Microscopy

		Microscopy

Microscopy

Familiarity with the tools and methods of any branch of science is essential for proper understanding of the subject. The aim of this practical is thus to introduce you to binocular microscopes in general. Cells, tissues and the detailed structures of organs cannot be resolved with our eyes. Microscopes are used to see these structures that are too small for our eyes to resolve. All microscopes are designed to magnify small objects, and come in a range of forms and specific functions. Our focus will be on the light microscope, but you will also learn about the dissecting microscope and the electron microscope.

You should be able to:

Describe the components of a generic microscope, a light microscope and an electron microscope.
Describe the function of each component of the microscope.
Adjust and use a light microscope correctly.
Describe the differences between a light microscope and an electron microscope.
Understand and explain the pathway of light through the microscope.
Describe how routine histological sections are made for light and electron microscopes.
Correlate the appearance of cells, cellular structures and tissues as seen with the light microscope (LM) with that seen with the transmission and scanning electron microscope (TEM and SEM).
Recognise and explain different magnifications, colours and shades of an image.
Explain how and why haematoxylin and eosin dyes (H&E) stains various components of cells and tissues.

The microscope

The light microscope is a precision instrument that enables the user to see the order and organization inside very small living structures. It plays an important role in understanding the compound picture of the structure and function of living material. In the effective use of the microscope it must be aligned correctly, handled carefully and kept clean at all times.

Components of the microscope

(See Microscopy section on Histoweb)

The stand, binocular tube, mechanical stage and light source comprise the following:

The microscope stand consists of:

Frame
Coarse and fine focus knobs
Light source
On/off power switch
Variable light control.

The integrated binocular tube consists of:

Eyepiece
Revolving nosepiece carrying the objectives
Very low magnification 4X
Low magnification 10X
High magnification 40X
Oil immersion 100X

The mechanical stage consists of:

Specimen holder with spring-loaded clamps and control knobs for moving the specimen.
Condenser

Function of each part of the microscope:

Frame: The various parts of the microscope are mounted on the frame.

Coarse and fine focus knobs: Adjustment wheels move either the stage or the microscope tube to focus the image seen at the ocular lens. At higher magnifications, only the fine focus knob is used.

Light source: Provides illumination of the specimen.

On/off power switch: Turns power to the light source on or off.

Variable light control: The variable light control adjusts the intensity of the illumination.

Eyepiece: The eyepiece or ocular lenses further magnifies and enhance the image from the objective lens and brings the image into focus for the eye. The final optical magnification of the specimen is the product of the magnification of the ocular and objective lenses. The eyepiece often contains a diopter adjustment ring which can be used to adjust the focus for the user’s own eyes.

Revolving nose piece: The revolving nose piece or objective turret carries the objectives and enables the user to easily change to a higher or lower magnification.

Objective lenses: Usually but not restricted to 4X, 10X, 40X and 100X (oil immersion) magnification. The objective lenses collect light from the sample and project an enlarged image in the direction of the ocular lens. Some types of objective lenses use oil or water placed on top the specimen or coverslip to obtain greater resolution at higher magnifications.

The mechanical stage: The stage below the objective lenses with a spring loaded clamp arrangement to support and move the specimen.

Condenser: The condenser focuses light from the light source in a cone onto the sample. It usually incorporates diaphragms and filters to manage both quality and intensity of illumination.

Using the light microscope

This is a very condensed version of the correct way to use a light microscope. Consult a manual or textbook if you plan to do serious work using a microscope.

Microscope quick checklist:
1. Condenser at the top
2. Iris open!
3. Focus with both eyes
4. Rheostat supply enough light
Using the microscope:
1. Handle the microscope by the stand only.
2. Remove the microscope from its box beneath the table and place it on the desk.
3. Ensure that there are no wires, books or pens under the base.
4. Turn the rheostat to 5.
5. Open the iris on the condensor all the way.
6. Turn the condensor as low as it can go.
7. Open the spring-loaded finger of the specimen holder and insert the slide. The specimen should be above the condenser in the path of the light. Ensure that the cover slip faces up.
8. Switch on the microscope.
9. Turn the condensor to directly beneath the slide.
10. Adjust your chair until the level of your eyes is slightly higher than the level of the oculars.
11. Look through any of the two oculars and adjust the light (using the rheostat) to a comfortable level (5-8).
12. Move the oculars apart.
13. Position your eyes approximately 2cm from the oculars.
14. Slowly move the oculars closer to each other until you see two circles or two half circles.
15. Move the oculars slowly until you see only one circle.
16. Close the left eye. Without moving your head, your right eye should see an image. The right ocular often contains a pointer. The pointer must be visible.
17. Close your right eye. Your left eye must see an image. If the right ocular has a pointer, it should not be visible.
18. Open both eyes. You should see one image with both eyes. If the ocular has a pointer, it should be visible with both eyes.
19. If you see one image with both eyes, you can start focusing the image.
20. Ensure that the 4x objective is in position above the slide.
21. Close your left eye.
22. Turn the course focus adjustment knob and focus the image in the right ocular.
23. Adjust the right image by turning the fine focus knob.
24. Close your right eye.
25. Focus the right image by turning the diopter adjustment ring.
26. Open both eyes and focus the image by turning the fine focus knob. After this it is not necessary to turn the diopter adjustment ring.
27. Turn the nosepiece until the 10x objective clicks into place above the condenser.
28. Focus the image only with the fine focus knob.
29. Turn the nosepiece until the 40x objective clicks into place above the condenser.
30. Focus the image with the fine focus knob.
31. Turn the nosepiece until the 10x objective clicks into place above the condenser.
32. Focus the image only with the fine focus knob.
33. Turn the nosepiece until the 40x objective clicks into place above the condenser.
34. Focus the image with the fine focus knob.

Microscope tasks

Page 1 - Make an annotated diagram of:: A light microscope; An electron microscope

Page 2 - Examples of microscopes and stains:: Find examples of a typical light and electron microscopic images. Print and glue on this page. Add descriptions, explaining what is shown on the photograph and how the light and electron microscopic image differ. Use your own words!; Find examples of different types of stains used for both light and electron microscopic work. Print and glue. Explain the various stains in your own words.

Page 3 - complete Slide 21: Amphibian blood: Find Slide 21 and complete the instructions as set out on the slide page.

Calibration

Make a line diagram of a light microscope.
Make a line diagram of an electron microscope.
Label each diagram, giving the parts as well as the functions of each part.
Describe the staining characteristics of cells and tissues.
Tabulate the difference between acidophilic and basophilic staining.
Tabulate the difference between haematoxylin and eosin staining.