Anatomy and Microscopy
Brendan Choat and Steven Jansen
The study of anatomy is concerned with the internal structure of plants. Anatomy initially included the morphology and architecture of plants but these are now considered to be separate branches of study. Because of the importance of internal structure to plant function from a biophysical, biochemical and phylogenetic perspectives, the study of plant anatomy is a central field in plant biology with a long and rich tradition. Most anatomical features are smaller than can be reliably described by the naked eye, which has a resolution of ca. 1/10 mm or 100µm. Therefore the development in the field of plant anatomy is intimately linked with the development of microscopy. Since pioneering work by early 17th century microscopists including Antonie van Leeuwenhoek, Marcello Malpighi, Nehemiah Grew, and Robert Hooke, microscopy has progressed to a point where structures may be viewed at the sub nanometer scale and detailed three dimensional structures of plants can be rapidly mapped.
In the context of plant physiological ecology and environmental sciences we are particularly concerned with how the anatomy of plants relates to plant function; how does variation in xylem anatomy influence a plants ability to survive drought? How does the arrangement of cells in a leaf influence light capture and photosynthesis? In this section we give a brief introduction to the various techniques used in the field of plant anatomy and microscopy, with progressively more detailed protocols linked to each section. Please note that these sections do not contain an extensive description of the microscopes and their components as these are covered in depth elsewhere. The major focus is on the applications of these instruments to plant physiological ecology and environmental studies and protocols used to achieve the best results.
The optical or light microscope was first developed in the 1500 and 1600’s with early use demonstrating the capacity of a simple (one lens microscope) to reveal the fine details of plant internal structure (Figure 1). Modern light microscopes are generally compound (many lenses) and fall into two major categories: the high power microscope and the stereo or dissecting scope (Figure 2). These microscope both have an ocular lens that typically magnifies 5x or 10x and a set of objective lenses. In high power microscopes, objective lenses range from 4x to 100x magnification, while dissecting scopes generally have objectives up to 40x magnification. As such, high power microscopes are generally used to view thin sections using transmitted light. Dissecting scopes are used to view whole mounts or coarser details in sections but because of the configuration of lenses, a three dimensional visualization of the specimen can be produced.
In anatomy, the majority of work is undertaken with high power microscopes, with dissecting scopes being used primarily to reveal fine details of morphology. Images (micrographs) produced by the light microscope are extremely useful in examining the basic structure of tissues and organs and some aspects of the cell. They have been used extensively in studies of plant development, taxonomy, comparative ecology and ecophysiology. Images of specimens are typically collected with a digital camera, which is interfaced with a computer. While there are a large range of digital cameras designed specifically for microscopy, standard digital cameras may also be easily modified to fit onto a microscope. Most modern digital cameras include software that allows for remote capture and real time viewing of the camera image on a computer. Sample micrographs showing transverse sections of xylem tissue and leaf tissue are shown in Figure 3.
It is important for measurements or image analyses that the exact magnification of images is known. Scale bars are visible in some ocular lenses and can be calibrated using a graticule. Most image capturing programs also have the option to burn a scale bar in micrographs.
In addition to the compound microscope or the transmitted light microscope, interference contrast microscopy, epifluorescence microscopy, and polarizing microscopy are useful methods in research of plant structure. Interference contrast microscopes amplify the small differences in refractive index of cellular components in weakly stained and unstained samples, which is especially useful when studying living cells. This method increases the contrast and definition of cytological structures such as vacuoles, nuclei and cytoplamic details. The epifluorescence microscope usually illuminates specimens with light of short wavelength from above with blue to ultraviolet light using excitation filters and an intense light source. In case the specimen is fluorescent, the light of short wavelength is absorbed and re-emitted by the sample as a light of longer wavelength. Many substances in plant tissues are autofluorescent, such as cutin, suberin, lignin, most phenolic compounds, and chlorophyll. Light microscopes equipped with a polarizing filter provide a very sensitive technique for observing calcium oxalate crystals, which rotate the plane of the polarizing light, allowing some of it to pass through a second filter. Crystals typically appear as light bodies on a dark field when using crossed polars.
While the upper limits of optical microscopy is around 2000x magnification, electron microscopes can achieve magnifications up to 1,000,000x and reveal much greater detail, particularly in cellular ultrastructure. There are two types of electron microscopes used commonly in plant anatomical studies: the transmission electron microscope (TEM) and the scanning electron microscope (SEM) (Figure 4).
Transmission Electron Microscope
The TEM can be used to observe thin sections (usually 50-100 nm thickness) of plant tissue at resolutions below 1 nm. The newer generation of high resolution TEMs can produce images with resolution below 0.5 Ångström. Electrons are emitted from an electron gun and focused by an electromagnetic lens so that they are transmitted through the sample or scattered by the parts of the sample that are not transparent to electrons. Transmission electron microscopes have been used extensively to study the ultrastructure and development of plant cells, for instance to detect differences in the structure of the chloroplast or mitochondria (Figure 5). Images are typically collected by a digital camera and recorded on a computer. Sample preparation for TEM is typically laborious, involving fixation, embedding in resin or plastic, sectioning with an ultramicrotome and staining.
Scanning electron microscope
The SEM differs from TEM in that it can produce high resolution three dimensional representation of the sample. Unlike TEM, electrons do not pass through the sample. Instead, a focused electron beam is rastered across the sample. Images are typically created as secondary electron are ejected or by detection of backscattered electrons from the original electron beam. While the SEM produces images at lower resolution than can be obtained with TEM, much larger samples can be examined with a large depth of field due to the very narrow electron beam (Figure 6). Because of this, the SEM is also of great use in examining micromorphological surface features on the leaf, shoot and roots. Samples can be whole mounts or sections through tissue and are usually coated with a very thin coating of metal (gold, gold/palladium alloy, platinum, or carbon). Normally, samples are observed under high vacuum and as such, must be dehydrated before observation. This can lead to the possibility of artefacts developing during preparation, which is a constant cause for concern when the dimensions of an object are important for analysis. To overcome this, samples with delicate structures (e.g., non-lignified cell walls) may be dehydrated using a critical point dryer, which removes the liquid by a process using high pressure liquid carbon dioxide. The liquid CO2 is heated until its pressure goes beyond the critical point. Alternatively, hydrated (fresh) samples can be observed using an environmental SEM (ESEM), although this technique is much more limited in resolving power. Sample may also be examined while frozen using a cold stage (cryoSEM), a technique which is of great value in observing hydrated biological specimens at high resolution.