Method for the focal plane determination of microscopic objects

Microscopy autofocus is designed to obtain the best average focus over the entire field of view, even though objects often reside at multiple foci. Here we report the design of a focus detection circuit for random access operation on any object in a given area-of-interest. A previously reported focus measurement technique based on the energy of the high frequency components of the video signal was modified to operate on a programmable area-of-interest. Then, the determination of the focal plane of the measured region corresponds to the position where the focus measure is maximized for the object. Resolutions of less than 10 μm have been obtained for the focal plane determination of contiguous objects. Besides, good flexibility on setting the size of the measuring window has been achieved. We expect that this development, coupled to additional image analysis automation, will provide tremendous savings in labor for complex micromanipulation.