A Rapid Image Acquisition Method for Focus Stacking in Microscopy

Introduction Modern image acquisition in microscopy bene"ts greatly from the use of well-known focus stacking photomontage procedures. #is o$en involves the taking of a set of images at successive focal distances, followed by mathematical processing and combining of the images to produce a resultant single image with an extended depth of "eld (DOF) [1]. #is is useful when viewing subjects whose visible features lie at depths greater than the DOF of the objective lens in use or when objects are tilted or have irregular surfaces. In the past, images to be included in a focus stack (stack) were taken one-at-a-time. A "rst image was taken, then the position of a subject relative to an objective lens was changed and a second image was taken, and so forth. #is process has worked well, however it takes time. #is article describes an improved system for rapidly obtaining a set of images at video rates, that is, at tens of images per second and at di&erent focal distances. #e subjectto-objective focal distance remains constant so there is no movement of the microscope stage or objective. #e new system greatly increases speed, convenience, and work'ow in obtaining images with an extended DOF. #e extended DOF brings life to moving and changing subjects and lets them be seen in the full context of their environment. #e new system also applies to cameras, telescopes, binoculars, and monoculars [2, 3].