Part I. Set up to access the virtual slides on campus

STEP 1: Plug in your computer (most laptops run faster when plugged in).

STEP 2: Disable your wireless connection.

• If you are unsure how to do this, ask for help from the LRC staff (or one of your colleagues).

STEP 3 (Windows users only): Create a mapped drive to the virtual slide file server for "on-campus" access if you have not already done so.

• Click here to download the "HistoDriveMap" batch file (click on the link and save the file to your desktop). Double click on the file on your desktop to create the mapped drive. YOU SHOULD RUN THE "HistoDriveMap" FILE EACH TIME YOU RESTART YOUR COMPUTER AND CONNECT TO THE CAMPUS NETWORK!

STEP 4 (Windows only): Confirm drive mapping.

• If you look in "My Computer," you should see an additional drive called "Images on 'ONStor EverON(172.20.57.42)’(Z:)"

STEP 5 (Windows Only): Confirm and optimize installation of ImageScope v9.

• Launch ImageScope from Start->Programs->ScanScope->ImageScope. The upper left-hand corner of the window should say "Aperio ImageScope v.9.1.19.1569"



• If you haven't installed the latest version of ImageScope v9, click here to download the ImageScope installer (save it to your desktop). After downloading, double-click on the file (ImageScopeInstaller.exe) which should launch and install it automatically.



• If you have a previous version of ImageScope installed, the installer may ask if you want to Modify, Repair, or Remove the previous version. Select "Repair," (see example) then click "Next."

To optimize performance of ImageScope, there are some adjustments to make to the default settings:

• Go to the "Tools" menu, then "Options". In the Options window, click on the "Performance" tab and UNCHECK the "Prefetching" option (see example).

PART II. The Basics

A. Accessing the Digital Slides

There are currently 3 different ways to access the slides, so you will see 3 different links for each digital slide in the online lab modules:

1. [WinLab]: use this link when ON CAMPUS (requires a Windows machine with ImageScope installed AND a mapped network drive to the high-speed file server)



2. [WinHome]: use this link when OFF CAMPUS using a Windows machine with ImageScope



3. [Mac]: this link will work on ANY machine ON or OFF CAMPUS, but it is really intended for those who are unable to run ImageScope (i.e. non-Intel Macs)

When in the lab, using the [WinLab] links is PREFERRED as this will ALWAYS deliver the images at the highest speed.

To help you remember, you will find the following table at the beginning of each lab module:

B. Viewing slides on the web server using [Mac] links.

• Click on this [Mac] link here to open slide 29 from the digital slide collection. A new window should open containing the digital slide. You can drag and re-size this window to your liking. Note that pop-up blockers (such as Google) may require you to allow pop-ups from this site. Take some time to use some of the basic features of the viewer such as panning and zooming.

C. Viewing slides on the local file server using [WinLab] links.

• Click on this [WinLab] link to open slide #29 in ImageScope. This link points to a small file (a .sis file) that should launch ImageScope and open the slide. If you are doing this for the first time, Internet Explorer may ask "Do you want to open or save this file?" You should uncheck the box that says, "Always ask before opening this type of file," then select Open.

• Once slide 29 has been opened in Imagescope, you may drag and re-size the window to your liking. Take some time to use some of the basic features of the viewer such as:
  
  
  1. panning and zooming
  2. filmstrip view (under the "View" menu)
  3. thumbnail view
  4. magnifier view
     

D. Viewing slides on the web server using [WinHome] links.

• Click on this [WinHome] link to open slide #176 in ImageScope. [WinHome] links are to be used when viewing this site from OFF CAMPUS or if you have any difficulty using the [WinLab] links. (The [WinHome] links will work on campus, but the performance will likely be slower.)



• Notice that your filmstrip now contains slide #29 (029-1_HISTO_40X.svs) and slide #176 (176_HISTO_20X.svs). You may switch between the two by clicking on the thumbnails in the filmstrip. You can also "tile" the two slides for side-by-side comparisons (under the "Window" menu).
  

