Junqueira's Basic Histology (15th ed.), Ch 1, Histology & Methods of Study

Junqueira's Basic Histology (15th ed.), Ch 2, The Cytoplasm
Junqueira's Basic Histology (15th ed.), Ch 3, The Nucleus

 

Goals:

1. Learn how to use your virtual microscope.
2. Study cells and cell organelles in tissue sections.

I.   Microscopy

Our slides have been scanned and digitized permitting analysis and viewing of the slides at high resolution on your computer (“Virtual Microscopy”). These webslides are delivered from a central server to your personal computer that acts as a digital microscope.

Helpful Points: 
(1) Open only one slide at a time to reduce the memory and processing load on your computer.
(2) An external mouse with a scroll wheel can help with panning and zooming on the VM.
(3) A links to a video tutorial on how to use DigitalScope is available in the drop down menu above or at the bottom of this page.

A. Using DigitalScope to view webslides

Below is a brief explanation of the toolbar shown in the DigitalScope viewer:

Link Tool options:

• Regions of interest/arrows – these options apply to annotated slides in which regions of interest can be selected on the slide or cells/structures indicated by an arrow
• Open Regions of Interest List – helpful if you have multiple regions of interest (e.g. a blood smear with multiple ROI’s outlining the various cell types)
• Open Navigation Window – this shows the navigator (zoom and pan) tool
• Open Miller Disc/Grid overlay – shows either a box with an inset or a grid overlay for cell or lesion counting
• Hide Link Tool – this shows the slide but HIDES the link tool
• Open Case History – automatically opens the case history if available
• Navigate to First ROI – takes the user to the first ROI on an annotated slide
• Navigate to Home – basically shows the “home” view with the slide sized to fit within the frame
• Navigate to Current Coordinates – takes the user to the specific area and magnification

Image Capture options:

The “Image Capture” tool will capture ALL of the pixels in a selected region in a .png file (i.e. at the resolution the slide was originally scanned, which is usually at 20x or 40x).  The resulting file can be very large, so it may be preferable to use the native screen capture function on your device (e.g. ‘Win’+Shift+S on a Windows device) which will capture at screen resolution.

To use the screen capture tool,
1. Select the preset option for the area to capture:

• Freeform – a rectangular area of any size expanded by clicking and dragging
• Square
• Vertical Rectangle – ‘portrait’ orientation
• Horizontal Rectangle – ‘landscape’ orientation

2. Select whether to include regions of interest or arrows (if the slide has annotations)
3. Click on the ‘Capture’ button to activate (it will turn blue)
4. Click on the viewer to either create the area (if in ‘Freeform’ mode) or to place the preset area on the slide; preset areas can be repositioned by clicking and dragging
5. Click on the check mark to capture or the ‘X’ to cancel

Click here for a video tutorial on using the DigitalScope viewer for virtual slides.

II. Cells and Organelles in Tissue Sections

Our server contains two types of webslides: (1) webslides scanned from thick tissue sections which provide a “low power” view of an entire tissue or organ and (2) webslides scanned from thin tissue sections which are useful for detailed high power analysis of cell structure. The appearance of tissues in section and the ability to resolve organelles is a function of the thickness of the section.  In the first part of this exercise two webslides of the liver will be studied, representing thick and thin sections. In comparing these two webslides you will be able to “see” the advantages of thick sections for low power views of the entire organ and the advantages of thin sections for the analysis of cytological fine structure. Because of their ease of preparation, most histological workups for pathological studies are done with thick sections.

"Thick" (10um) section: Webslide 0084A_J: Liver and gall bladder, monkey, Masson  [DigitalScope]

As should be done each time you view a slide, start with low magnification to scan the entire structure and to inspect the edges of the specimen to distinguish cut edges from natural surfaces. Next, use higher objective settings to scan the brown-staining portion of the slide. Locate large cuboidal cells that appear in section as rows or “cords” of cells that alternate with emptied sinusoids which normally carry blood. These large cuboidal cells are hepatocytes, the main functional (parenchymal) cells of the liver. With the highest objective setting, study these hepatocytes, noting the nuclei that are more darkly stained than the cytoplasm. Although plasma membranes cannot be resolved, boundaries between cells can be detected.

