Light microscopy and staining methods




Introduction to histology

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.

The cartilage matrix is also a common basophilic structure as well.

On the other hand, eosin is a negatively charged acidic dye that stains positively charged or eosinophilic structures red or pink.

Some of the commonly eosinophilic structures include collagen, cytoplasmic proteins, and mitochondria.

Mitochondria are especially eosinophilic and stains strongly to eosin, giving cells with a lot of mitochondria, like cardiac muscle cells, a dark pink coloration.

As you might expect, other stains can be used in order to help visualize and examine specific structures that might not be as easily seen with H&E.

The Periodic acid-Schiff or PAS stain is also commonly used.

It stains complex carbohydrates dark red or magenta, making it useful for identifying glycogen in cells such as hepatocytes and muscle cells.

In this image of hepatocytes, the tissue was stained with PAS and also counter-stained with hematoxylin.

This allows us to see the dark magenta glycogen, as well as the nuclei of the hepatocytes, which are stained purple by hematoxylin.

The sugars found within mucus are also stained well by PAS, such as the mucus found lining the stomach, which is intensely stained a dark magenta in this image.

The PAS stain will also stain connective tissue and basement membranes, such as the glomerular basement membrane and the basement membrane of bowman’s capsule found in the kidneys.

This section was taken from the small intestine and stained with masson trichrome.