Microscopy (plants)

Categories: Cellular biology; methods and techniques

Light Microscopes

The first microscopes used a beam of light to form an image and were probably invented in the Netherlands, where the devices were used in the manufacture of spectacles and cloth. Dutch microscopist Antoni van Leeuwenhoek (1632-1723) improved upon the cloth merchants’ microscopes and used his version to study small objects from nature, such as single-celled organisms and red blood cells. English scientist Robert Hooke (1635-1703), using his own simple microscope, discovered “cells” in a slice of cork.

The human eye by itself is able to resolve images of about 100 micrometers (0.1 millimeter). This means that two objects (such as lines or dots) less than 100 micrometers apart will appear to blur into one object. The highest resolution available in light microscopes will improve upon the human eye five hundred times, allowing it to distinguish objects that are 0.2 micrometer (200 nanometers) apart. Resolution by a light microscope is limited because the shortest wavelength of visible light itself is about 0.4 micrometer (400 nanometers). This limits the effective magnification of a light microscope to about 2,000 times. At this magnification most bacteria are readily visible, as are a variety of organelles within plant cells, such as vacuoles, nuclei (including chromosomes during cell division), chloroplasts, and mitochondria. Smaller structures, such as ribosomes and microtubules (as well as other components of the cytoskeleton), are not visible using a light microscope.

Electron Microscopes

The electron microscope was invented in 1931 by Ernst Ruska, who won the Nobel Prize in Physics for this effort in 1986. Since the time of its invention, several different types of electron microscope have been developed. Electron microscopy uses a beam of electrons instead of a beam of light to form an image. Light is a form of electromagnetic radiation, a process that transfers energy as a wave without transferring matter. An electromagnetic wave can be visualized in terms of a wave traveling on the surface of a pool of water: The undulating pattern of peaks and troughs constitutes the wave. In the case of electromagnetic radiation, the undulations that constitute the wave occur in electric and magnetic fields that are perpendicular to each other.

Waves and Wavelengths

It is usual to consider light as a wave, or a ray, and to consider electrons as particles. According to quantum physics, however, waves and particles are two aspects of the same phenomenon. Light can be considered as a stream of particles, and a stream of electrons can be considered as a wave. This behavior is usually referred to as wave-particle duality.

One property of a wave is its wavelength. The wavelength of a wave is the distance between adjacent peaks (or adjacent troughs) in the waveform. Wavelength is important because when a wave interacts with matter, any structures that are smaller than one-quarter of the wavelength are “invisible” to the wave. In approximate terms, the smallest object that a wave can be used to image is equal in size to the wavelength of the wave used to form the image. The wavelength of visible light is between approximately 400 nanometers and 700 nanometers (a nanometer is one one-billionth of a meter).

A stream of electrons has a smaller wavelength than a beam of light, and electrons can therefore be used to form images of small objects—such as cells. If distinct images of separate objects are formed, the images are said to be resolved. Because the wavelength of an electron is much smaller than the wavelength of light, the electron microscope can resolve images of objects that are a million times smaller than the objects seen in traditional optical microscopes. The resolving power of a scanning tunneling microscope (STM) is sufficient to render it capable of determining the positions of individual atoms in the surface layer of a material.

There are several different types of electron microscope. The basic types include the transmission electron microscope, the scanning electron microscope, and the scanning tunneling microscope. The specimen in an electron microscope is usually observed in a vacuum in order to prevent scattering of the electrons by air molecules; the need for a vacuum presents the greatest difficulty in the application of electron microscopy to biological systems.

Transmission Electron Microscopes

The simplest electron microscope is the transmission electron microscope (TEM). Electrons are produced by an electron gun and are accelerated by a potential difference (voltage). The electrons from the electron gun pass through a condenser lens and are then used to illuminate the specimen. The electrons which pass through the specimen are then allowed to pass through an electron lens objective. The objective magnifies the image, and then a second electron lens, which plays the role of the eyepiece in the standard microscope, is used to focus the image for observation. The “lenses” used in electron microscopes are not lenses in the usual sense; instead, they are electric and magnetic fields, and they are accordingly referred to as electrostatic or magnetic lenses.

The image can then be formed on a photographic plate or observed on a fluorescent screen, or the electrons can be collected by a charge-sensitive device to produce an image on a cathode-ray tube. Higher magnification can be achieved by using more lenses.

