An automatic focus/hold system for optical microscopes

Abstract

A system for maintaining long‐term focus of samples under high‐magnification quantitative observation in an epi‐illumination optical microscope is described. A negative feedback signal is generated from focus‐dependent changes in the backreflection of an off‐axis HeNe laser. This reflection is intercepted by a small prism downbeam from the standard trinocular head, and detected by a small two‐photodiode array. Spontaneous drifts in sample focus (presumably due to thermal and mechanical relaxations) are detected as a nonzero difference signal, which is used to drive a dc motor mechanically coupled to the fine‐focus knob of the microscope. This system has several advantages: (1) it is completely compatible and nonobstrusive with concurrent data acquisition of sample intensities; (2) it requires no alteration of the sample, sample stage, or objective; (3) it monitors the focal position of sample areas very near to those under observation; (4) it is inexpensive. In an experimental test, the system can hold a thin glass coverslip sample (a common substrate for biological cell cultures) to within 0.5 μm of its preset focus position, well within the depth of focus of the microscope. Without the system, such samples typically drift several micrometers over periods of 10 min. In response to a disturbance of the focus knob, the system can restore the focus to within 0.5 μm of the preset position.