Dynamic 4-dimensional microscope system with automated background leveling.

This paper describes recent advances in developing an automatic background leveling algorithm for a new, novel interference microscope system and presents images and data of live biological samples. The specially designed optical system enables instantaneous 4-dimensional video measurements of dynamic motions within and among live cells without the need for contrast agents. "Label-free" measurements of biological objects in reflection using harmless light levels are possible without the need for scanning and vibration isolation. This instrument utilizes a pixelated phase mask enabling simultaneous measurement of multiple interference patterns taking advantage of the polarization properties of light enabling phase image movies in real time at video rates to track dynamic motions and volumetric changes. Optical thickness data are derived from phase images. This data is processed with an automatic background leveling routine which separates the objects from the background by thresholding the calculated gradient magnitude of the optical thickness data. Low-order Zernike surfaces are fit to the unmasked background pixels and the resulting background shape is removed. This method effectively eliminates background shape for datasets containing both large and small objects. By applying this method to many sequential frames, it results in all the frames having the same mean background value across all frames which is essential for quantitatively montoring time-dependent processes.