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Review article

Scanning electron microscope: a new approach of an old issue

Shoukry, Youssef

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The Egyptian Journal of Histology: June 2011 - Volume 34 - Issue 2 - p 179-181
doi: 10.1097/01.EHX.0000398103.69273.b3
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Abstract

Introduction

Decades have passed since a new visual world was introduced to the biomedical field by the application of the scanning electron microscope (SEM). During this period, a tremendously large variety of tissues, cells, and other objects have been observed and a vast number of studies demonstrating their SEM images have been documented [1].

Practically, SEM observation was hindered either by charging of the specimens or by the thick metal coating applied to prevent it. Nevertheless, the three-dimensional visualization of the objects, (which is easily attained by the SEM) turned out to be very useful for the studies, and valuable results rapidly accumulated in different research fields [2].

Using new and different approaches in preparation and examination of the specimens, the SEM does not only provide the ultrastructural view but also in addition can provide a new facility in the medical research field, starting a new era of achievements to review the entire microfabric of the body through the eye of the SEM.

Techniques

Maceration technique

A very useful and simple technique was introduced to reveal the depth of the specimens by macerating the surrounding connective tissue. After fixing the collected specimens in 2.5% buffered glutaraldehyde, they are incubated in 6N NaOH at 60°C for 25 min. The digestion time is determined according to the nature of the specimen, ranging from 30 min to 24 h. After chemical treatment, specimens are then processed (dried and coated) for SEM examination [3] (Figs 1 and 2).

Figure 1
Figure 1:
A scanning electron micrograph (SEM) of the ampulla of the rabbit fallopian tube after the maceration technique, showing the thin peritoneal covering (P) with underlying outer longitudinal muscle fibers (colored in red) below which plexiform inner muscle bundles are present (arrow head). SEM ×216. Note: the area in the inset is magnified at the right bottom to show the regular longitudinal muscle bundles (arrows). SEM ×863 (adopted from [8]).
Figure 2
Figure 2:
Skeletal muscular tissue treated for 30 min with 6N NaOH at 30°C for removal of the collagen fibers and the basement membrane showing a bundle of motor nerves (N), which end in two motor end plates (E). The terminal portion of the nerve fibers is arborized into anastomosing and twisting end feet. Note; a Schwann cell (S), blood capillary (C), and one of its pericytes (P).SEM ×1650 (adopted from [9]).

Cracking technique

The natural surface of a specimen is only a small part of the aim of SEM observation. By cutting open the specimens, the world of SEM images has been tremendously expanded. We can see the surface structures of cells hidden deeply in the tissue. In addition, advanced fracture techniques have included the interior structures of cells within the range of high-resolution SEM. The simplest, and yet very useful, cracking method is to break the tissue by hands or by the forceps. However, the fracture surface as a whole is quite rough and often contaminated. To create a clean and precise fracture surface, the frozen liquid cracking method is the best. After fixing the specimens, they are immersed in ethanol and then frozen in liquid nitrogen and cracked by a forceps or a chisel. The tissue is then processed for SEM examination [4] (Figs 3–5).

Figure 3
Figure 3:
A frozen-liquid fracture scanning electron micrograph (SEM) of the ampulla of the rabbit fallopian tube, showing the lining epithelium of two mucosal folds (colored in blue) lying on a delicate connective tissue C.T core of the lamina propria (colored in green). Notice the bundles from the inner muscular coat that invaginate into the core of one fold (colored in red). SEM ×617 (adopted from [8]).
Figure 4
Figure 4:
Scanning electron micrograph (SEM) showing the epithelium covering an intestinal villus, revealing both surface and fracture views. Fracture occurred along the cell boundaries showing the prism of absorptive cells. Three goblet cells (arrows) are indicated as SEM ×300. The area in the inset is magnified SEM ×8500 to show the upper half of the goblet cell filled with granules of mucous secretion, whereas the arrow is noting tortuous interdigitations of the microvilli of the absorptive cell (adopted from [1]).
Figure 5
Figure 5:
A frozen-liquid fracture scanning electron micrograph (SEM) of the rabbit ovary, showing the secondary oocyte nucleus (S), covered by the zona pellucida (Z) and surrounded by the corona radiata (CR) cells. SEM ×964 (adopted from [8]).

Resin-corrosion cast technique

It is designed to reveal the three-dimensional structures of the vascular framework of organs after dissolving the tissue and examining the remaining cast. Our knowledge of the microcirculation in different organs is markedly advanced by this method, not only the normal microcirculation but also in different pathological conditions, such as tumors [5] (Fig. 6).

Figure 6
Figure 6

The technique itself begins by washing the feeding vessel of the specimen with heparin to prevent clotting of the blood. The specimen is then washed by saline, followed by injection of glutaraldehyde (fixative) and then injection of resin. Water bathing of the specimen in hot water (60°C for 2 h) is done, followed by several baths of 15% KOH in room temperature for 4–5 days. The specimen is then dehydrated; dried, coated, and finally examined [6].

In addition, the SEM can provide the facility of examining the deep insights of highly specialized cells, tissues, organisms, and insects. One of these is the bacterial biofilm (Fig. 7), which explains the resistance of some bacterial infections to standard treatment [7]. Moreover, the SEM can be used as simple and effective method of teaching, especially in the field of embryology [8].

Figure 7
Figure 7:
Scanning electron micrograph of a bacterial biofilm over the dentine tubules of an extracted tooth (B). Area in the inset is magnified to show the clusters of the cocci (adopted from [7]).

References

1. Fujita T, Tanaka K, Tokunaga J SEM atlas of cells and tissues. 19822nd ed Tokyo Igaku-Shoin
2. Tanaka K, Naguro T. High resolution scanning electron microscopy of cell organelles by a new specimen preparation method. Biomed Res. 1981;2(Suppl):63–70
3. Takahashi Iwanaga H, Fujita T. Application of an NaOH maceration method to a scanning electron microscopic observation of Ito cells in the rat liver. Arch Histol Jpn. 1986;49:349–357
4. Hamano M, Otaka T, Nagatani T, Tanaka T. A frozen liquid cracking method for high resolution scanning electron microscopy. J Electron Microsc. 1973;22:298
5. Walocha JA, Litwin JA, Miodonski AJ. Vascular system of intramural leiomyomata revealed by corrosion casting and scanning electron microscopy. Hum Reprod. 2003;18:1088–1093
6. Sangiorgi S, Manelli A, Congiu T, Bini A, Pilato G, Reguzzoni M, Raspanti M. Microvascularization of the human digit as studied by corrosion casting. J Anat. 2004;204:123–131
7. El Nabawy OM. Detection of microbial bio-film on central venous catheters removed from intensive care unit patients. Department of Clinical Pathology, Faculty of Medicine, Ain Shams University. 2011. MD thesis at the library of Ain Shams University.
8. Shoukry Y. Cyclic changes of the rabbit fallopian tubes and the effect of the unilateral tubal ligation. Department of Anatomy, Faculty of Medicine, Ain Shams University. 2008. MD thesis at the library of Ain Shams University.
9. Uehara, Dezaki J, Fujiwara T. Vascular autonomic plexuses and skeletal neuromuscular junctions: a scanning electron microscopic study. Biomed Res 1981 (Suppl 2):139–143.
    Keywords:

    approach; corrosion cast; maceration; scanning electron microscope

    © 2011 The Egyptian Journal of Histology