Laboratory Exercise 2 “Microscopy”

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

Laboratory Lecture

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)

-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

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

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

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

(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

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)

Resources available… Anatomy and Physiology Laboratory Study Pages; submenu: Microscope http://ctle.hccs.edu/biologylabs/AP1/AP1index.html

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!

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