Part III. 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 you should have open already. If not, click on this [WinLab] link (if your computer cannot connect to the file server due to drive-mapping issues, you may use this [WinHome] link instead).



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 to placement of the annotation graphic, it may be moved by pressing the Ctrl key and then clicking on the graphic with the mouse.



5. Entering text 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 (e.g. 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".



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 [WinLab] link or this [WinHome] link. 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.

Objectives:

1. Be able to classify epithelial tissues.
2. Know the structure and function of junctions.
3. Know the structure of apical specializations and their functions.
4. Be able to correlate different types of epithelia to their functions.

In epithelia, cells are organized in sheets, either a single layer thick (simple epithelia) or made up of multiple layers (stratified epithelia). Be able to identify the classes of epithelia underlined in the text below, and give some thought to why these different classes of epithelia have such different morphologies.

The glass slide sets occasionally contain different stains or even different slides of the same tissue in place of the slide that you have in your set. Certain stains are much more instructive than others and different (alternate) tissues often help to explain functional changes. When these differences occur, the lab guide will usually refer to the particular slides as being in either "even" or "odd"-numbered slide collections. If you are studying from the glass slide material try to look at the alternates by borrowing from your lab mates.

I. SIMPLE EPITHELIA

A. Simple columnar epithelium (W pg. 84, 5.3; pgs. 277-8, 14.20, 14.22; pg. 283, 14.29)
slide 29 (small intestine) [LINK] --please try out this test link first, if it fails to work use the backup links below:
slide 29 (back-up links) [WinLab] [Mac] [WinHome]
slide 176 (colon) [WinLab] [Mac] [WinHome]

Remember that epithelia line or cover surfaces.  In slide 29 and slide 176, this type of epithelium lines the luminal (mucosal) surface of the small and large intestines, respectively.  Refer to the diagram at the end of this chapter for the tissue orientation and consult the atlas (W pg 277 14.20; pg 278, 14.22) for the cell types that make up the epithelium.  First, examine slide 29 at low power and note finger-like projections of intestinal villi. Also, note that, in some areas, these villi are transversely sectioned and appear instead as circular profiles. The villi are lined by a simple epithelium, and, therefore, you should ideally see a single row of dark nuclei toward the base of the cells as, in W pg. 84, 5.3 (you may see more if the epithelium is cut tangentially).  Also, it is a columnar epithelium, so the cells should be taller than they are wide.  Look around on the slide until you find a region of epithelium having this appearance.

Note the presence of goblet cells (W pg 278, 14.22), which look like balloons suspended in the epithelium. These cells secrete mucus; the clear "balloon" in the apical region of each goblet cell is where the stored mucus is located (mucus does not react with H&E stain). Look at the columnar epithelium in slide 176 and note that it contains a very large population of goblet cells relative to that found in slide 29. In the colon, villi are absent and, instead, a simple columnar epithelium forms the intestinal "glands," which are invaginations made up of mostly goblet cells (W pg 283, 14.29).

Identify the microvillous border at the apex of the epithelial layer at 40x magnification in slide 29. This region appears as a darker staining line at the top of the cells. In a very good section, it may appear striped, or "striated", because it is made up of finger-like projections called microvilli (seen at the electron microscopic level in EM #128, also in W pg 280, 14.25 a&b).

B. Simple cuboidal epithelium (W pg. 84, 5.2; pg. 321, 16.21a)
slide 9 (digital slide 9N-1, kidney, H&E) [WinLab] [Mac] [WinHome]
slide 210 (kidney, H&E) [WinLab] [Mac] [WinHome]

These slides show simple cuboidal epithelium, lining tubules in the kidney. The tubules are cut in all different orientations; look for a region toward the middle of the slide where the tubules are cut more or less in longitudinal section in slide 9 or slide 210, and appear as parallel wavy rows (at 4x magnification). Look for a favorable area where you can see a space (the tubule lumen) lined on either side with simple cuboidal epithelium. Note also that there is very little other tissue between tubules, so that you often see two rows of cuboidal epithelia from adjacent tubules back to back. In other parts of the section, look for tubules in cross-section in slide 9 or slide 210 where the lumen will be surrounded by a circle of cells.

