Introduction to Histology and Virtual Microscopy

OBJECTIVES

From the lecture

1. Understand the processes of preparing and viewing tissues by light and electron microscopy.
2. Understand the physical bases for the appearance of tissues in the light and electron microscopes (e.g. What is basophilia and what causes structures to be basophilic? What creates the contrasting light and dark regions in an electron micrograph?)

From the lab session

3. A brief listing of some common stains is present at the end of this section. You should have a general familiarity with H&E (Hematoxylin and Eosin), Masson, PAS, and elastic stains.
4. Become familiar with the various ways to access and view images in the Michigan virtual slide collection

General And Connective Tissue Stains

Hematoxylin and Eosin
Hematoxylin is the most commonly used nuclear stain in histology and pathology although, despite its long use and honorable history, the chemistry of the dye is still not fully understood. Essentially, hematoxylin is a basic dye and complexes with nucleic acids (DNA and RNA in the nucleus; RNA in the cytoplasm) or other negatively charged molecules (such as sulfate groups). Structures that bind hematoxylin are therefore termed “basophilic” (base loving).

Eosin is an acidic dye and the basic structures it stains are termed “eosinophilic” or less commonly “acidophilic” (acid loving). It stains membranes and most proteins. Cells that have large quantities of folded membranes stain intensely with eosin, because of basic amino acids in the membranes (e.g. macrophages contain lots of membrane in the form of phagocytic vesicles as well as basic lysosomal enzymes within those vesicles that stain with eosin). Collagen is generally stained some shade of red/orange whereas actin (such as in smooth muscle cells) is a bit more pink. Elastin, when present in relatively large amounts (such in the walls of blood vessels, in elastic cartilage, and in the esophagus and trachea), will appear glassy red.

A note about acids/bases and their charges: It always seems to a point of confusion as to how it is that an acid such as DNA can have a negative charge when we generally think of something that is acidic as being positively charged (i.e. a solution with lots of H+ ions is “acidic”). However, the better way to think of acids is as proton donors –in solution, an acid such as DNA donates H+ protons (which makes the solution acidic). Upon donating protons, the DNA therefore becomes negatively charged and it is in this state that it binds hematoxylin.

Masson Triple Stain (or “Trichrome”)
This dye combination stains mucus as well as collagenous and reticular fibers blue (aniline blue) or green (fast green) depending on the mixes of dyes used; muscle red; nuclei red (they are black if preceded by an iron hematoxylin). This is a commonly used connective tissue stain in both histology and pathology. On your slides the stain is designated “Masson” or “Mass”; but the blue or green collagen is the tip-off.

Elastic stains

• Aldehyde fuchsin
  • Aldehyde Fuchsin is a deep purple dye. It stains elastic fibers and granules of beta cells in the islets of Langerhans, cartilage matrix, and stored neurosecretory product in the hypophyseal pars nervosa, among other things. In some of your slides, it is the only stain and therefore only elastin is demonstrated. Other times it is combined with Masson’s trichrome.
• Weigert’s stain
  • Uses a different kind of fuchsin (basic fuchsin), but the result is similar: elastic fibers stain a deep purple color.
• Verhoeff/van Gieson elastic tissue stain
  • Verhoeff’s hematoxylin contains ferric chloride and iodide which causes it to stain elastic fibers deep purple/black. Frequently counterstained van Gieson’s solution with which stains collagen red/orange and cytoskeletal elements (such as actin) yellow-brown.

Silver Stain
In this case silver nitrate is reduced to metallic (black) silver. The process of development and fixation is similar to developing a photograph (stains reticular fibers).

Periodic Acid Schiff (PAS)
This is an extremely useful technique for demonstrating glycoproteins, mucins and some proteoglycans -anything that contains a relatively high amount of sugar groups. It involves the generation of dialdehydes from hexoses (present as the carbohydrate portion of the aforementioned compounds. One of its main uses is the demonstration of basement membranes, especially in the kidney, and/or in sections with epithelia atypia, where breech of the basement membrane is suspected in early carcinomas. An excellent example is slide 210 from the kidney WebScope ImageScope where PAS staining demonstrates the basement membranes (pink lines) of the simple cuboidal epithelium lining the tubules and squamous epithelium in the glomeruli (the round tangles of cells). Note that PAS staining also shows the glycocalyx associated with microvilli (appears as a fuzzy pink border) on epithelia lining some of the tubules.

Click the”Virtual Micrscopy Link” in the menu bar to find more information about virtual slides: Set up to access the virtual slides on campus; Installing Aperio ImageScope; Navigating digital slides using Aperio Imagescope; Using Aperio Imagescope Annotations; Using WebScope to Create “Bookmarks” to Regions of Interest

Getting Started: Using Annotations in ImageScope

One of the main advantages of viewing the virtual slides in ImageScope is the ability to annotate the images in a “layer” that you can then save to your own computer (the original image on the server is not altered in any way).

1. To begin making annotations, go to a slide of interest, in this case we will use slide #29, which may be opened by clicking on this ImageScope .
2. Begin by opening the “Annotations” window (under the “View” menu or by pressing Crtl+N)
3. Go to an area of interest, then select an annotation tool from the toolbar ( see example )
4. Once an annotation tool is selected, the pointer will change to a small “pen” icon. Click and drag to draw the the annotation on the image; finish by releasing the mouse button. If you do not like the placement of the annotation graphic, it may be moved by pressing the Ctrl key and then clicking on the graphic with the mouse. Or you can delete the annotation by clicking on the “X” in the annotation window ( see example ).
5. Corresponding text can be entered in the “Annotations” window to go along with the annotation graphic:
   • in the “Text” column: text entered here will also be displayed on the image
   • in the “Description” column: text here is NOT displayed on the screen
6. After generating additional annotations, you may quickly move from one annotated region to another by clicking on rows within the “Annotations” window. You can also “hide” the annotations by clicking on the “Show/Hide Layers” icon (it looks like an eye).
7. Save the annotation file by Exporting it to your computer’s hard drive. Be sure to use the “Export Annotations To File” icon ( see example ) and name the file accordingly –BE SURE to include the slide number in the filename.
8. Now, CLOSE slide #29. You should get an alert box saying “One or more annotation layers have been modified… Do you want to save the changes?” Select “No”. ( click here for an explanation )
9. Now, we’ll see how to use the annotation files once they’ve been saved to your computer. Re-open slide #29 using this ImageScope . Click on the “Import Annotations To File” button in the “Annotations” window ( see example ) and select the file that you just saved; you should see the annotations that you generated in steps 4 through 6 above.
10. One thing to note is that ANY annotation file can be imported and overlaid on ANY slide –this is why it is important to include the slide number the annotation filename, so you’ll know which file to import for a given slide. To illustrate this point, try opening slide #176 using this ImageScope and then import the slide #29 annotation file that you generated in the steps above. Notice that the annotations are overlaid on the slide, but probably in a manner that doesn’t make sense.