1. Laboratory Exercise 2
“Microscopy”

2. Care and Use of the Microscope
In this exercise, we will study the application of a compound microscope, know its parts and function, and learn to use and take care of it.
These terms should be familiar to you: magnification, resolution, stereoscopic- (dissection), simple-, compound-, and electron- microscopy (scanning or transmission).
This laboratory exercise correlates with Chapter 3 of your lecture textbook; Exercise 2 of your laboratory manual

3. Laboratory Lecture

4. Concept: Cell Biology tools - microscopy & chemistry
The quality of an image depends on
Magnification, the ratio of an object’s image size to its real size
Resolution, the measure of the clarity of the image, or the minimum distance of two distinguishable points
Contrast, visible differences in parts of the sample (can be enhanced by stains/markers)

5. -Electron Microscope 2 nm
Scale of Resolution
-Naked Eye .2 mm
-Light Microscope .2 mm
-Electron Microscope 2 nm
10 m
Human height
1 m
Length of some nerve and muscle cells
0.1 m
Unaided eye
Chicken egg
1 cm
Frog egg
1 mm
100 µm
Most plant and animal cells
Light microscope
10 µm
Nucleus
Most bacteria
1 µm
Mitochondrion
Figure 6.2 The size range of cells
Smallest bacteria
Electron microscope
100 nm
Viruses
Ribosomes
10 nm
Proteins
Lipids
1 nm
Small molecules
0.1 nm
Atoms

6. Light Microscopy
In a light microscope (LM), visible light passes through a specimen and then through glass lenses, which magnify the image
Various techniques enhance contrast and enable cell components to be stained or labeled
Most subcellular structures, including organelles (membrane-enclosed compartments), are too small to be resolved by a Light Microscope

7. Viewing Techniques for: Imaging
Naked Eye
Light Microscope
Electron Microscope
TECHNIQUE
RESULTS
(a) Standard/Brightfield
(unstained specimen)
Imaging
50 µm
(b) Standard/Brightfield
(stained specimen)
Imaging w/ stain
(c) Phase-contrast
Imaging & density
The microscope manipulates optics to improve contrast between the structures
(d) Differential-interference-
contrast (Nomarski)
Imaging & optics
(e) Fluorescence
Imaging w/ Labeling
Figure 6.3a-d Light microscopy
50 µm
(f) Confocal
Imaging & focal planes
50 µm

8. Electron microscopy
Two basic types of electron microscopes (EMs) are used to study subcellular structures
Scanning electron microscopes (SEMs) focus a beam of electrons onto the surface of a heavy metal stained specimen, providing images that look 3-D
Transmission electron microscopes (TEMs) focus a beam of electrons through a heavy metal stained specimen, used mainly to study the internal structure of cells

9. (b) Transmission electron
TECHNIQUE
RESULTS
Cilia
1 µm
(a) Scanning electron
microscopy (SEM)
SURFACE
Longitudinal
section of
cilium
Cross section
of cilium
1 µm
Figure 6.4 Electron microscopy
(b) Transmission electron
microscopy (TEM)
SECTION OR SLICE

10. What procedures are we doing?
We will know the parts of-, the proper use of- and the clean up procedure for a bright-field compound and dissection microscope.
You will examine a slide with the letter “e” (or words affixed to it), and a prepared slide (any tissue) using a compound microscope.
DO NOT PREPARE A WET MOUNT FROM THE CELLS OF YOUR INNER CHEEK (ignore the procedure on p. 26)

11. Resources available…
Anatomy and Physiology Laboratory Study Pages; submenu: Microscope

12. Lets get familiar with each other
We will be working in groups to complete exercises (3 groups of 8 – groups A, B and C or 6 groups of 4 – group 1, 2, 3, 4, 5 and 6)
[Lets look in the laboratory manual...] Within your group, who is the group leader for the exercise? Who is going to do which experiment(s) of that exercise? You must decide amongst yourselves.
Next step: decide who is going to get the materials to complete the exercise. This is collaboration!

13. Compound Microscope
Immersion oil
“e” slide
Prepared tissue slide
Cleanser
Fine (Lens) and Coarse paper towels