Simple way of pinpointing the three-dimensional position of biomarkers in fluorescence microscopy using a through-focus exposure method

The author proposes a method to identify the three-dimensional positions of fluorescent biomarkers by recording just two images. In the proposed method, the x and y positions of all fluorescent markers are recorded in the first exposure, and the z positions are obtained from a blurred image in the second exposure. The author has verified this method using a specimen with 1 μm deep grooves and applied it to measuring chromatic aberration and the separation between two biological probes in fluorescence in situ hybridization cells. The method offers the advantage of greatly reduced data storage requirements.