System and method for high numeric aperture imaging systems

An imaging system for acquiring multiple images of a target object, the imaging system including an autofocus ability, the imaging system comprising:

• (a) a first imaging sub-system for generating a first image of the target object onto a first focus area using unaltered light;
• (b) a second imaging sub-system for generating a second image of the target object onto a second focus area using defocused light;
• and
• (c) a processor logically coupled to the first and second focus areas, the processor configured to implement an autofocus technique using the functions of:
• (i) analyzing the frequency content of the first and second images;
• (ii) if the frequency content for the first and second images are not balanced, calculating a focal error signal;
• (iii) if frequency content is not balanced, determining a focal shift;
• and
• (iv) adjusting refocusing optics based upon the determined focal shift and the focal error signal.

A non-transitory memory medium having machine instructions stored thereon for implementing an autofocus technique in an imaging system generating a first image in a first focus area and a second image in a second focus area, the machine instructions, when implemented by a processor, carrying out the functions of:

• (a) analyzing the frequency content of the first and second images;
• (b) if the frequency content for the first and second images are not balanced, calculating a focal error signal;
• (c) if frequency content is not balanced, determining a focal shift;
• and
• (d) adjusting refocusing optics based upon the determined focal shift and the focal error signal.