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.
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. This stain is used to visualize supporting tissue better, particularly collagen.
With Masson trichrome, connective tissue will stain blue, as we can see clearly in the submucosa of the small intestine.
If we zoom in closer, we can see that nuclei stain dark red or purple, and cytoplasm will stain red or pink. Keratin, erythrocytes, and muscles will also stain red or pink as well.
At this magnification, we can also see the lamina propria in blue, which is the connective tissue that supports the epithelium.Alcian blue stains acidic polysaccharides blue, such as the polysaccharides found in cartilage and certain types of mucin.
It’s often used along with other stains such as H&E or van Gieson. The mucin contained within goblet cells can be easily seen in this image of the nasal mucosa, although the stain appears almost green or teal because van Gieson was also used to stain the tissue, making it easier to see the tissue surrounding the goblet cells.
Van Gieson is often used to stain blood vessels and the skin. This stain in particular can be useful to visualize an abnormal increase in collagen or to help differentiate smooth muscle from collagen within a tissue sample.
It stains collagen pink; elastic fibers black; nuclei blue; and erythrocytes, muscle, cytoplasm, and fibrin yellow.Giemsa stain is a standard stain used for blood and bone marrow smears.
Erythrocytes will appear pale pink; cytoplasm a pale blue; and nuclei a dark blue or violet color. Sudan black and osmium are dyes that will stain lipid-containing structures a brown or black color, such as the myelin around nerves and adipocytes.
Also, this stain can be useful in diagnosing metabolic diseases that result in increased intracellular cholesterol, phospholipids, or glycolipids.Clinically, frozen sections are used for much faster results, when needed.
For example, during surgery, if the surgeon is removing a tumor, the surgeon may need to make sure all the unhealthy tissue is removed by having tissue samples analyzed quickly by a pathologist before completing the surgery.
To do this, the margin of the surgically resected mass is sent to the pathology lab, where the mass is embedded in a compound called optimal cutting temperature compound or OCT for short.
It’s then rapidly frozen in liquid nitrogen. This will quickly preserve and harden the tissue so that it can be sectioned in a machine called a cryostat, which is a type of microtome where the tissue can be maintained at a subfreezing temperature.
The slides are most commonly stained with H&E, methylene blue, and/or PAS stains. This method is used primarily due to its fast turn-around time, which can be as fast as 10 minutes if performed by an experienced pathologist.
Frozen sections are usually only used for urgent analysis, because the quality of slides produced from frozen sections are not as good as the typical formalin-fixed and paraffin-embedded tissue, which can also be called permanent sections.
The permanent sections can take about 12 hrs to 2.5 days to complete, depending on the tissue size and staining method used.
These images of necrotic fibroadipose tissue with fungal organisms show an example of the potential difference in image quality between a frozen section and a corresponding permanent section, both stained with H&E.
##SummaryAlright, as a quick recap. The most common form of light microscopy is bright field microscopy, which most commonly utilizes H&E stains.
Hematoxylin will stain nuclei, ribosomes, rough endoplasmic reticulum, and cartilage matrix a purple or dark blue color.
If urgent analysis of a tissue is required, frozen sections can be used in order to provide results much faster. Other stains can be used along with H and E in order to help visualize and examine specific structures that might not be as easily seen with H and E alone If urgent analysis of a tissue is required frozen sections can be used in order to provide
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