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Concept
Focus stacking
Summary
Focus stacking (also known as focal plane merging and z-stacking or focus blending) is a technique which combines multiple images taken at different focus distances to give a resulting image with a greater depth of field (DOF) than any of the individual source images. Focus stacking can be used in any situation where individual images have a very shallow depth of field; macro photography and optical microscopy are two typical examples. Focus stacking can also be useful in landscape photography.
Focus stacking offers flexibility: since it is a computational technique, images with several different depths of field can be generated in post-processing and compared for best artistic merit or scientific clarity. Focus stacking also allows generation of images physically impossible with normal imaging equipment; images with nonplanar focus regions can be generated. Alternative techniques for generating images with increased or flexible depth of field include wavefront coding, light-field cameras and tilt.
The starting point for focus stacking is a series of images captured at different focus distances; in each image different areas of the sample will be in focus. While none of these images has the sample entirely in focus they collectively contain all the data required to generate an image which has all parts of the sample in focus. In-focus regions of each image may be detected automatically, for example via edge detection or Fourier analysis, or selected manually. The in-focus patches are then blended together to generate the final image.
This processing is also called z-stacking, focal plane merging (or zedification in French).
Getting sufficient depth of field can be particularly challenging in macro photography, because depth of field is smaller (shallower) for objects nearer the camera, so if a small object fills the frame, it is often so close that its entire depth cannot be in focus at once. Depth of field is normally increased by stopping down aperture (using a larger f-number), but beyond a certain point, stopping down causes blurring due to diffraction, which counteracts the benefit of being in focus.
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Optical microscope
The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast. The object is placed on a stage and may be directly viewed through one or two eyepieces on the microscope.