2 Using a light microscope
The remainder of this week focuses on the use of a light microscope for histological purposes. You will have plenty of opportunity to put your learning into practice by using the virtual microscope tool to explore some real samples, and later on to take measurements from them.
Histology departments use light microscopes with transmitted light. Microscopes come in many different types, each of which has distinct functions and applications. However, in a routine histology department, virtually all of the work is done using a light microscope with transmitted light. This means that the light passes through the section to the objective lens, as shown in Figure 9.
Here, light from the lamp is focused on the specimen by the substage condenser. The objective collects light from the specimen and focuses it to form an image within the barrel of the microscope. The eyepiece allows this image to be viewed as if it were a projection in the plane of the section.
It is important to understand some of the limitations of a light microscope.
First, the absolute limit of resolution of a light microscope is determined by the wavelength of light being used (λ) and the properties of the optical components of the microscope in question (). The limit of resolution is defined as the smallest distance () at which it is possible to distinguish two separate items.
Optics tells us that the relationship between these properties is:
= λ /(2 x )
Using the best optical components the value of is approximately equal to 1. Consequently, the resolving power of a microscope is approximately equal to of the wavelength of light passing through the sample.
So for example, green light has a wavelength of 550 nm. This means that structures such as the nucleus (~5000 nm), mitochondria (~1000 nm) and large vesicles (>200 nm) can be resolved by a light microscope. Smaller structures, however, cannot be resolved.
Another limitation of a light microscope is the degree of magnification. No matter how the optical system is configured, increasing the overall magnification beyond 1000x cannot resolve further detail of the cell structure. For this reason, the highest objective found on light microscopes is typically 100x. When used in association with a 10x eyepiece, it gives the maximum of 1000x magnification.
Finally, be aware that the quality and thickness of the section affects what can be resolved. In general, thicker sections allow lower resolution than thin sections. The sections produced for diagnostic pathology are generally about one cell thick, which allows a clear view of the structure of a single cell.