable, damage can be minimized by holding the tissue in chilled normal saline for a short time. However, the tissue must not be frozen at any point.
Fixation
The fixatives used in TEM generally comprise a fixing agent in buffer (to maintain pH) and, if necessary, various additives to control osmolarity and ionic composition. Other factors which affect fixation include fixative concentration, temperature, and the duration of fixation. The standard protocol involves primary fixation with an aldehyde, usually glutaraldehyde, to stabilize proteins, followed by secondary fixation in osmium tetroxide to retain lipids ( ), this is termed ‘double fixation’.
Fixative concentration
Glutaraldehyde is effective at a concentration of between 1.5 and 4%, with 2.5% the simplest to prepare from commercially available 25% stock solutions. Osmium tetroxide is usually used at a concentration of 1 or 2%.
Temperature
Tissues may be placed in cold primary fixative solution, but this is not essential. Fixation at room temperature improves the penetration rate, particularly of aldehyde fixatives, and reduces the time required for fixation, although it may also increase the risk of autolytic change. Osmium tetroxide is generally used at room temperature.
Duration of fixation
The time required for optimal fixation depends on a range of factors. These include the type of tissue, the size of the sample, and the type of fixative and buffer system used. In most circumstances immersion of 0.5–1.0 mm 3 blocks of tissue in 2.5% glutaraldehyde fixative for 2–6 hours is sufficient. It is recommended that punch biopsies of skin taken for the diagnosis of CADASIL should be fixed overnight to ensure adequate preservation, particularly if they are left whole (i.e. not dissected) and sent to a distant laboratory for processing and screening ( ). Secondary fixation in 1% osmium tetroxide for 60–90 minutes is usually effective but much longer times are required if osmium tetroxide is the primary fixative. The use of microwave irradiation can accelerate fixation times in aldehyde fixative to as little as 5–10 seconds ( ), after which the sample may be stored in buffer or processed immediately.
Buffers
Fixatives are normally prepared in buffer (the fixative ‘vehicle’) which is adjusted within a range of pH 7.2–7.6 ( ). Ideally the osmolarity and ionic composition of the buffer should mimic that of the tissue being fixed. Generally this is not a major requirement but, an osmolarity slightly hypertonic to or equivalent of plasma (300-330 mOsm) is suitable for most circumstances. Non-ionic molecules such as glucose, sucrose or dextran are used to adjust tonicity as these will not influence the ionic constitution of the buffer. The addition of various salts, particularly calcium and magnesium, is thought to improve tissue preservation, possibly by stabilizing membranes ( ). This is unlikely to have a major effect in routine diagnostic applications.
Phosphate buffers
Phosphate buffers ( ) are the buffer of choice as they are non-toxic and work well with most tissues but have two disadvantages which restrict their use. Firstly phosphate buffers are a good growth medium for molds and other microorganisms; secondly most metal ions form insoluble phosphates with this buffer. The phosphates of sodium, potassium and ammonium however, are soluble.
Phosphate buffer (0.1 M, pH 7.4)
Stock reagents
Solution a
| Disodium hydrogen phosphate (Na 2 HPO 4 anhydrous) | 14.2 g |
| Distilled/deionized water | 1000 ml |
Solution b
| Sodium dihydrogen phosphate (NaH 2 PO 4 ·2H 2 O) | 51.6 g |
| Distilled/deionized water | 1000 ml |
Method
Mix 40.5 ml of solution a with 9.5 ml of solution b . The pH should be checked and adjusted if necessary, using 0.1 M hydrochloric acid or 0.1 M sodium hydroxide.
Alternative buffers
Other buffers which have been recommended for use in TEM include cacodylate ( ), HEPES ( N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic acid), MOPS (3-( N -morpholino) propanesulfonic acid) and PIPES (piperazine- N, N ′-bis2-ethanesulfonic acid) ( ).
Aldehyde fixatives
Glutaraldehyde
Although glutaraldehyde is the most widely used primary fixative in TEM, its fixation reactions are poorly understood. The most important reaction of glutaraldehyde, stabilizing proteins, is thought to occur via a cross-linking mechanism involving the amino groups of lysine and other amino acids through the formation of pyridine intermediaries. Lipids and most phospholipids (those not containing free amino groups) are not fixed and will be extracted during subsequent processing without secondary fixation ( ).
