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Home > Nanotechnology Columns > FEI > New Developments in Electron Microscopy Enable Key Trends in Nanobiological Research

Matt Harris
Vice President, Worldwide Marketing & Business Development
FEI Company

Abstract:
Microscopes have long been among the most important tools in the biologist's kit, starting with Leeuwenhoek's observations of single-celled "animicules," and continuing to the present day with instruments capable of resolving the shapes of individual proteins and other biological macromolecules. Recent developments in electron and ion microscopy are revolutionizing many areas of biological investigation. For simplicity, we can organize most of these developments into three categories that reflect major trends in biological sciences at the nanoscale: the continuing drive to transform the scale of our understanding from individual cells to individual molecules, the need to move from two-dimensional descriptive imaging to three- (and four) dimensional structural and mechanistic modeling, and the requirement for fast and efficient analysis of large sample sets and huge volumes of data that comprehend the vast complexity of biological systems.

April 24th, 2007

New Developments in Electron Microscopy Enable Key Trends in Nanobiological Research

Microscopes have long been among the most important tools in the biologist's kit, starting with Leeuwenhoek's observations of single-celled "animicules," and continuing to the present day with instruments capable of resolving the shapes of individual proteins and other biological macromolecules. Recent developments in electron and ion microscopy are revolutionizing many areas of biological investigation. For simplicity, we can organize most of these developments into three categories that reflect major trends in biological sciences at the nanoscale: the continuing drive to transform the scale of our understanding from individual cells to individual molecules, the need to move from two-dimensional descriptive imaging to three- (and four) dimensional structural and mechanistic modeling, and the requirement for fast and efficient analysis of large sample sets and huge volumes of data that comprehend the vast complexity of biological systems.

Cellular to Molecular
Prior to the widespread commercial availability of electron microscopes in the last fifty years, microbiological investigations were limited primarily by the resolution of optical microscopes, roughly a micrometer or so. Electron microscopes pushed the resolution limit down to the nanometer range, but their applicability to biological systems was limited by other factors, such as the fragility of biological specimens in the microscope environment and the low image contrast offered by many biological materials. In response, microscopists developed sophisticated fixing and staining techniques, but these introduced questions about the fidelity of the observations and added significant overhead for sample preparation. Still, biologists made significant advances in understanding biological systems on a smaller and smaller scale.

Certainly one of the most important discoveries of the period (though unrelated to electron microscopy) was the molecular basis for the transmission of genetic information, and the resulting perspective of biological systems as complicated molecular machines. The recent decoding of the genomes of humans and many other organisms is now fueling an explosion of interest in molecular biology as scientists seek to understand how the products of gene transcription function to create life. These developments in molecular biology have been paralleled by equally dramatic improvements in electron microscopy. Aberration-corrected TEMs are now available with sub-Ångstrom image resolution, capable of resolving individual atoms. In biological applications, aberration correction permits the use of lower beam voltages that reduce damage and improve contrast, without sacrificing resolution. Equally important developments have occurred in SEMs. For instance, ESEM (environmental scanning electron microscope) technology permits the imaging of wet, non-conductive samples with little or no preparation, and DualBeam instruments that combine FIB cross sectioning with SEM imaging can be used to reveal subsurface structure.

Two to Three Dimensional (and Four Dimensional) Imaging
The functional mechanisms of biological machines are difficult to discern from flat two-dimensional images. Advanced techniques in electron microscopy can now provide three-dimensional representations of complex biological structures. At the molecular scale, TEM-based electron tomography (ET) can resolve the tertiary and quaternary structure of protein molecules and complexes, and other biological macromolecules. Electron tomography often complements the atomic scale information provided by NMR and XRD. In other cases, it provides information about molecules that cannot be analyzed with NMR or XRD because they are too large or cannot be crystallized. Because electron tomography looks at only one molecule at a time, it can analyze changes in shape over time—the fourth dimension. By comparing the shapes of different molecules having the same composition, it can determine the range of motion of flexible proteins or changes in conformation that occur as proteins bind and interact. Another approach to three dimensional analysis, known as single particle analysis, correlates individual images of a large number identical but randomly oriented particles, such as viruses, to derive detailed structural information. At a larger scale, researchers are using DualBeam instruments to analyze intra- and intercellular structure with a technique known as Slice & View. Using an iterative procedure, the SEM images the sample surface and the FIB then removes a layer to reveal a new surface. Computer software then combines the stack of images into a detailed three-dimensional model of the sampled volume.

High Volume Analysis
The sheer volume of information required to completely describe complex biological systems is mind boggling. Capturing that complexity requires the ability to analyze large sample sets and even larger data sets. Advanced automation capabilities address a number of issues related to high volume analysis on at least three levels. At the most basic level, careful attention to mechanical, electronic, thermal, and environmental stability, combined with fully digital control of all operational parameters, eliminates most of the setup and constant adjustment required by previous generations of instruments. This is important not only in shortening the time required to obtain first result, but also in maintaining repeatability among results acquired over an extended period of time. At the next level, automation efficiently handles many aspects of data capture, management and reconstruction, for instance, the unattended acquisition of a tomographic tilt sequence, or a series of images for single particle analysis, or the execution of a Slice & View procedure. Finally, automation can reduce the overhead associated with sample handling and preparation. For example, consider the task of mapping neuronal pathways in a block of tissue. A microliter of tissue may contain well over one kilometer of pathways. Mapping these pathways requires careful cross sectioning of the tissue block, followed by imaging, and then correlation of features through the image sequence. Systems that can automate the entire procedure will soon be available.

The potential benefits that will accrue from our growing understanding of nanobiology are likely to exceed even our wildest imagination. It is conceivable that we will one day have a complete and detailed description of all biological systems at the molecular level. Much closer are molecular descriptions of targeted disease processes that can serve as the basis for rational drug design. Device manufacturers are investigating nanoengineered materials that will improve the performance of implanted devices and prosthetics. Pharmaceutical researchers are looking at highly selective drug delivery vehicles, perhaps functionalized nanocapsules that will deliver a therapeutic agent to cancer cells identified by the presence of a specific protein in the cell membrane. Whatever the benefits, electron microscopy will certainly play a key role.

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