Rapid polymerisation with microwave irradiation for transmission electron microscopy

Successful results of microwave polymerisation of different epoxy formulations have been reported in the literature. The present study was intended to shorten the time needed for polymerisation of epoxy resin by the use of a microwave technique. A standard double fixation and tissue processing was applied to samples of rat kidney tissue. Tissue samples from the control group were polymerised in a conventional oven at 60 degrees C for 48 h, while tissue from the experimental group was irradiated in a microwave oven, initially at 900 W for 10 min and then at 360 W for another 100 min. During this irradiation, the sealed BEEM capsules were submerged in a water bath, so that the temperature rise was uniform and constant. This resulted in a homogeneous and rapid polymerisation. The cutting properties of the blocks in both groups were similar and no noticeable difference in the quality of the sections was evident when evaluated with TEM. The results showed that the use of a microwave oven reduced the time needed for the polymerisation of Epon blocks without any loss in quality.     

A new method for studying human oocytes by light and electron microscopy

Since ovarian follicles appear to be randomly oriented with respect to the plane of the section, the method of sectioning and examining follicles at their maximum diameter described here allows direct comparison between oocyte populations of women and small differences can be detected. Re-sectioning for EM allows selected follicles of interest to be examined at a higher resolution.     

An alternative method to prepare samples of the pathogenic yeast Cryptococcus neoformans for scanning electron microscopy analysis

In this work, an alternative to conventional preparation procedures for scanning electron microscopy (SEM) analysis of Cryptococcus neoformans was performed. The cells were fixed directly in the agar culture. This method is simpler than others already reported and the morphology of the cells was well preserved.     

Rapid fixation and embedding for electron microscopy

A method has been developed for rapid processing of animal tissues for electron microscopy. The whole process of fixation staining dehydration, infiltration and embedding including polymerization is completed in less than 4 hr. A variety of human and animal tissues such as liver, spleen, muscle, kidney and embryonic chick heart were processed by this method and the results were excellent. The rapid fixation and embedding method is strongly recommended when relatively soft tissues are to be studied. This method is especially useful for examining pathological tissues for rapid diagnostic purposes.     

Conventional transmission electron microscopy

Researchers have used transmission electron microscopy (TEM) to make contributions to cell biology for well over 50 years, and TEM continues to be an important technology in our field. We briefly present for the neophyte the components of a TEM-based study, beginning with sample preparation through imaging of the samples. We point out the limitations of TEM and issues to be considered during experimental design. Advanced electron microscopy techniques are listed as well. Finally, we point potential new users of TEM to resources to help launch their project.Transmission electron microscopy (TEM) has been an important technology in cell biology ever since it was first used in the early 1940s. The most frequently used TEM application in cell biology entails imaging stained thin sections of plastic-embedded cells by passage of an electron beam through the sample such that the beam will be absorbed and scattered, producing contrast and an image (see

An improved method for chromosome preparations from preimplantation mammalian embryos, oocytes or isolated blastomeres

A method is described for making chromosome preparation from mammalian oocytes or preimplantation embryo, with or without the zona pellucida, or from isolated blastomeres. It is more robust and requires less skill and experience than previous techniques, yet chromosome structure is well preserved and very high quality preparations can be made. The method, which involves use of cold hypotonic solution and very cold fixative, reduces turbulence and allows even single blastomeres to be located and handled with relative case, while the duration of hypotonic treatment becomes noncritical. The softening solution recommended contains no lactic acid and hence does not harm the chromosomes.     

Improved method to prepare RNA-free DNA from mammalian cells

To isolate DNA for nucleoside analog incorporation studies, many investigators use RNase A to remove RNA from total cellular nucleic acid. We observed persistence of ribonucleotides from RNA in nucleic acid samples treated with RNase A alone. Although incubation of [5-³H]uridine-labeled nucleic acid with 50 μg/ml RNase A decreased tritium by 97%, HPLC analysis of the resulting DNA preparation digested to nucleosides revealed high levels of ribonucleosides. Increasing RNase A 10-fold (500 μg/ml) effected only a 1.7-fold reduction in ribonucleosides. Overall, the level of ribonucleosides was one-fourth that of the deoxynucleosides, primarily due to the high levels of guanosine. It was hypothesized that the ribonucleosides originated from guanosine-rich tracts of RNA since RNase A cuts preferentially 3′ to pyrimidine monophosphates and to some extent after AMP. The addition of 0.05 μg/ml RNase T1, which preferentially cleaves RNA 3′ to GMP, decreased total ribonucleosides by nearly 20-fold. In conclusion, we have developed a rapid method which removes greater then 99% of cellular RNA from nucleic acid extracts and a reversed-phase HPLC procedure that detects RNA contamination more sensitively than [5-³H]uridine labeling. These methods are useful for the determination of analog incorporation into DNA, especially for agents which incorporate into both DNA and RNA.     

