Fluorescence spectroscopy and laser light scattering will be used to quantify the radiation induced heating and cell damage effects that may occur during the optical micromanipulation of liposomes and sperm cells using optical tweezers. Heating and thermal effects, if induced, may produce photochemical and photobiological changes that can ultimately result in altered cell structure and function. To assess the degree to which optical trapping is a non-invasive and non-destructive process, an optical laser trap is to be integrated, for the first time, with a flourescence detection and forward light scattering measurement system, for the simultaneous confinement, micromanipulation, and light excitation/emission study of liposome test particles, and starfish and human sperm cells. The research premise is that, as focused laser radiation is absorbed by a cell, its temperature increases, and can produce a shift in the fluorescence emission spectrum of liposomes and cells containing temperature-dependent membrane and nuclear fluorescent probes. A liposome test cell is to be tagged with a fluorescent dye; confined by an optical tweezer; fluorescence excited and detected; and measurements made as functions of the optical tweezer parameters, including power, wavelength, and exposure time. Departure from normal cell structure and function will be used as assays for cell heating and damage effects.
The specific aims for this project include: integration of the trapping and diagnostic measurement capabilities; fabrication and calibration of liposomes as model cell samples and hosts for fluorescent membrane probes; measurement of light scattering profiles and temperature dependent fluorescence emission spectra as a function of laser trapping parameters; analytical modeling of the laser-cell interaction; and, application of the same techniques to the assessment of heating and cell damage in starfish and human sperm, using motility and sperm velocity, flagellar beat frequency, membrane fluorescence, nuclear fluorescence of DNA and chromatin structure, and reproduction capability as a measure of potential damage. The techniques described herein will provide a basis for understanding optical tweezer radiation effects on liposomes and sperm cells, biological systems that function as important transport vehicles for drugs, enzymes, and genetic material within the human body.
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| Chapman, C F; Liu, Y; Sonek, G J et al. (1995) The use of exogenous fluorescent probes for temperature measurements in single living cells. Photochem Photobiol 62:416-25 |
| Liu, Y; Cheng, D K; Sonek, G J et al. (1995) Evidence for localized cell heating induced by infrared optical tweezers. Biophys J 68:2137-44 |
