Recent advances in confocal microscopy have demonstrated the potential of optical imaging to provide molecular images with sub-cellular resolution. In this proposal, we extend our work in high resolution confocal microscopy, based on a much simpler alternative, based on our observation that high resolution optical images of the top 1-2 layers of cells can be obtained without the need for a confocal imaging gate if the cells are placed in direct contact with a flat optical window. Here we propose a new class of optical imaging device - the integrated optical needle (ION) - which can be inserted into a small gauge needle and advanced through tissue to acquire images of tissue at the distal tip of the needle with sub-cellular resolution in real time; the use of targeted contrast agents or vital stains can yield additional contrast as well as functional and/or molecular imaging capability. The first version of the optical needle, described in Aim 1, is based on a flexible, coherent fiber-optic bundle coupled to macroscopic imaging optics and a CCD image sensor. The under-sampling associated with the structure of the fiber bundle used in Aim 1 limits the spatial resolution. The second approach, an integrated optical needle (ION) described in Aims 2 and 3, uses LIGA technology to integrate miniature lenses fabricated using grayscale lithography, together with the light source and image sensor to yield a miniature microscope which can be inserted through a needle. This ION consists of a NA = 0.4 microscope that can image with 1 micron lateral resolution from a 250 um field of view; it is designed to fit within a needle that has an inner diameter of 1 mm. We will carry out a variety of preclinical tests using cell culture, tissue culture, animal models and pilot clinical trials to evaluate the relative performance of these systems. The question to be addressed is whether imaging without a pinhole or other depth-sectioning technique yields images of sufficient quality to appreciate cellular detail required clinically, and to determine whether out-of-focus and scattered photons reduce contrast to too large a degree. Our preliminary results indicate that it is possible to obtain good images of cellular detail in human subjects using contrast agents in routine clinical use. However, to judge image quality with the various devices and contrast agents, we will use quantitative measures of image resolution, image contrast and image SNR. Research proposed here will develop a new tool for image guided tissue assessment in real time, and confirmatory biopsy at image directed locations. The ION can image a broad range of contrast agents enabling molecular information from specific sites. The cost of the integrated optical needle will be low even in small quantities (below $2,300) but there is great potential to further reduce this cost when manufactured in quantity. ? ? ?
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