Light microscopy and staining methods

Histology is the study of tissues and organs using microscopy.

Similar to the phrase “form follows function”, the structures of human tissues are closely related to their functions.

By integrating histology with other disciplines, such as biochemistry, cell biology, and physiology, we can gain a much better understanding of how the body functions.

While there are many types of microscopy, light microscopy or LM is the most common, largely because of how practical it is in both the clinical setting, as well as in research.

Other types of microscopy include scanning probe, ultraviolet, virtual, and electron microscopy or EM.

Light microscopy can be further subdivided based on the specific technique that’s used, such as bright field, immunofluorescence, and dark field microscopy.

We’ll be focusing primarily on bright field microscopy and some of the various staining methods that go along with it.

Bright field microscopy is not only the simplest type of light microscopy but also the type that most people are familiar with, utilizing ordinary light to examine stained tissue at high magnification.

In order to visualize the tissue, it needs to be properly prepared first.

The most common method of tissue preparation has three main steps.

First is to fix, or preserve the tissue using formalin.

The next step is tissue processing, which is the step that removes water from the tissue.

This is done by using ethanol, which replaces the water in all the cells, and xylene, which removes ethanol by dissolving it, making it easier to embed the tissue in paraffin.

Embedding the tissue in paraffin wax is the third step, which removes xylene from the tissue and also solidifies into a “paraffin block” that makes it easier to section, or cut the tissue into very thin slices.

This is done using a machine called a microtome, which most labs use to cut the paraffin block into slices 4 μm in thickness.

Next, the paraffin slice is mounted on a glass slide and the tissue is stained to make it easier to see under the microscope.

The most common stain is the combination of hematoxylin and eosin or H&E for short.

This image of a pancreatic duct was stained with H&E and the image on the right was stained with only hematoxylin.

Hematoxylin is a positively charged basic dye and will stain negatively charged, structures purple or dark blue.

Because of this characteristic, tissue components that strongly stain with basic dyes are often called basophilic structures.

Nuclei, ribosomes, and rough endoplasmic reticulum are all basophilic structures because of the negatively charged DNA and RNA that they contain.