It is, of course, always desirable to acquire images in perfect optical focus. But sometimes it isn’t possible, and software must be called in to help out. One situation arises when the optics have insufficient depth-of-field to provide sharp focus for the entire scene or sample. Historically, microscopists would adjust focus up and down through the necessary range and then make a sketch showing the entire specimen, based on their mental compilation of in-focus information.

There is a way to achieve the same effect with a series of digital images, acquired as the focus setting of the microscope is varied through its range. From each individual image, just those pixels that are in focus are kept, and combined with those from others in the series to produce a result that is in focus everywhere. The criteria for selecting the best focus is the statistical variance of the brightness values in a small neighborhood around each pixel (a 5 pixel wide circle in the example shown). When the region is not in sharp focus, the variance decreases significantly. This interactive tutorial illustrates combining multiple images to form an extended focus composite.

The tutorial initializes with a randomly selected specimen imaged in the microscope appearing in the Specimen Image window. The Choose A Specimen pull-down menu provides a selection of specimen images, in addition to the initial randomly chosen one. Clicking on the Through-Focus Series button allows using the Focus Depth slider to scan through the series of images, either by moving the slider control or by clicking on the arrow buttons at either end. Clicking on Best-Focus Composite displays the result of combining all of the individual frames in the series.

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