C. Simple squamous epithelium (W pg 83, 5.1)
slide 30 (mesentery, H&E) [WinLab] [Mac] [WinHome]

Simple squamous epithelial cells are flattened, i.e., wider than they are tall. A simple squamous epithelium, called "endothelium," lines blood vessels, lymphatic vessels, and the chambers of the heart.  When sections through endothelial cells are viewed with the light microscope, the cytoplasm cannot be seen, because the flattened cell is so thin.  Thus, endothelium is generally identified on the basis of the structure and position of nuclei alone; that is, the nuclei are also often flattened and elongated, and are found lining the lumen of the vessel (W pg 83, 5.1). Observe the endothelial lining of blood vessels in the mesentery. Sometimes the blood vessels contain red blood cells and can be identified that way. Otherwise, look for tubular or circular profiles at low power and examine the endothelial lining of these vessels at high power. Note that the endothelium may be damaged during processing such that it separates from the vessel wall or it may slough off entirely and not be visible at all. In areas where you can find an endothelium, note that the nuclei do not always look flattened in vessels that have contracted. Now, move to the periphery of the tissue section and observe a simple squamous epithelium (flattened cells) covering the surface of the mesentery. This epithelium --also called mesothelium-- and the irregular connective tissue immediately underneath together make up the peritoneum that lines body cavities and most of the viscera contained therein. The portion applied against the walls of the cavities is known as parietal peritoneum whereas the portion covering the organs (and their associated mesenteries) is the visceral peritoneum.

II. STRATIFIED EPITHELIA

A. Stratified squamous epithelium (W pg 86, 5.6)

This type of epithelium covers surfaces that are subjected to abrasion.  The epithelium is constantly replacing itself by division of the basal layer of cells.  These cells change morphology as they move toward the surface and are ultimately sloughed off.  They are called "stratified" because there are multiple cell layers, and "squamous" because the outermost layer of cells is flattened.  There are two subclasses:

1. Stratified squamous nonkeratinizing epithelium (W, pg 86 5.6a/b; pg 267 14.5; pg 377 19.26)
slide 153 (esophagus, H&E) [WinLab] [Mac] [WinHome]
slide 250 (vagina, H&E)[WinLab] [Mac] [WinHome]

This type of epithelium covers some internal surfaces that are kept moist by mucus or other fluids.  Thus, these epithelia do not need to keratinize to avoid desiccation.  The lubrication provided by mucus helps to protect against abrasion.  Study this type of epithelium in the esophagus (slide 153) and vagina (slide 250). Again, cell morphology changes from base to apex of the epithelium, the outermost being "squamous" in appearance whereas the basal cells appear more cuboidal or low-columnar.   The orientation of the tissue can be confusing because of connective tissue projections that push up into the epithelium.   The importance of looking at this tissue sectioned in the proper plane is discussed in the Introduction lesson of the MMH CD. Unlike keratinizing epithelium, nuclei are still present in most surface cells (although they are often difficult to see in sectioned tissue.)

2. Stratified squamous keratinizing epithelium (W pg 86, 5.6c; pg 169, 9.1; pg 169-170, 9.2, 9.3)
slide 106 (plantar skin, H&E) [WinLab] [Mac] [WinHome]
slide 112 (plantar skin, H&E [WinLab] [Mac] [WinHome]

This epithelium is found at the surface of the skin and is known as the epidermis. As protection against desiccation, it undergoes a process known as cornification or keratinization. As cells move toward the surface, they differentiate and eventually die, leaving an outermost layer of dead cells filled with keratin. The absence of nuclei in this layer shows that it is devoid of live cells. In some slides, the keratinized region is gray, but occasionally it has been penetrated in places by red stain. Note the differences in morphology of the cells as they move toward the surface. You will learn the names of these layers when we study the skin. Toward the middle of the epithelium, try to detect a spiny appearance of cells (this looks like little lines between cells, and can be difficult to see); these are sites where desmosomes attach the cells to one another (W pg 170, 9.3).

