Need for Speed
Tips & Tricks for Rapid Preparation of Tissue Samples for TEM
- Fig. 1: Microwave-assisted tissue processing. (A) Schematic of the protocol with incubation times, temperature conditions, and the different irradiation modes used (Cont: continuous radiation; pulse: 10 s on/ 50 s off; slope: temperature reached at the end of incubation period). A microwave processor with a monomode chamber was used (AMW, Leica Microsystems). Curing of embedded samples can be performed either with the microwave processor or, as shown here, conventionally in an oven at 100°C. Total time: 5h 33’. (B-D) Examples of tissues processed this way. (B) Hepatocytes (HC) in the mouse liver. (C) HC at higher magnification with bile canaliculus (BC) at center of the image. (D) Pancraetic acinus, the apical parts of 4 cells (1-4) surrounding the acinal lumen (Lu) are visible. Futher abbreviations: G, Golgi field; M, mitochondrium; MvB, multivesicular body; Nuc, nucleus; rER, rough endoplasmic reticulum; ZG, zymogen granule.
- Fig. 2: Rapid sample processing with reduced incubation times. (A) Schematic of the protocol with incubation times and conditions. All steps are performed at room temperature unless indicated otherwise. Total time: 7h 31’. (B-E) Examples of tissues processed this way. (B,C) Mouse exocrine pancreas. (B) Part of a cross-sectioned interlobular duct with duct cells (DC), zymogen filled lumen (Lu), and adjacent connective tissue with fibrocytes (Fib) and collagen fibers (Col). (C) Details of exocrine pancreas cells with rough endoplasmic reticulum (rER), mitochondria (M), and zymogen granules (ZG). (D,E) Mouse intestine. (D) Intestinal mucosa with enterocytes (En) and a goblet cell (GC). (E) Apical junctional complex (TJ, tight junction; AJ, adherens junction; D, desmosome) between two enterocytes. Further abbreviations: Mv, microvilli; Nuc, nucleus; V, vesicle.
Chemical fixation followed by epon embedding and thin sectioning still is a powerful approach for the ultrastructural analysis of a wide variety of biological and clinical samples. To achieve good preservation, most protocols, however, are labour intensive and time-consuming, usually taking 3-4 days. Here, we describe alternative approaches that provide samples with reasonable ultrastructural preservation and tissue contrast within one day, using either a microwave-assisted procedure or a protocol with reduced incubation times.
For EM of tissues time-consuming procedures are performed. The samples are fixed in aldehydes, postfixed/contrasted with heavy metals (osmium tetroxide (OsO4), uranyl acetate (UA)), dehydrated in graded series of organic solvents (e.g. ethanol, acetone), infiltrated in resins (e.g. epoxy or methacrylate resins), and polymerized by heating, before they can be sectioned, stained, and imaged with a TEM. Usually such a protocol takes 3-4 days. To speed up the procedure, two different routes have been taken, microwave (MW)-assisted sample processing [1-4], or sample processing with reduced incubation times and increased polymerisation temperatures [5-8].
Materials and Methods
Microwave-assisted Sample Processing
MW-iradiation acts by dielectric heating, which increases the temperature simultaneously throughout the sample, thereby speeding up all chemical processes. It is widely used for a number of histological applications and for fast EM preparation [1,2]. Here, an automated tissue processor equipped with a monomode chamber for homogenous irradiation of the samples (Leica EM-AMW) has been used. The AMW is suitable for fixation and processing of difficult-to-fix samples , for epon embedding of different tissues [11,12], and for fast histological preparations of samples embedded in methacrylate resin [12,13].
Tissues are fixed in a mixture of glutaraldehyde (GA) and paraformaldehyde (PFA) (2% GA /2% PFA in 50-100 mM HEPES or in 100-150 mM cacodylate buffer). They are fixed either in the AMW or collected and fixed conventionally and subsequently processed further in the instrument.
After fixation, the samples are washed several times in 100 mM buffer, postfixed in 1-2% OsO4/water, washed in water, dehydrated in a graded series of ethanol or acetone, and infiltrated/embedded in resin (e.g. epon 812). The curing is performed either in the MW-processor or conventionally in an oven. Ultrathin sections are prepared with an ultramicrotome, collected on formvar coated grids and stained with lead citrate  and 2% UA/water. A typical protocol including MW-assisted fixation is shown in figure 1A. The results of such a procedure (fig. 1B-D) show that tissue preservation is very good, with dense cytoplasm and strong contrast.
Rapid Sample Processing by Reducing Incubation Times (“Quick’n Dirty”)
Alternatively, the samples are processed with short incubation times, especially during dehydration, and increased incubation temperatures, for example during osmium postfixation and polymerisation [5-8]. Some protocols use low viscosity resins to facilitate fast infiltration . Here, the protocol of Hayat and Giaquinta  has been used, prolonging some incubation periods and adding additional osmium-contrasting steps that were developed for serial-block-face SEM  (fig. 2A).
The tissues were fixed in GA/PFA as described above, washed in water, postfixed in 2% aqueous OsO4 containing 1.5% potassium ferrocyanide and 2 mM CaCl2, washed in water, incubated in 1% thiocarbohydrazide (TCH)/water, washed, and contrasted again in 2% OsO4/water (O-T-O). Samples were washed in water, en-bloc contrasted with 1% UA/water, washed, dehydrated in a graded series of acetone, and infiltrated/embedded in epon 812 (fig. 2A). Finally, they were cured at 100°C, sectioned, and stained with lead citrate and UA. The protocol takes about 8 h and gives excellent ultrastructure and contrast, especially of membranes (fig. 2B-E).
Both approaches provide good preservation and contrast in one day. They are useful for many routine applications or the urgent diagnosis of pathological conditions. Advantages of the MW procedure are short process time (about 6 h) and high throughput of samples. Drawbacks are the fixed need of 10 ml aliquots for each reagent, even for a few samples, and the high costs for the instrument and consumables. The conventional rapid processing takes more time and effort, but does not need sophisticated instrumentation and saves expensive consumables (e.g. OsO4 and UA), if only a handful of samples are processed.
We thank DFG, CRTD, and the European Fund for Regional Development (EFRE) for financial support.
Susanne Kretschmar1 and Thomas Kurth1
1 TU Dresden, Center for Molecular and Cellular Bioengineering (CMCB), DFG-Center for Regenerative Therapies Dresden (CRTD) and BIOTEC Center, Dresden, Germany
Dr. Thomas Kurth
TU Dresden, Center for Molecular and Cellular Bioengineering (CMCB),
DFG-Center for Regenerative Therapies Dresden (CRTD) and BIOTEC Center,
Joint Electron Microscopy and Histology Facility
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