Three-dimensional (3-D) imaging is a formidable task in optical microscopy. Optical sectioning microscopy (OSM) and a radically new microscope design, scanning confocal microscopy (SCM), are the two common techniques available in practice. In this proposal, a novel optical scanning microscope system is introduced and studied. The system consists of two stages. In the first recording or coding stage, the 3-D specimen is two-dimensionally scanned by a time-dependent Fresnel zone plate (TFZP). The TFZP is created by the superposition of a plane wave and a spherical wave of different temporal frequencies. While the specimen is scanned, a photodetector collects the light transmitted through the specimen and delivers a heterodyne current as an output. The current, which contains the FZP-coded information of the specimen, is then mixed down to become a demodulated signal and stored on a computer. The stored image is an on-axis FZP coded image or essentially a hologram which contains the 3-D information of the specimen. In the reconstructing or decoding stage, the stored hologram is outputted to a display and photographed as a transparency. A laser then illuminates the transparency to reconstruct a 3-D image. The coded image can also be reconstructed digitally. To reconstruct the image at some depth, the coded image is simply treated with a filter matched to the depth required.
Specific aims i n this proposal are: 1) To design and build a prototype. 2) To demonstrate the 3-D reconstruction capability of the proposed system. Specifically, two planar objects at different depth can be scanned simultaneously and stored in a computer. Reconstruction of the two images at two locations would then reveal the 3-D imaging capability. 3) To investigate multiplexing of the proposed system. Specifically, the heterodyne current could be mixed down by a quadrature reference electronic signal instead of an in-phase signal. In this way, the image is effectively coded with a FZP which is 90 degree out of phase with respect to that of the one achieved with the in-phase reference. The two corresponding coded images are then combined to achieve off-axis FZP coding. The major advantage of the proposed system is that 3-D information is achieved with a 2-D scan and 3-D reconstruction can be performed optically or digitally. In OSM, although only a 1-D scan is required, precise alignment of the 2-D images throughout the depth is crucial in order for any digital reconstruction algorithms to work at all. In SCM, time-consuming 3-D scanning and the stringent instrumental tolerances are required. The proposed system could prove to be more robust and practical than currently available methods for 3-D imaging.
