User:Brian P. Josey/Notebook/2009/08/27

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Notes on Microscopy

On Andy's suggestion, I spent the first part of the day reviewing basic microscopy. The things that I looked at included Koehler illumination, bright field, DIC and fluorescence. I also looked up the basic components of a microscope. They are:

  • Eyepiece- Two or more lenses that you look into to see the image.
  • Objective lenses- One or more lenses that collect light from the sample.
  • Stage- A platform used to hold the sample.
  • Illumination source- Simply gives light to the microscope.
  • Condenser- A lens used to concentrate light to be collected in the objective.
  • Diaphragm- An opague structure with an opening in its center that can be adjustable. This opening is called the aperture.

The basic techniques and types of microscopes are:

  • Bright Field- Bright field is the most basic type of microscopy. Essentially light from either a light bulb, or reflected from a mirror, is collected in a diaphragm and condenser. This concentrates the light on to the objective holder or stage, where the slide rests. The light then passes through the object being studied and is collected in the objective lenses, is focused and moves on to the eyepiece.
  • Differential Interference Contrast (DIC)- The two microscopes that are used in the lab use DIC, which relies on polarized light to create a finer image, based on the optical properties of the sample. The basic path of the light is:
    • Unpolarized light enters the microscope and is polarized at 45 degrees.
    • The light then enters a Wollastom prism and is split into two sets of waves that are polarized to each other at 90 degrees. These two set of waves are the sampling and reference rays.
    • The rays are focused by a condenser to pass through a sample. When they do pass through the sample, two corresponding waves are offset from each other by about 0.2μm.
    • They pass through the sample at two different, adjacent areas of the sample. This in essence creates two adjacent bright field images that are slightly offset from each other.
    • The light then travels through the objective lens and focused before entering a second Wollaston prism.
    • In the prism, the light is recombined at a polarization at 135 degrees. This creates interference patterns, lightening and darkening the images at points corresponding to the different optical qualities of the sample.
  • Fluorescence Microscope- A fluorescence microscope uses fluorescence, and phosphorescence instead of or in addition to reflection and absorption. To do this, fluorescent molecules are attached to the subject. A particular wavelength of light is filtered out from a light source and then falls on the sample. The fluorescent molecules then absorb the light, and release light with a longer wavelength. This new set of light is then collected in the same way that all light is collected, creating an image of the sample.
  • Kohler Illumination-Kohler illumination is a technique used to illuminate a sample. The stepwise procedure is:
    • Focus on the specimen
    • Close the field diaphragm to the most closed state, so that its edges appear in the field of view.
    • Use the condenser focus knobs to bring the edges of the field diaphram into the best focus possible.
    • Use the condenser-centering screws to center the image of the closed field diaphragm in the field of view.
    • Open the field diaphragm just enough so that its edges are just beyond the field of view.
    • Adjust the condenser diaphragm to introduce the proper amount of contrast to the sample.
    • Adjust as needed.

After looking up these things I began to read "Mechanical Design of Translocating Motor Proteins" by W. Hwang. It is a review paper on how both kinesin and myosin, among other motor proteins work. Notes on this paper will come in a later entry.