9217217 Lanni The goal of this project is the construction of a fluorescence microscope for high-speed, high-resolution, three-dimensional imaging of biological specimens. The approach to be taken is a direct extension of Standing-Wave Fluorescence Microscopy, demonstrated in this laboratory previously. The proposed instrument will incorporate a laser-based optical system for excitation field sYnthesis, in which standing wave fields are time-multiplexed in the specimen to create an effective intensity field that is peaked at the in-focus object plane. Fluorescence emission will therefore be excited maximally from only in-focus object features. Serial focal plane images will then be recorded in the usual way, by use of an electronic (CCD) camera. The optical transfer function for this field-synthesis fluorescence microscope (FSFM) covers a band of axial spatial frequencies not accessible through classical optical sectioning microscopy. In principle, this instrument has the speed and transverse resolution of an imaging system, and axial resolution comparable to or better than a scanning confocal microscope. The significance of this project in the biological sciences can be stated concisely. Microscopes are most severely limited in axial, as opposed to transverse, resolution. This problem is rectified by confocal scanning, but with a loss of the fast, efficient information transfer that occurs in direct image formation. In many biological problems, particularly in studies of live cells, speed and photometric efficiency are primary constraints. The field-synthesis fluorescence microscope is designed specifically to give improved axial resolution while still retaining the high-performance characteristics of an imaging system.
