(This application is in response to NIMH program announcement PA-99- 007, """"""""Probes and instruments for micro-imaging the brain."""""""") We have devised a novel strategy for optical sectioning of three- dimensional microscopic specimens that is based on wide-filed optical principles and LED illumination. The overall structure of a high-contrast specimens (such as a stained neuron with a brain slice) will be imaged as a dark silhouette displayed against a colored (blue) background. Structures within the plane of focus will be imaged in an opposing color (yellow) and displayed as a thin optical section through the dark silhouette. The structures within the optical field will be imaged at high resolution, whereas structures outside the section will be imaged with great depth of field. This method will complement confocal microscopy in many situations. By providing useful depth-of-field information, our approach will allow each optical section to be interpreted in the context of the overall three-dimensional structure. With the use of the wide-field approach for light gathering, image acquisition can be completed in real time and images will not require further digital processing. Moreover, because implementation can be achieved with relatively inexpensive modifications to existing microscopes, high-resolution optical sectioning of microscopic specimens an be made more accessible to a greater number of scientists.
The ability to optically section a three-dimensional structure with high resolution in real time while preserving useful depth-of-field would interest a major portion of the biomedical community who use optical microscopes. Such an option would likely be incorporated in s significant number of new microscopes with is a $500 million a year business. An even larger market may be the retrofitting of existing microscopes.
