Focus-Assist Device for a Flying-Spot Microscope

Uncertainty in locating optimum focus can be a major problem when a scanning microscope is used as an input to a quantitative image-analysis system. Our novel solution to this problem is to maximize the integral of the video light-loss signal above a threshold, when the threshold is arbitrarily set above background and within the range of grayness of the image. This algorithm has been incorporated into a real-time analog device to monitor the quality of focus. The focus-assist device uses IC operational amplifiers and has a bandpass of 0.5 MHz. When the device is used with images of metaphase chromosomes, the standard deviation of optimum focus is reduced to 0.09 μm as contrasted to the unassisted value of 0.37 μm. The device is simple, fast, reliable, and extremely sensitive. It should have general application to both macro- and microscopic scanning systems.