B. Pseudostratified epithelium (W pg 85 5.5; pg 238-9, 12.7-9)
slide 20 (trachea, H&E) [WinLab] [Mac] [WinHome]
slide 40 (trachea, H&E) [WinLab] [Mac] [Winhome-v]

"Pseudostratified" is a term applied to epithelia which appear to be stratified (i.e., have nuclei at various levels), but in which all component cells reach the base of the epithelium and are attached to the underlying basement membrane.  The basement membrane l ooks like a pink line at the base of the epithelium, which is rather easily seen in places on this slide. The basement membrane is not always this thick in other epithelia (Note that "basal lamina" is a term that refers to an ultrastructural feature while "basement membrane" refers to a light microscopic feature.  Ultrastructure refers to structures seen at the electron microscope level.  See the MMH epithelial lesson on the basement membrane.)  Only some of these cells reach the free surface of the epithelium, where it is generally ciliated and contains goblet cells.  Since this type of epithelium lines the respiratory tract, it is referred to as the “respiratory epithelium”.  The cilia appear as hairlike projections at the cell apex. What substructures form the core of each cilium? (EP1) There appears to be a dotted line at the base of the cilia; this is due to the presence of basal bodies in this region.

C. Transitional epithelium (W pg 87, 5.9; pg 326-7, 16.24-7)
slide 19 odd (digital slide 19-2, non-distended ureter, H&E) [WinLab] [Mac] [WinHome]
slide 19 even (digital slide 19-1, distended ureter, H&E) [WinLab] [Mac] [WinHome]

("even" and "odd" refer to the box number on the glass slide collections --if you are studying this material using your glass slides, be sure to ask your neighbor for the alternate version)

Transitional epithelium is confined to the urinary tract and is adapted for extensibility and water impermeability, as when the bladder fills with urine. We will compare the epithelium lining the lumen of a distended (glass slide 19 even or digital slide 19-1) and a non-distended ureter (glass slide 19 odd or digital slide 19-2). In the non-distended ureter, note the shape of the large surface cells, which are frequently dome-shaped and may bulge out into the lumen. Now, compare with the distended ureter (this tissue was artificially over-distended to make a point about the capacity of this epithelium). The surface cells have been stretched thin, as have the other layers, and there actually appear to be fewer layers, as the cells can slide past one another to a certain degree.

Electron Micrograph Wall Charts

While understanding the light microscopic structure of tissues and organs is important for interpreting pathological change, much of the really interesting biological side of medicine now involves understanding cell structure in more detail. Therefore, we feel you should be comfortable with interpreting electron micrographs by the time you complete this course, as they are becoming more important in diagnosis and many of the micrographs in your professional literature will be of this sort. We assume that you already have a general knowledge of cell ultrastructure and can recognize the nucleus, mitochondria, cell membranes, endoplasmic reticulum, and ribosomes. Your Wheater's Atlas contains an excellent review of cell ultrastructure in Chapter 1 (pages 2-32) as does the Ross text (Chapter 2). Please review these structures in these sources and the electron micrographs listed below.

EM#1 [WinLab] [Mac] [WinHome]
EM#2 [WinLab] [Mac] [WinHome]
EM#4 [WinLab] [Mac] [WinHome]
EM#11 [WinLab] [Mac] [WinHome]

Use these micrographs to review the structure of organelles. Be sure you can recognize favorable sections of the nucleus, mitochondria, and rough ER.

EM# 0-A [WinLab] [Mac] [WinHome] and EM#0-B CELL JUNCTIONS [WinLab] [Mac] [WinHome]
Review the structure of occluding and communicating junctions.

