Fluorescence microscopy allows for spatial and temporal resolution of systems which are inherently fluorescent or which can be selectively labeled with fluorescent molecules. Temporal resolution is crucial for imaging real time processes in living samples. A common problem in fluorescence microscopy of biological samples is autofluorescence, fluorescence inherent to the system, which interferes with detection of fluorescence of interest by decreasing the signal to noise ratio.
Two current methods for improved imaging against autofluorescence are two-photon excitation and total internal reflection microscopy. Two-photon excitation occurs when two longer wavelength photons are absorbed quasi-simultaneously by a single fluorophore. For this to take place there must be a photon density on the order of 1030 photons/(cm2)(s), which is achieved through use of a femtosecond pulsed laser and a high magnification microscope objective. Two-photon excitation then only occurs at the focal spot, significantly reducing the focal volume and therefore background autofluorescence.
The second method, total internal reflection, is based on evanescent wave excitation, which decreases exponentially in intensity away from the imaging surface. This allows for excitation of a thin (~200 nm) slice of a sample. Since only a narrow region of interest is excited, an optical slice can be imaged, decreasing excitation of out-of-focus autofluorescence, and increasing the signal to noise ratio.
By coupling total internal reflection with two-photon excitation, an entire cell can be imaged while still maintaining the use of lower energy photons to irradiate the sample and achieve two-photon excitation along the length traveled by the evanescent wave. This system allows for more sensitive detection of fluorescence of interest from biological systems as a result of a significant decrease in excitation volume and therefore a decrease in autofluorescence signal. In the two-photon total internal reflection microscopy setup detailed in this work, an excitation area of 20 μm by 30 μm is achieved, and used to image FITC-stained actin filaments in BS-C-1 cells
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/34786 |
| Date | 04 May 2009 |
| Creators | Ogden, Melinda Anne |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Detected Language | English |
| Type | Thesis |
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