The sample thickness will affect the resolving ability of the TEM. Usually, at least in the case of biological samples, the sample should be no more than ten times thicker than the structures that are to be analyzed. The resolving power of the TEM is such that it can observe structures that are slightly larger than atoms, but since the development of other systems, it has become less used, even though its resolution often exceeds that of the scanning electron microscope. Typical resolutions are in the subnanometer range, with magnifications of up to 500,000 times.

The interpretation of the electron micrographs produced by a TEM is sometimes difficult. The major source of difficulty is that the image is produced by transmitted radiation. The eye is accustomed to interpreting images that are produced by reflection. In the absence of a sample, a TEM beam would saturate a film plate used to record an image. The sample prevents some of the electrons from reaching the film plate; the image produced by a TEM is somewhat similar to a negative produced in a normal camera. Much of the difficulty can be removed by photographing the micrograph and converting it to a positive image—there are, however, some residual interpretation difficulties caused by shadows.

High-Voltage and Scanning Electron Microscopes

The high-voltage electron microscope (HVEM) is a variant of the TEM. The conventional TEM works best on particles less than 0.5 nanometer thick; electrons of higher speed can be produced by increasing the voltage used to accelerate them, and thicker samples can then be analyzed. The wavelength of the electrons decreases as their speed (hence, their kinetic energy) increases. These short-wavelength electrons are less likely to collide with atoms as they pass through the specimen, and they are therefore able to render a sharper image of a thicker sample.

The scanning electron micrscope (SEM) works on a different principle. Electrons are again produced and accelerated by an electron gun, but in an SEM the beam is focused by electron lenses and used to scan a sample. The scanning will result in two different electron beams being emitted by the sample, a primary beam of backscattered electrons produced by reflection and a secondary beam of electrons emitted by the atoms of the sample. By scanning the entire sample and collecting the primary and secondary electrons, the operator can produce an image of a sample on a cathode-ray tube.

Scanning Tunneling Electron Microscopes

The scanning tunneling electron microscope (STM) works on a completely different principle. It was developed by Gerd Binnig and Heinrich Rohrer at an International Business Machines (IBM) research laboratory in the 1980’s. Binnig and Rohrer shared the 1986 Nobel Prize in Physics with Ernst Ruska for their work. The STM uses quantum tunneling. Quantum tunneling is the penetration of a barrier by a particle that, when analyzed by classical physics, does not have enough energy to pass through the barrier. Quantum mechanics predicts that there is a finite probability of a particle passing through a barrier, even if it lacks the energy to pass over it. The number of particles passing through the barrier will vary with the barrier thickness and the particle energy.

This principle is used in the STM by allowing electrons to tunnel across a vacuum, from a stylus to a sample. The quantum tunneling of electrons sets up a “tunneling current” that increases as the vacuum gap between the stylus and the sample decreases. The variation in tunneling current can be used to map the surface of the sample as the stylus moves across its surface. The STM is capable of detecting structures 0.1 nanometer in size in the direction parallel to the motion of the scanning stylus. This means that atoms can be easily detected. The performance in the vertical direction is even more impressive: The STM can detect irregularities on the order of 0.01 nanometer. Thus, the STM, with its higher resolving power than the SEM or TEM, has little difficulty in the imaging of atoms.

Preparing Samples

The principal accommodations that must be made in the examination of biological samples using electron microscopy occur in the preparation of the sample. A commonly used method is the construction of replicas, made by the vacuum deposition of thin layers of carbon, metals, or alloys on the surface of the sample. These films provide a replica of the surface, which can be scanned. Another useful technique, which is the standard technique of sample preparation used in optical microscopy, is the sectioning of samples and their impregnation with stains. The stains that are useful in electron microscopy are usually chemical compounds of heavy metals. These are effective stains because they strongly scatter electrons.

Many methods of sample preparation have been developed to enable electron microscopy to be more widely used on biological samples. Freeze-fracture and freeze-etch are methods of sample preparation that have been widely used. Freeze-fracture involves the freezing and splitting of a water-containing sample. Freeze-etch is a second step, in which ice is allowed to sublime (vaporize without forming a liquid) before the sample is analyzed in an electron microscope. Both techniques are used to examine the internal structure of materials without subjecting them to chemical changes. Freeze-fracture and freeze-etch allow the interior layers of water-containing samples to be investigated without the straining and chemical preparations that are needed to render internal structures visible in a standard light microscope. In particular, these techniques allow the observation of cell walls and cell membranes in a state which is as close as possible to the living state.

