This photomicromosaic showing a collection of diatoms at a magnification of 200 is composed of approximately 160 individual exposures. Diatoms are aquatic, photosynthetic plants comprising a major group of algae. They’re also one of the simplest forms of phytoplankton. Diatoms are frequently used to monitor environmental conditions and water quality.
At a magnification of 200, it’s not possible to have three-dimensional objects like diatoms in sharp focus from top to bottom, so the technique of focus-stacking is employed. In focus stacking, the top layer of an object is tightly focused and an image taken. The focus is then slowly and finely moved down to the bottom of the object with an image taken at each fine step. Finally, all the images are stacked together using focus-stacking software, I use Helicon Focus for this purpose, and the result is a highly magnified image of an object which is fully in focus from top to bottom. The resulting image is very similar to a scanning electron microscope (SEM) image in which highly magnified objects can also be seen entirely in focus. However, a major difference using focus-stacking and an SEM image is that the focus-stacked image results in a real color image. Also, because at a magnification of 200 this slide was far greater than the field of view of the microscope, not only was each frame focus-stacked, but eight individual frames had to be taken and stitched together, forming a final image that’s a focus-stacked photomicromosaic.