The development of instrumentation for microscopy has advanced enormously in recent years and one of the critical advancements has been the development of low voltage scanning electron microscopy (LVSEM) that allows high-resolution imaging of delicate biological structures with minimal coating requirements. LVSEM provides resolution at levels that previously could only be achieved with transmission electron microscopy (TEM).

In addition to imaging fine details on surfaces of intact biological material, LVSEM also allows imaging of isolated cell structures with remarkable detail. Three-dimensional images can be generated to clearly identify interactions of macromolecular complexes without confusion of structural overlap; specific molecular components can be identified by using colloidal-gold labelling techniques which is particularly useful when molecular components are present only in low quantities which would be difficult to quantitate in sectioned material using conventional TEM or negative staining. Furthermore, the ability to tilt samples and perform three-dimensional stereo analysis of thick material yields spectacular images with more precise information on structural and molecular interactions than possible with TEM.

Historic Perspectives, the Emergence of LVSEM and Advantages over Conventional SEM

Development of scanning electron microscopy began in Germany in the 1930's and was further pursued in the United States. Scanning electron microscopes have evolved as valuable research tools from 1965 to the present time and new developments continue steadily. In 1965 the first SEM, the Stereoscan 1SEM by Cambridge Instrument Company in the United Kingdom, became commercially available followed by JEOL's JSM-1 [reviewed in 1]. Although research to develop the first LVSEM started in 1960, only recently has LVSEM found wider use, perhaps aided by the remarkable advances in specimen preparations that include improved methods for critical point drying, specimen coating, and new methods for sample preparation. Breakthrough developments in LVSEM came with the development of field-emission sources that made it possible to form an intense beam of low voltage electrons with a small diameter.

The low accelerating voltage allowed effective scanning with good contrast, reduced radiation damage, and reduced specimen charging [reviewed in 2]. It has allowed imaging of gold-metal particles in specimen with low contrast [reviewed in excellent detail in 3] and imaging of flat samples with minimal coating (1-2 nm). Presented below are examples of biological material for which detailed imaging could only be achieved by utilising the advantages provided by LVSEM.
Improved specimen preparation has added to the range of applications and imaging details which includes ultra-rapid freezing and freeze-drying to eliminate the need for chemical fixation and reduce some artifacts that may be associated with conventional preparation techniques.
While immunofluorescence and fluorescene microscopy of fixed and live cells has advanced our knowledge in cell and molecular biology, it has limitations and is oftentimes primarily focused on the molecules of interest while cellular interactions are not readily analysed. Correlated microscopy has recently emphasised the use of several microscopy approaches to correlate the data on different levels and with higher resolution. Compared to TEM, the present LVSEM instruments are most suitable for such approaches and allow faster high resolution analysis than TEM.

Specific Biological Applications

Numerous biological applications have benefited greatly from using LVSEM, some of which have recently been presented as book chapters by experts in their respective field who contributed specific expertise [4] and demonstrated the versatility and diversity of applications using LVSEM. The present article will highlight a few selected applications that have been elaborated and pursued by the late Dr. Hans Ris, a pioneer in novel preparation techniques for cell biological applications. Because specimen preparation is one of the critical aspects for producing high-quality results, the following will focus on one particular form of specimen preparation and LVSEM imaging that clearly demonstrates the unique capabilities and data acquisition that have only been possible with LVSEM imaging. The method presented here can be applied to numerous research areas and have been used to generate novel data for cellular structures including nuclear pore complexes, mitotic spindles, the microfilament network in bone cells, the actomyosin organisation in insect muscle, and others [5]. The method relies on visualisation of resin-extracted de-embedded thick sections of biological material as a unique approach to examine the interior of cells and tissue in remarkable detail. The procedure is briefly described as follows. Cells or tissue are embedded as for TEM using Epon as embedding medium. Semi-thick sections of ca. 200 nm are cut, picked up on a copper wire loop and transferred onto coverglass chips. Resin is extracted by using an extraction kit available from Polysciences (Polysciences Inc.), and sections are then prepared for SEM, critical-point dried and coated with a thin layer (ca. 1 nm) of Pt deposited by Argon ion-beam sputtering. The de-embedded critical point-dried sections are then viewed using an FESEM instrument operating at 1.5kV accelerating voltage. Examples of such preparations are presented in figures 1-4 displaying the parasite Toxoplasma gondii shortly after host cell invasion (figs. 1 and 2). In figure 1 we see a parasite within the parasitophorous vacuole (PV) displaying structural interactions with its host cell which would be difficult to capture in thin-section TEM. Figure 2 shows more elaborate structural interactions of the PV with the host cell. These structural interactions are thought to serve nutritional exploitation of host cells by Toxoplasma. The technique was excellently applied by the late Dr. Hans Ris to analyse nuclear pores in Xenopus oocytes which is displayed in figure 3. Figure 4 represents a different view of thoracic flight muscle from Drosophila and shows a de-embedded sample in which the hexagonal arrangement of thick and thin filaments are clearly visible.
While only a few examples are depicted here, this technique can be applied to a variety of other cells or tissues in which structural detail is being studied.