"Thin" (0.5um) Section: Webslide 0011_J: Liver, guinea pig, thin section, AF-TB   [DigitalScope]

Use the low power objective setting to find a well-stained region, then switch to the highest objective setting.  For hepatocytes find: 

1. Nuclei.  Note the heterogeneous appearance of chromatin. What is the molecular basis for this heterogeneity?
2. Nucleoli. Dark staining, circular structure near center of nucleus.
3. Mitochondria. Compact, smooth-bordered circles or rods in cytoplasm, about 1 µm in size.
4. Lakes of glycogen.  Uniform, pale regions in cytoplasm.  After examining several hepatocytes, draw a typical hepatocyte, including a scale marker in µm.

B.  Pancreatic Acinar Cells

Webslide 0039_J: Pancreas, monkey, AF-TB   [DigitalScope]

Webslide #39 is a thin section of the pancreas, which contains nearly circular clusters of cells called acini.  Each acinar cell secretes precursors of digestive enzymes that are released into ducts for transport to the duodenum.  Scan the slide with a low power setting to find uniform, well-stained regions of the section.   Next change to a higher magnification setting and locate the circular acini, which are typically about 25 to 40 µm in diameter (measure some with the Ruler Tool).   Note how many cells are in each acinus.  Finally switch to the highest magnification setting for a careful examination of typical acinar cells.  Identify the cell margin, the apex and base (top and bottom) of the cells.  How can you identify apex from base?  (Hint: where should the secretory granules be located?)  Note the size, density, and abundance of these secretory granules.  Locate cell nuclei and examine for nucleoli.   Determine the dimensions of a typical nucleus and nucleolus. Can you find any pale staining Golgi regions between the nucleus and the secretory granules?  Find the location of the rough ER, which corresponds to the diffuse, palely basophilic region between the base of the cell and the nucleus. Sketch a typical acinar cell, labeling all the organelles you can identify.  Include a scale marker in µm.

C. Cerebellar neurons

Webslide 0048_S: Cerebellum, human, H&E and Luxol fast blue [DigitalScope]

Webslide #48 is a section through the cerebellum of the brain prepared by routine H&E staining that stains basophilic structures in neurons such as the nucleolus and the rough endoplasmic reticulum dark purple and acidophilic structures such as cytoplasm and extracellular proteins pink. Another staining component, Luxol fast blue, stains myelin found in white matter tracts deep blue.

The neurons can be found at the junction between the light and dark staining layers of the cerebellum. They are distinguished from other cell types due to their very large size and unique staining. Locate a large neuron where the pale cell nucleus and large, prominent nucleolus are in the plane of section. In the cytoplasm of the cell body, most of the dark, purple-staining densities represent rough endoplasmic reticulum. Notice that these densities are found throughout the neuronal cell body, but not in the processes (long projections extending from the cell body). Measure the sizes of the cell bodies and nuclei of several typical neurons, and include these dimensions in a diagram below.  Compare to the dimensions you obtained above for the liver hepatocytes and pancreatic acinar cells.

III. Pathology Correlate - changes in cell staining resulting from cell death

Slide UMich 008_40x (kidney infarction)  [DigitalScope]

This specimen of kidney was obtained from a patient that had a small clot in the aorta that broke loose and lodged into a small branch of a renal artery causing the infarction of a triangle-shaped wedge of tissue.

The healthy tissue is on the right side of the slide and should be examined first [example]. Zooming in on the healthy area you should see numerous tangles of capillary loops called glomeruli [example] where blood is initally filtered and tubules [example] lined by simple cuboidal epithelium that modify the filtrate (mostly by resorbtion) to produce urine. All of these cells contain eosinophilic cytoplasm and basophilic nuclei.

The infarcted areas are in the upper areas of the slide [example]. Zooming in on the infarcted tissue, notice that you can still see the eosinophilic components of the glomeruli [example] and tubules [example] but note that these cells no longer exhibit any basophilic nuclei (the irregular, basophilic nuclei that are present belong to inflammatory cells).

What you are seeing in the cells in the infarcted areas is a process known as karyolysis, or dissolution of the nucleus, that occurs as part of cell death which activates DNAses and RNAses that quickly break down the chromatin in the nucleus and therefore no longer picks up any of the hematoxylin stain. The proteins of the cell cytoplasm, however, are stable for some time even after cell death and therefore will still pick up the eosin stain, thus explaining what is observed here.

These changes that occur in H&E staining are just some of the features that a pathologist might use to evaluate dysfunction of an organ and hopefully illustrate the value of understanding what structures in a healthy cell are typically basophilic and which are typically eosinophilic.