EM#70 SIMPLE SQUAMOUS EPITHELIUM [WinLab] [Mac] [WinHome]
The endocardium, the simple squamous epithelial lining of the heart, can be seen at the top of this section. Under it, you can see connective tissue, which we will study next time. Note how thin the epithelium is.

EM#17 SIMPLE COLUMNAR EPITHELIUM [WinLab] [Mac] [WinHome]
You can see that this is a simple epithelium, as it is one cell layer thick, and columnar, as the cells are tall. Note the basal lamina at the base of the epithelium. You can see the apical area where junctions are located. This cell also has very short apical microvilli.

EM#128 SIMPLE CUBOIDAL EPITHELIUM [WinLab] [Mac] [WinHome]
Here we see a kidney tubule cut in cross section. Fingerlike microvilli can be seen. A similar structure in the intestine gives rise to the "striated border" we saw in the LM.

EM# 18 STRATIFIED SQUAMOUS KERATINIZING EPITHELIUM [WinLab] [Mac] [WinHome]
You can appreciate that this epithelium (skin) is stratified (has multiple layers of cells), and that the layers near the surface (at the top of the micrograph) have keratinized (lost their nuclei, and become a layer of keratin). The spiny appearance of cells deeper in the epithelium can also often be seen in the light microscope. They are points of cell-cell attachment, made more obvious by shrinkage during preparation. What junctions are found here? (EP2)

EM#94 STRATIFIED SQUAMOUS NONKERATINIZING EPITHELIUM [WinLab] [Mac] [WinHome]
Compare this micrograph to the previous one. This is the lining of the mouth, where it is no longer necessary to have an outer keratinized layer to protect against desiccation, as it was for skin. Thus, the outermost layer is still cellular and contains a nucleus. Note again the spiny appearance of the cells, due to the desmosomal attachments.

EM#101 PSEUDOSTRATIFIED EPITHELIUM [WinLab] [Mac] [WinHome]
The definition of a pseudostratified epithelium is one in which there are multiple levels of nuclei, but all cells extend to the base of the epithelium. In this micrograph, you can find cells with nuclei at different levels which can be traced down until they are at least close to the base of the epithelium before some of them go out of the plane of section. Note also the apical cilia and basal bodies. You can see that the goblet cells are not ciliated, and are polarized for secretion, i.e., the nucleus is basal to the clear secretion granules, which will be released into the lumen at the top of the micrograph.

EM#102 PSEUDOSTRATIFIED EPITHELIUM [WinLab] [Mac] [WinHome]
This is the apex of the epithelium seen in EM #101, cut in a different plane of section, as indicated by the line in #101. You can see cross sections of the cilia, and also the secretion granule-filled apex of an occasional goblet cell.

Review Question Answers

EP1: What structures form the core of each cilium?

answer

EP2: What junctions are found in the spinous layer of stratified squamous keratinizing epithelium

answer

Practice Questions

Click on either the MAC link or the PC link to open the image.

1. The type of epithelium indicated by the arrow lines:

1. skin
2. mucosa of the esophagus
3. respiratory tract
4. urinary tract
5. mesentery

ANSWER

Click here to view image

2. The type of epithelium shown is:

1. simple cuboidal
2. simple columnar
3. stratified columnar
4. pseudostratified columnar (respiratory)
5. transitional

ANSWER

3. Which of the following statements regarding microvilli is FALSE?

1. They contain a core of keratin intermediate filaments.
2. They are anchored to the cell by a terminal web of intermediate and actin filaments.
3. They are immotile (they do not move on their own).
4. They facilitate absorption.
5. They are generally considered to be localized on the apical surface of epithelial cells.

ANSWER

Click here to view image

4. The type of intercellular junction as viewed by routine transmission electron microscopy in panel A and in a freeze-fracture preparation in panel B:

1. extends as a zone around the apical perimeter of adjacent cells.
2. posseses dense plaques that are anchored to intermediate filaments.
3. permits the passage of ions from one cell to another.
4. requires calcium to bind adjacent cells.
5. mediates adhesion of cells to an underlying basal lamina.

ANSWER