Conceptual Overview
The development of concepts regarding plant anatomical structure has been inextricably linked to the development of various forms of microscopy. Plant anatomy is typically regarded as the microscopic study of plant tissues and cells, and the development of light and electron microscopes has had a major impact in elucidating our knowledge of structure. It is, therefore, important that we have some idea regarding the development, design and use of these types of instruments.
Light microscopy enables us to resolve detailed structures of about 0.2 µm which is not fine enough to visualize cell membranes and many subcellular units. However, in the range in which it works, and given the use of color and special imaging techniques which are possible, it has become the most valuable tool of the plant anatomist. Realizing that, it is useful to understand some basics of light microscopy starting with image formation.
Many students in elementary or secondary school have designed and built pin-hole cameras. These are very simple devices which have a small circular pin-hole in a piece of foil through which light rays pass and are projected onto the back of a light-tight box where there is a piece of photographic film attached. An image of a well-lighted scene can be projected onto the film through the pin-hole due to the rectilinear propagation of light (that means that light travels in straight lines). The size of image points produced on the film depends on the diameter of the pin-hole and the distance between the hole and the film. The larger the hole, the more blurred is the image. If the hole is too small, brightness and diffraction become limiting factors.
The image produced on the piece of film can be greatly improved by using a glass lens in place of the pin-hole. A lens has the extremely valuable property of refracting light, thus counteracting the principle of the rectilinear propagation of light. It also can be of much larger diameter than a pin-hole, and thereby capable of collecting much more light to make a much shorter exposure time on the film. Now suppose that you are photographing a potted plant at some distance. The light travels from any point on the plant in all directions, but a certain solid angle of the light is intercepted by the lens of the camera, refracted (bent), and recombined at the film plane. Normally, the image on the film plane will be much smaller than the original object, and the distance from the center of the lens to the image is much shorter than the distance from the object to the lens center. Actually, this sets up a consistent ratio in which we can say that A/a = B/b where A is the height of the object (the potted plant), and where a is the distance to the lens from the object. B is the height of the image on the film plane, and b is the distance from the center of the lens to the film plane. Thus, the ratio of A to a is always equal to the ratio of B to b when the image is in focus.
With this concept in mind, it is now possible to consider how a microscope may function. Naturally, a microscope magnifies; therefore, the camera which gives reduced image sizes is not a suitable basis for demonstrating how a microscope functions. Instead, a better example might be the familiar slide projector. Since a slide does not emit light, it must be illuminated from behind. Therefore, a lamp is placed behind it. However, the slide is much larger than the lamp filament and will not be fully and uniformly illuminated unless a lens (called a condenser lens) is inserted between the light source and the slide. Such a lens (usually plano-convex, i.e. flat on the side towards the slide) will project the light source efficiently and uniformly through the transparent slide. These rays, which are nearly parallel, are then cast into a projection lens which inverts the image, and projects it a long distance away to a viewing screen. The ratio of A/a = B/b is the same as in the camera described above, except that the object (slide) is small, and the image is large. Likewise, the distance from the slide to the projection lens is small, but the distance to the screen from the lens is large.
Now let us imagine that we can replace the projection screen with a semi-transparent ground-glass plate, and use a magnifying lens to see more detail on the surface of the plate. If we stand in front of the ground-glass plate we will block the light and not see the image. But if we move to the opposite side we do not interfere with the light projection, and we can focus our magnifying lens onto the ground glass to see more detail. Not only that, but the magnifier can increase the angle of view for more convenient observation.
This now, is essentially the design of a light microscope. There is a light source that projects rays through a condenser lens which in turn casts the light through the specimen slide and into a projector lens (in a microscope we would call this the objective lens). The objective lens focuses and casts an inverted image in a plane where an aerial image exists. The aerial image is real and can be visualized if a screen (or even a piece of paper) is positioned at that point, but the magnifying lens (or ocular lens in a light microscope) can pick up the aerial image, magnify a part of it and cast that image onto a film surface, or the retina of your eye. Think carefully about this analogy and consider the basic optical design of a light microscope.
Imagine again observing the image of a projected slide on a screen from far behind the projector. The magnification observed is low because the angle of view is quite small. As you move closer to the screen the magnification increases and you begin to recognize details which originally escaped your attention. But finally a point is reached where higher magnification (moving closer) does not yield better results, but just a very grainy image. Thus, increasing magnification beyond a certain point gives no new detail, and can be called empty magnification. In a light microscope, the same situation exists. The aerial image from the projector is equivalent to the image formed by the microscope objective lens. Thus, there is a limit for the enlargement of the projected aerial image where useful magnification ends and empty magnification begins.
The early designers of the transmission electron microscope used the same optical principles as used in light microscopy. However, electrons will not penetrate through any significant mass, including air, and therefore must be projected within a vacuum. This also means that glass lenses cannot be employed. Instead, hollow electromagnetic lenses are used in the electron microscope since electrons, being charged particles, can be refracted by conical magnetic fields in much the same way as glass lenses influence the pathway of light photons. In a transmission electron microscope, the design is similar to that of an inverted light microscope with the illumination source (an electron gun assembly) at the top of an optical column. The emitted and accelerated electrons are projected by one or two condenser lenses through a very thin specimen (remember that electrons cannot penetrate through very thick objects), and into an objective lens where focusing of the electron beam takes place and projection of a real image some distance away occurs. Then, one or more projector lenses cast a final magnified image onto a sheet of film, electronic camera, or a viewing screen. When electrons strike objects they give up their energy by generating X-rays that can be damaging to the operator. Thus, the operator must observe images through a thick leaded-glass window which also separates the vacuum of the column from the room outside of the microscope. Electrons cannot be directly visualized, so they are only observed on film, or on viewing screens (within the vacuum) that are coated with a phosphorescent paint that glows when excited by the energy of the electrons.
The scanning electron microscope has many features similar to the transmission electron microscope up to a point. To a degree it may be thought of as the "top half" of a transmission electron microscope column. In essence, the electron beam is reduced in size by one or more condenser lenses, and then it is moved in a raster pattern, across a solid specimen surface in a series of lines, each of which is composed of many image points. The electron beam dwells for a very short time at each image point (e.g. a millionth of a second or less), during which time it excites outer shell electrons out of the specimen (called secondary electrons). These are low-energy electrons (typically <50 electron volts of energy), and are therefore capable of being attracted to a positively charged (usually ~+250 eV) detector, where they create an electrical signal that is proportional to their numbers from any given site on the specimen. The strength of this signal regulates the intensity (brightness) of a corresponding electron beam in a cathode-ray tube (a television or monitor-like screen). Thus, the amount of electrons emitted from the surface of a specimen at any one point determines the intensity of the signal on the electronic viewing screen. Both electron beams move in synchrony. However, the ratio of the length of the electron beam scan across the specimen, to the scan of the one across the viewing screen determines image magnification. The smaller the sweep across the specimen, the greater the displayed magnification. Thus, this instrument is more like an image mapping system compared to the optical projection of the transmission electron microscope.
In summary, the transmission electron microscope, like the light microscope, projects an optical image on a viewing or recording plane from a thin specimen. On the other hand, the scanning electron microscope generates an image map of the surface of a specimen. It can readily be calculated that the limit of resolution of the light microscope is approximately 0.2 µm, that of the transmission electron microscope is approximately 0.1 nm, and that of the scanning electron microscope is approximately 1.0 nm. Thus, based on these resolution potentials, the highest useful magnification of the transmission electron microscope is about 500× greater than the light microscope and 10× greater than that of the scanning electron microscope.
