Automatic on-the-fly focusing for continuous image acquisition in high-resolution microscopy

With the typically narrow depth-of-field microscope optics, biological specimens do not lie in a single focal plane across the slide and this complicates automated scanning for image cytometry. An on-the-fly autofocus system for high-resolution image cytometry is presented which keeps the image sharply focused during continuous stage travel. To track possible foci, an image volume is acquired by concurrent optical sectioning of the specimen with a dedicated imaging array. This volume scanning camera was designed for adjustment of the optical path lengths to allow simple adaptation to objectives with different depths-of-field and magnifications. The computational demand of calculating and adjusting focus dynamically is absorbed by an array of parallel autofocus circuits that measure the 3D image in real time. In conventional optical sectioning microscopy, where the image data is acquired by sequential sectioning, a priori knowledge of the specimen and its boundaries exists. In continuous volume scanning, this is usually not the case and variations in specimen thickness and information content are routine. This makes implementation of fully automatic, high-speed, high-resolution image cytometry a challenge. A model of the combined specimen and optical system was developed to evaluate strategies for tracking focus. Data from this model is described along with the volume scanning camera array design.