Microscopy & Cell Organelles

Atlas:
Ross & Pawlina (6th ed.), Plates 1-3, pgs. 152-157
Text:
Ross & Pawlina (6th ed.), Ch 4, pgs. 98-103, Tissues: concept and classification
Ross & Pawlina (6th ed.), Ch 5, pgs. 105-151, Epithelial Tissue

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. On our histology website (www.duke.edu/web/histology) most of the slides will have two links to choose from, Aperio WebScope and Aperio ImageScope.

Helpful Points: 
(1) Please open only one slide at a time to prevent crashing your computer.
(2) Using a hardwire connection will speed loading of the webslides.
(3) Bring a mouse, which makes use of the VM easier and faster.
(4) Links to video tutorials on how to use Aperio WebScope and ImageScope are available in the drop down menu above or at the bottom of this page.

1. PC Users:
Aperio WebScope will open Aperio slides in any flash-enabled browser. WebScope allows for simple viewing of the slides as well as webslide annotation (labeling of structures). However, annotations can only be saved using ImageScope, not WebScope.

Aperio ImageScope: ImageScope should be loaded on your computer. If you need a copy, the ImageScope viewer is available for free from http://www.aperio.com/download-imagescope-viewer.asp. Follow the directions to download the self-executable installation file to your computer. Once downloaded, launch the .exe file and follow the on-screen instructions to complete the installation.

2. Mac users:
For exams we suggest that you use Aperio WebScope, not ImageScope. Aperio WebScope will open Aperio slides in any flash-enabled browser and allows for simple viewing of the slides as well as webslide annotation (labeling of structures) without requiring Windows. However, annotations can only be saved using ImageScope, not WebScope.

Aperio ImageScope will only work in a Windows operating system. Use VMware fusion found in your Application folder to start Windows, and then from Windows go to our histology website (www.duke.edu/web/histology). ImageScope should be loaded on your computer. If you need a copy, the ImageScope viewer is available for free from https://web.duke.edu/histology/siteParts/ImageScope_v11.2.0.780.exe (RIGHT click on the link and use 'Save As' to save the file to your desktop). Once downloaded, launch the .exe file and follow the on-screen instructions to complete the installation.  

 

WebScope will open Aperio slides in any flash-enabled browser (on a PC or a Mac). WebScope allows for simple viewing of the slides as well as webslide annotation (labeling of structures). However, any annotations cannot be saved using WebScope, only in ImageScope.

Moving the Slide:  Place the mouse pointer over the image.  Click and drag the image by holding down the left mouse button as you move your mouse. Alternatively, the slide can be moved by clicking to the portion of the thumbnail that corresponds to the part of the slide you would like to view. If the thumbnail is not visible click the Thumbnail icon on the toolbar.

Changing Objectives:  You can change the magnification quickly by scrolling with your mouse wheel. Alternatively, clicking on the webslide will increase magnification. The Zoom Toolbar also allows users to quickly change magnification. If the Zoom Toolbar is not visible, click the Zoom Toolbar icon on the main toolbar. Maximizing the window so it fills the screen will allow you to see more of the slide at any one time.

Measuring Dimensions:  To measure distances click the Ruler Tool icon on the toolbar. Place the cursor at the edge of the object to be measured, click and hold down the left mouse button, extend the cursor to the opposite side of the object and release the mouse.  Read the displayed distance measure.  Please remember that dimensions will be extremely important for identification of specific cells, tissues, or organs.

Image Adjustment: For pale slides, it is sometimes useful to adjust the brightness and contrast for the image. These can be adjusted by clicking the Brightness/Contrast icon on the toolbar.

Creating Annotations: Structures of interest can be labeled using the Rectangle, Ellipse, Arrow, and Pen Tool icons on the toolbar. In addition, text boxes can be added using the Annotations icon on the toolbar and entering text into the text column that corresponds to the region that was labeled. Remember that the annotations created in WebScope cannot be saved like they can in ImageScope. If you try to save annotations by clicking the Save Annotations icon you will get a message saying the connection failed.

