The preparation of a histologic slide requires a series of steps beginning with the collection of the specimen and ending with the placement of the coverslip. During each step, an artifact (an error in the preparation process) may be introduced. In general, artifacts that appear on the finished glass slide are linked to methodology, equipment, or reagents used during preparation. The inferior purity of chemicals and reagents used in the process (fixatives, reagents, and stains), imperfections in the execution of the methodology (too short or too long intervals of fixation, dehydration, embedding, staining, or careless mounting and placement of the coverslip), or improper equipment (e.g., a microtome with a
FIGURE 1.9 • Diagram comparing the optical paths in different types of microscopes. For better comparison between all three types of microscopes, the light microscope (left) is shown as if it were turned upside down; the TEM (middle); and the SEM (right). Note that in both the TEM and the SEM, specimens need to be inserted into the high vacuum (10 4 to 10 7 Pa) environment.
image in electron eye detector with CCD camera defective blade) can produce artifacts in the final preparation. It is important for students to recognize that not every slide in their slide collection is perfect and that they should be familiar with the most common artifacts found on their slides.
Besides bright-field microscopy, which is commonly used for routine examination of histologic slides, other optical systems (described below) are used in clinical and research laboratories. Some of them are used to enhance the contrast without staining (such as phase contrast microscope), whereas others are designed to visualize structures using specific techniques such as immunofluorescence (fluorescence and confocal microscopes).
The phase contrast microscope enables examination of unstained cells and tissues and is especially useful for living cells.
The phase contrast microscope takes advantage of small differences in the refractive index in different parts of a cell or tissue sample. Light passing through areas of relatively high refractive index (denser areas) is deflected and becomes out of phase with the rest of the beam of light that has passed through the specimen. The phase contrast microscope adds other induced, out-of-phase wavelengths through a series of optical rings in the condenser and objective lenses, essentially abolishing the amplitude of the initially deflected portion of the beam and producing contrast in the image. Dark portions of the image correspond to dense portions of the specimen; light portions of the image correspond to less dense portions of the specimen. The phase contrast microscope is therefore used to examine living cells and tissues (such as cells in tissue culture) and is used extensively to examine unstained semithin (approximately 0.5-m) sections of plastic-embedded tissue.
Two modifications of the phase contrast microscope are the interference microscope, which also allows quantification of tissue mass, and the differential interference microscope (using Nomarski optics), which is especially useful for assessing surface properties of cells and other biologic objects.
In dark-field microscopy, no direct light from the light source is gathered by the objective lens.
In dark-field microscopy, only light that has been scattered or diffracted by structures in the specimen reaches the objective. The dark-field microscope is equipped with a special condenser that illuminates the specimen with strong, oblique
FIGURE 1.10 • Example of sections from an orange and a kidney renal corpuscle. The dotted lines drawn on the intact orange indicate the plane of section that correlates with each cut surface. Similarly, different sections through a kidney renal corpuscle, which is also a spherical structure, show differences in appearance. The size and internal structural appearance are reflected in the plane of section.
light. Thus, the field of view appears as a dark background on which small particles in the specimen that reflect some light into the objective appear bright.
The effect is similar to that of dust particles seen in the light beam emanating from a slide projector in a darkened room. The light reflected off the dust particles reaches the retina of the eye, thus making the particles visible.