The objective of the proposed research is to expand the current capabilities of the two-photon microscope (TPM) system constructed in the LAMMP facility to image cellular activity within tissues, especially cutaneous wounds, to depths of at least 500 (m. One of the main advantages the TPM has over a confocal microscope is that the system can be used to look at any specific excitation in the region 350-450nm due to the tunability of the excitation laser. Another advantage of this TPM is the unique capability to obtain fluorescent images from two different wavelength regions e.g. red and green, simultaneously. We propose to extend this capability by integration of a cooled CCD spectrometer which will allow for spectral measurements in the range 400-800nm to pixel size resolution.
The specific aims of this work are 1) Optimization of the TPM for depth and resolution using Monte Carlo simulations and imaging through phantoms of turbid media to determine the principal factors in image degradation, 2) Image cells and tissues in vitro, especially those involved in wound healing, using exogenous localized photosensitizers (PS) and endogenous fluorophores (auto fluorescence) to provide contrast, 3) Determination of PDT dose for relevant cells using the two-photon system, 4) Image cells and tissues in vivo using animal models to demonstrate depth imaging and the monitoring of wound repair, 5) Obtain real time images of FRET signals and determine excitation wavelengths that optimize energy transfer.
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