Cell volume regulation in mammalian oocytes and preimplantation embryos

The earliest stages of preimplantation embryos are particularly sensitive to increased osmolarity, even within the physiological range. This sensitivity contributed to persistent developmental arrest, even when embryos were cultured in vitro in older, conditioned culture media, and seems to arise when embryos at the 1‐ and 2‐cell stages accumulate inorganic ions used for cell volume homeostasis at too high a level, through activation of coupled Na⁺/H⁺ and HCO/Cl^? exchange. Such accumulation of inorganic ions can be disruptive since, above a certain level, the increased ionic strength disrupts cellular biochemistry and macromolecular functions and alters membrane potential. To counter this, embryos have evolved mechanisms of cell volume regulation that are unique to early preimplantation embryogenesis. The primary role of these is glycine accumulation via the GLYT1 transporter, with a secondary contribution by betaine accumulation via the SIT1 transporter. Independent cell‐volume regulation first arises in the oocyte only after ovulation is triggered, when the strong oocyte‐zona pellucida adhesion present in germinal vesicle stage oocytes in the ovarian follicle is released and GLYT1 becomes activated to begin accumulating glycine. Open questions still remain regarding how these processes are regulated. Mol. Reprod. Dev. 79: 821–831, 2012. © 2012 Wiley Periodicals, Inc.     

A simplified method for the preparation of rotifers for transmission and scanning electron microscopy

Tor TEM and SEM prepatations, rotifers are placed in little panels made of plastic Beem capsules normally used for embedding, with their conical parts cut off and closed by plankton filter cloth. Thus, the risk of losing animals is considerably reduced.     

Biological specimen preparation for transmission electron microscopy

Biological Specimen Preparation for Transmission Electron Microscopy (1998). A.M. Glauert, P.R. Lewis. In: A.M. Glauert (Ed). Practical Methods in Electron Microscopy, Vol 17. London: Portland Press, 326 pp. £39.50 paperback; ISBN 1 85578 060 7     

Integrated fluorescence and transmission electron microscopy

Correlative microscopy is a powerful technique that combines the strengths of fluorescence microscopy and electron microscopy. The first enables rapid searching for regions of interest in large fields of view while the latter exhibits superior resolution over a narrow field of view. Routine use of correlative microscopy is seriously hampered by the cumbersome and elaborate experimental procedures. This is partly due to the use of two separate microscopes for fluorescence and electron microscopy. Here, an integrated approach to correlative microscopy is presented based on a laser scanning fluorescence microscope integrated in a transmission electron microscope. Using this approach the search for features in the specimen is greatly simplified and the time to carry out the experiment is strongly reduced. The potential of the integrated approach is demonstrated at room temperature on specimens of rat intestine cells labeled with AlexaFluor488 conjugated to wheat germ agglutinin and on rat liver peroxisomes immunolabeled with anti-catalase antibodies and secondary AlexaFluor488 antibodies and 10nm protein A-gold.     

Development of membrane excitability in mammalian oocytes and early embryos

Developmental aspects of the capability to generate an action potential have been studied in mouse oocytes and early embryos, taking the peak level and the rate of rise of the action potential as parameters of cell excitability. We report here that experimentally induced membrane excitability in mouse oocytes appears throughout oogenetic growth, further develops throughout meiotic maturation, does not undergo main changes at fertilization, and slightly decreases throughout early cleavage, at least up to the four-cell stage of embryonic development. Calcium is involved in the rising phase of action potential throughout oogenesis, maturation, and early embryonic development.     

Rapid detection by transmission electron microscopy of mycoplasma contamination in sera and cell cultures

Transmission electron microscopy has been employed for the rapid detection of mycoplasma in sera and cell cultures. High speed centrifugation of sera or low speed centrifugation of cell debris, followed by negative staining of the resuspended pellet, detected mycoplasma contamination more frequently than a culture method followed by direct fluorescence (DAPI), which was used as a control procedure. The appearance of the mycoplasma cell border and content gives some information about particle viability.