A method for mapping and correcting optical distortion conferred by live cell specimens in microscopy that cannot be overcome using optical techniques alone can be used both for light microscopy and confocal microscopy. The system determines the 3D refractive index for the samples, and provides a method for ray tracing, calculation of 3D space variant point spread, and generalized deconvolution.
Microscopy: The method was developed and applied for light microscopy, and is of critical importance for detection of weak fluorescently labeled molecules (like GFP fusion proteins) in live cells. It may be applicable also to confocal microscopy and other imaging methods like ultrasound, deep ocean sonar imaging, radioactive imaging, non-invasive deep tissue optical probing and photodynamic therapy. Gradient glasses: The determination of the three-dimensional refractive index of samples allows testing and optimization of techniques for production of gradient glasses. Recently continuous refractive index gradient glasses (GRIN, GRADIUM) were introduced, with applications in high quality optics, microlenses, aspherical lenses, plastic molded optics etc. Lenses built from such glasses can be aberration-corrected at a level, which required doublets and triplets using conventional glasses. Optimized performance of such optics requires ray tracing along curved path, as opposed to straight segments between surface borders of homogeneous glass lenses. Curved ray tracing is computation-intensive and dramatically slows down optimization of optical properties. Our algorithm for ray tracing in gradient refractive index eliminates this computational burden.
A computerized package to process three-dimensional images from live biological cells and tissues was developed in order to computationally correct specimen induced distortions that cannot be achieved by optical technique. The package includes: 1. Three-dimensional (3D) mapping of the refractive index of the specimen. 2. Fast method for ray tracing through gradient refractive index medium. 3. Three-dimensional space variant point spread function calculation. 4. Generalized three-dimensional deconvolution.