Formaldehyde
Commercially supplied formaldehyde solutions (formalin) normally contain some formic acid and considerable quantities of methanol and are poor cytological fixatives not suitable for TEM. In contrast, formaldehyde which has been freshly prepared from paraformaldehyde powder is adequate for TEM as it lacks impurities and also penetrates faster than glutaraldehyde. Paraformaldehyde has been used in conjunction with glutaraldehyde and may be useful for electron immunohistochemistry as tissue epitopes are less likely to be significantly altered during fixation and, if required, antigen unmasking is more effective.
Aldehyde combinations
The use of an aldehyde mixture may offset the disadvantages of glutaraldehyde (a slow penetration rate) and formaldehyde (less stable fixation) when applied individually ( ).
Paraformaldehyde (2%) and glutaraldehyde (2.5%) fixative (buffered) (based on )
Stock reagents
| 0.2 M buffer, pH 7.4 (phosphate, cacodylate) | 50 ml |
| Paraformaldehyde | 2.0 g |
| 25% aqueous glutaraldehyde | 10 ml |
| Distilled/deionized water to | 100 ml |
Method
- 1.
Completely dissolve paraformaldehyde in buffer using heat and with continuous stirring. It may be necessary to add a few drops of 1.0 M sodium hydroxide to clarify the solution.
- 2.
Cool the solution rapidly under running water.
- 3.
Add aqueous glutaraldehyde. Check the pH of the mixture and adjust if necessary to pH 7.4.
- 4.
Add distilled water to make 100 ml.
Note
Adding 0.2 ml of 1.0 M calcium chloride is thought to have a membrane stabilizing effect but this may precipitate if phosphate buffer is used.
Osmium tetroxide
The use of osmium tetroxide fixation to preserve lipids is fundamental to TEM ( ). Whilst primary fixation in osmium tetroxide is effective, its extremely slow penetration rate can give rise to autolytic changes. For this reason, osmium tetroxide is usually used as a secondary fixative, termed ‘post-fixation’, after primary fixation in aldehyde. The penetration rate of osmium tetroxide is also higher in stabilized tissue and immersion for 60–90 minutes is sufficient for most specimens.
Osmium tetroxide is usually supplied in crystalline form, sealed in glass ampoules. Extreme care should be exercised when preparing this material, gloves and eye protection should always be worn. It is essential to only handle osmium tetroxide in a fume-hood, as the vapor will also fix other tissues, including the eyes and nasal tissues of the handler.
Specimens fixed in aldehyde solutions should be washed thoroughly in buffer before post-fixation in osmium tetroxide to prevent interaction between the fixatives which can cause precipitation of reduced osmium. Osmium tetroxide can be prepared as an aqueous solution, although it can also be made in the same buffer used to prepare the primary fixative. Osmium tetroxide should be avoided if electron immunogold labeling studies are to be performed, as it has the potential to alter protein structure significantly, rendering epitopes unreactive.
Osmium tetroxide fixative (2% aqueous)
Stock reagent
| Osmium tetroxide | 1.0 g |
| Distilled/deionized water | 50 ml |
Method
- 1.
In a fume-hood, clean then score the glass ampoule with a diamond pencil and place in a dark glass storage bottle.
- 2.
Break the ampoule with a glass rod and add water. It may take 24 hours or longer for the osmium to dissolve completely unless ultrasonicated.
- 3.
Prepared solutions may be stored for short periods at room temperature in the dark in a well-sealed bottle (double wrap the bottle in aluminum foil); for long periods store at 4°C. All containers of osmium solutions should be stored inside a second well-sealed container to prevent the leakage of osmium fumes; milk powder can be placed in the second container to absorb fumes which leak from the primary container. Aqueous osmium solutions which are prepared and stored in a clean container should last for approximately 1 year; solutions in buffer may only last a few days before they deteriorate.
- 4.
For a 1% working solution combine 1:1 with water or buffer.
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