First, let's look at the electromagnetic spectrum. If we are going to "look" at the details of proteins and other biologically interesting molecules, we must use a "ruler" of appropriate size.

1. Optical Techniques

• Near Field Scanning Optical Microscopy
• Confocal Laser Scanning Microscopy and Single Photon Fluorescence. (fig. 9-18a, Lodish) (fig. 9-18b, Lodish)
• Recently, a paper was published in Nature Methods that describes the use of confocal microscopy with fluorescent probe molecules to distinguish between two hypotheses of how the Golgi apparatus processes proteins.: "Microscopy matures, and so does the Golgi"
• Multiphoton excitation fluorescence microscopy, like confocal microscopy, can produce three-dimensional images.
• Deconvolution Optical Microscopy. (fig. 9-19a, Lodish) (fig. 9-19b, Lodish)

2. More Uses of Fluorescence in Cell Biology

• Cell sorting
• Microarrays (DNA chips)

3. Electron microscopy (fig. 9-20, Lodish)

• We will discuss limits on resolution of electron microscopy - How the properties of electrons affect the ability of electron microscopy to resolve very small structures.

4. X-Ray Diffraction

• X-ray diffraction (fig. 3-42a, Lodish) permits us to determine the dimensions of periodic (repeating) structures, such as a helical protein or nucleic acid or a crystal. If the crystal contains a non periodic structure, such as globular protein at each point in the crystal structure, then the array of diffraction spots will be modulated in "brightness" by the details of the globular protein structure. (fig. 3-42b, Lodish)

5. SDS-Polyacrylamide Gel Electrophoresis

• SDS-PAGE uses a detergent to denature proteins, then electrophoresis causes some chains to move at a faster rate due to a smaller unwound size. Proteins are denatured by the SDS detergent into a random configuration. It can be shown that the average radius of this unwound ball of string is related to the molecular weight. So, SDS-PAGE can not only be used to separate proteins but also to measure the molecular weight. (fig. 3-36b, Lodish)

All text and images, not attributed to others, including course examinations and sample questions, are Copyright, 2009, Thomas J. Herbert and may not be used for any commercial purpose without the express written permission of Thomas J. Herbert.