This work will develop an epi-illumination fiber optic microprobe that could be used to analyze metabolic events at microscopic sites within biological tissues. Monochromatic light from a tunable dye laser will be coupled into an optical fiber and guided to the opposite end where there is a microscopic tip (5 um diameter) that can be inserted within a biological target tissue. The light will be used to stimulate fluorescence of intrinsic molecules, artificial fluorochromes or to monitor absorbance changes of electron carriers and metabolic intermediates. We propose to develop and test the instrument within plants but in principle, the epi-illumination probe could be used in a wide variety of biological systems. Specific developmental efforts will be directed toward (1) analysis of the specialization of different absorbance changes of photosynthetic electron carriers; (2) the development of fiber optic chemical senors that can be used to monitor metabolic changes, such as CO2 concentration, within cells and tissues. This will be pursued by attaching polymeric films with embedded fluorochromes to the probe tip that undergo changes in fluorescence in response to changes in the probe environment. The epi-illumination fiber optic probe will provide information both in vivo and in real time within living tissues. The response time is rapid (ns) and the probe will have high spatial resolution so that it will be possible to measure events at microsites within light-scattering tissues, cells and to examine the physiological specialization of small numbers (3-4) of organelles such as chloroplasts.
