Observing the Actin Cytoskeleton by Fluorescence Microscopy
Background on Fluorescence Microscopy
A substance is said to "fluoresce" when it emits light following excitation by light or another energy source. In the last laboratory session, you labeled actin with rhodamine, pinocytotic vesicles with Lucifer Yellow, and cell nuclei with DAPI. Today you will have the opportunity to use fluorescence microscopy to detect the labeled cytoskeletal proteins and to localize them in the cell relative to the nucleus.
The fluorescence microscope is specially designed to filter light, since fluorescent molecules absorb and emit light at different wavelengths (Table 1; Figure 1). The light source passes through one filter that allows only the wavelength that is absorbed by the fluorescent molecule of interest to pass through to the sample. This excites the fluorescent dye. A second filter allows only the emitted wavelength to pass through to the eyepiece and thus the eye of the observer.
Visualization of the three fluorescent dyes will require using three different filter systems. Your instructor will demonstrate how to use the fluorescence microscope during lab and how to take digital photographs of representative fields for both the untreated and treated fibroblast cells. The files for these digital photos will be made available for you to use in your lab reports and class presentations.
Figure 1. Optical System of a Fluorescence Microscope (Lodish H, et al. Molecular Cell Biology, p.140.)
Instructions for using the Fluorescent Microscope: Media:Using the Nikon 80i Fluorescence Microscope and Camera.doc
Beltz, B.S. and G.D. Burd. (1989) Immunocytochemical Techniques: Principles and Practice. Blackwell Scientific Publications, Cambridge, MA.
Lodish H., Berk A., Zipursky S., Matsudaira P., Baltimore D. and Darnell J. (2000) Molecular Cell Biology, 4th ed. W.H. Freeman and Co., New York.
Watson, S.J. and H. Akil. (1981) Immunocytochemistry: Techniques, trials, and tribulations. Neurosci. Comm., 1: 10-15