Optical Sectioning Microscopy by Wavelength Scanning Digital Interference Holography

University Of South Florida, Tampa FL

Investigators

Abstract

This award will develop a simple yet novel and versatile digital holographic microscope device for imaging three-dimensional microscopic volume objects. The resultant images obtained from this device will have submicron resolution in both the lateral and longitudinal directions. Moreover, the blurring and degrading of images due to out-of- focus object planes are completely suppressed. The device is based on numerical reconstruction and interference of a number of digitally recorded holographic images, while the wavelength of the illuminating laser is scanned. Because a hologram retains all of the amplitude and phase information of the light field and because of the freedom of digital manipulation of the holograms, it is possible to carry out an interference process in the numerical virtual space that is quite impossible in physical space. When a number of holographically recreated light fields of varying wavelengths are superposed, and if the wavelengths are regularly spaced, then the interference among them result in the light intensity being concentrated at object points only - propagation of light through space is in effect eliminated. When focused, numerically, on an image plane, only those points corresponding to any discontinuities or irregularities, such as cellular surfaces and structures, of the object render bright image spots. The resulting effect is analogous to scanning confocal microscope images, but this system presents significant potential advantages. It has no mechanical moving parts. The image acquisition consists of N exposures of two dimensional images, instead of pixel-by-pixel build-up of 3D volume. The range of physical sizes and resolution of objects that can be imaged is readily controlled by proper choice of the wavelength intervals. And with the holographic phase information readily available, further interferometric or holographic image processing is possible. Furthermore, the optical sectioned images of the awarded system can be reassembled into a three-dimensional digital model that can be further manipulated for specific applications, such as correction of scale distortion, arbitrary section and cut-away views, and automatic feature enhancement and identification. This system has a potential to impact the general field of microscopic optical imaging by providing a simple and versatile mode of acquiring and manipulating three-dimensional digital model of microscopic objects.