Exploiting chromatic aberration for image-based microscope autofocus

Autofocus is a critical aspect of any automated microscopy application. Here we report on the successful exploitation of chromatic aberration to speed autofocus for biological microscopy. Using pairs of lenses aligned nominally as infinite conjugates, the chromatic aberration produced by a microscope can be manipulated to achieve multiplanar imaging covering a wide range of spacings. Using both three-chip CCD and Bayer-array color cameras, chromatic aberration-based multiplanar imaging is used to reduce by a factor of 3 the number of mechanical movements of the biological sample necessary to determine best focus. Chromatic aberration-based autofocus is validated using a 20×0.75 NA objective while scanning over 6000 fields of National Institutes of Health 3T3 cells cultured on a coverglass. The combined precisions (standard deviations) of 23.7 nm for a three-chip CCD camera and 23.3 nm for a Bayer-array camera are obtained. These precisions are approximately 1/20th of the depth of field of the objective.