Uses

While light microscopy remains important in the identification, location, and observation of cells both singly and in groups, the electron microscope has revolutionized scientists’ understanding of the microscopic world in the biological, medical, and physical sciences. Although it is not possible to observe living materials using electron microscopy, freeze-fracture and freeze-etch have allowed the observation of biological materials in an almost natural state. Correlative microscopy, an increasingly popular method, involves the examination of a sample using both light microscopy and electron microscopy; this method allows the acquisition of a variety of views of the same structure, and it removes the ambiguities that may result from views produced by a single microscope.

The transmission electron microscope (TEM) was the first electron microscope to be developed, and it is the most common type. The TEM has more in common with light microscopes than it does with any other electron microscope. It also shares the chief disadvantage of the optical microscope—that is, it gives little impression of the vertical scale of the specimen under observation. This means that the structures present in the specimen are imaged, but the subtleties of the surface of the specimen are lost. Furthermore, the TEM imposes severe constraints on the type of specimens which can be analyzed. The sample must be thin enough to permit the beam of electrons to pass through it, and it must be resilient enough to resist being damaged by the imaging electrons.

Most biological materials are too thick to be observed under the TEM, so it is necessary to prepare ultra-thin sections of samples prior to their analysis. Both plant and animal samples have been examined using the TEM, and their analysis has led to the discovery of a variety of internal structures. The internal structure of the mitochondria, which had previously been discovered with light microscopy, has been probed. The TEM has also been used to examine the interiors of the nuclei of cells. The examination of the cell nuclei has enabled the investigation of chromosome organization and gene structure. This examination of the microstructure of cells has contributed to the development of molecular biology and genetic engineering.

The TEM has also been used to examine bacteria and viruses. The TEM detected the presence of nucleic acids in bacteria and produced the first images of viruses; most viruses are too small to be resolved in light microscopes. In plants, the TEM has been used in the study of chloroplasts and the walls of cells. The observation of chloroplasts led to the discovery of the internal membranes called thylakoids, which absorb light for the process of photosynthesis. The discovery of the thylakoids has enhanced the understanding of photosynthesis.

The scanning electron microscope (SEM) has been widely used in the biological sciences. Physical laws impose no constraints on the size of the sample to be examined—they are, instead, imposed by the available sample chambers. The SEM is usually used in the magnification range of 10 to 100,000 times. When compared with the light microscope, the main advantage of the SEM is that it is able to produce three-dimensional images. These images are possible because the entire sample can be observed in focus at the same time, and the sample can be observed from a variety of angles.

The SEM has allowed images to be formed of algae, bacteria, spores, molds, and fungi. These images have enabled the structure and function of these samples to be determined. The xylem and phloem cells that transport water through the stems of plants have been examined in the SEM, thus allowing the water transportation process to be better understood. The SEM also has applications in exploring the pathology of cells. Many structures formed by living cells are better understood because sample preparation techniques such as freeze-fracture and freeze-etch have allowed the examination of lifelike samples under the SEM.

Bibliography

Bozzola, John J., and Lonnie D. Russell. Electron Microscopy: Principles and Techniques for Biologists. 2d ed. Boston: Jones and Bartlett, 1999. Covers the theory of scanning and transmission electron microscopes, specimen preparation for both, digital imaging and image analysis, laboratory safety, and interpretation of images and provides an atlas of ultrastructure.

Harris, Robin, ed. Electron Microscopy in Biology: A Practical Approach. New York: IRL Press, 1991. Part of the Practical Approach series; includes bibliographical references and index.

Hey, Tony, and Patrick Walters. The Quantum Universe. New York: Cambridge University Press, 1988. This work, written for the layperson, is devoted to quantum physics. All physical processes necessary for a good understanding of electron microscopy are covered, and there is a section devoted to microscopy. The main ideas are developed pictorially rather than through the use of mathematical equations.

Koehler, James K. Advanced Techniques in Biological Electron Microscopy. 3 vols. New York: Springer-Verlag, 1973-1986. As the title suggests, this is an advanced work, intended for the reader who wishes to learn to use the electron microscope. An invaluable reference for those who wish to know how samples are prepared for use in an electron microscope.

Shipman, James T., Jerry D. Wilson, and Arron W. Todd. An Introduction to Physical Science. 6th ed. Boston: Houghton Mifflin, 2000. For readers who lack an understanding of wave-particle duality and quantum physics. It is an elementary college-level text requiring no mathematical prerequisites. Recommended for those who wish to develop a thorough understanding of the advantages of electron microscopy.

Watt, Ian M. The Principles and Practice of Electron Microscopy. 2d ed. New York: Cambridge University Press, 1997. An easily understood survey of the entire field of electron microscopy. This book is a good resource for those who need more information about the field. Many references for further study are provided.