Bookmarking: Even though annotations cannot be saved in WebScope, a specific view can be captured using a bookmark. Click the arrowhead on the toolbar to expose the Image tab and select Custom Bookmark. A new window will appear that gives you a link that can be saved to return to the same view that is currently on your virtual microscope. Saving the text of the link and putting it into an application such as Microsoft Word allows you to quickly return to the same view.

Viewing Annotations: Annotations that have been saved to our servers and accompany a slide should be visible at low magnification and can be clicked on twice to view in more detail. Alternatively, click the Annotations icon on the toolbar and select the region of interest.

ImageScope will only operate in a Windows operating system (and with the ImageScope viewer installed).

Moving the Slide:  Place the mouse pointer over the image.  Click and drag the image by holding down the left mouse button as you move your mouse. Alternatively, the slide can be moved by clicking to the portion of the thumbnail that corresponds to the part of the slide you would like to view. If the thumbnail is not visible click the View tab and select Thumbnail.

Changing Objectives:  You can change the magnification quickly by scrolling with your mouse wheel. Alternatively, double-clicking on the webslide will increase or decrease magnification. The Zoom Toolbar also allows users to quickly change magnification. If the Zoom Toolbar is not visible, click the View tab and select Zoom Slider. Maximizing the window so it fills the screen will allow you to see more of the slide at any one time.

Measuring Dimensions:  To measure distances click the Ruler Tool icon on the toolbar. Place the cursor at the edge of the object to be measured, click and hold down the left mouse button, extend the cursor to the opposite side of the object and release the mouse.  Read the displayed distance measure.  Please remember that dimensions will be extremely important for identification of specific cells, tissues, or organs.

Image Adjustment: For pale slides, it is sometimes useful to adjust the brightness and contrast for the image. These can be adjusted by clicking the Image Adjustments icon on the toolbar.

Capturing Views:A specific view on the virtual microscope can be captured using the Snapshot icon (camera) on the toolbar which allows users to save the view as a TIFF or a JPEG file.

Creating Annotations:Structures of interest can be labeled using the Rectangle, Ellipse, Arrow, and Pen Tool icons on the toolbar. In addition, text boxes can be added using the Annotations icon on the toolbar and entering text in the text column that corresponds to the region that was labeled. In order to save your annotations to your computer, click the Export Annotations to File icon in the annotations window. Be sure to include the slide number in your file name to remember which slide the annotation is associated with. The Disk icon on the annotation window is for saving annotations to the server so you will not be able to use this feature. To open your annotation, open the slide in ImageScope and then click the Import Annotations from File icon.

Viewing Annotations: Annotations that have been saved to our servers and accompany a slide should be visible at low magnification and can be clicked on twice to view in more detail. Alternatively, click the Annotations icon on the toolbar and click on the region of interest.

 

Click here for a video tutorial on using the Aperio ImageScope client to view virtual slides (NOTE: the ImageScope client is a WINDOWS-only application).

Click here for a video tutorial on using the Aperio WebScope viewer for virtual slides (NOTE: this viewer will work in any FLASH-enabled browser in either the Windows or the Mac OS).

 

II. Cells and Organelles in Tissue Sections

A.  Liver Cells

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 Section: Webslide 0084A_C, Liver and Gall Bladder, Masson
[Aperio ImageScope] [Aperio WebScope]

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 Section:  Webslide 0011_C, guinea pig, TB-AF
[Aperio ImageScope] [Aperio WebScope]


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, monkey, TB-AF
[Aperio ImageScope] [Aperio WebScope]

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 0085_C, cat, LFB-CFV
[Aperio ImageScope] [Aperio WebScope]

Webslide #85 is a section through the cerebellum of the brain prepared by a procedure that stains basophilic structures in neurons such as the nucleolus and the rough endoplasmic reticulum. 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 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 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.

 

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