Self-calibrating and directional focusing systems and methods for infinity corrected microscopes

Abstract

Systems and methods for autofocusing an objective lens in a microscope system. A self-calibrating autofocus system includes a light source, a decentered aperture, and image-capturing device. The system may be connected to the microscope system so that the light source generates a light on an optical path that includes the objective lens and a plate having a reference surface proximal to a sample holding component. The image capturing device images a reflection of light from the reference surface as the objective lens moves to a series of z-positions. A reference calibration slope is generated for the objective lens by determining positions of the images taken at the z-positions of the objective lens. At least one image having a particular attribute corresponds to a best focus position. The objective lens is moved to predicted best focus or preferred offset from calibrated best focus using the reference calibration slope.