![]() We want to use magnification in the microscope objective range, ×5 and above, so our ability to use conventional macro lenses is limited. This subject is small so magnifications above 1:1 will be needed to reveal its detail, but it is significantly thick and three dimensional so we cannot simply put it on a slide, squash it with a coverslip and examine it with the microscope. Let us suppose we wish to take a highly detailed picture of the head and eyes of a dead fly. This site is primarily for those interested in all aspects of the microscope and microscopy rather than the world of macrophotography, but increasingly these two interests are overlapping and coalescing as macrophotographers tackle smaller subjects and higher magnifications. In principle any semi-transparent structures which lie above or below other similar structures, can create headaches in stacking.īefore moving to consideration of the choice of stacking software, it is worthwhile to mention stacking in high magnification macrophotography. There is no substitute for retouching in this case, and Zerene Stacker’s ability to retouch the output from single frames from the stack is a powerful help. The result can be loss of detail and semi-transparent hairs instead of solid ones. A notorious example is fine overlapping hairs, when the software finds it difficult to decide what is above and what below. ![]() Certain kinds of structure cause problems to all stacking software.Stacking with objectives above ×40 requires a precise fine focus and a steady hand. Start with a subject that is suitable for a moderate magnification objective, ×10 – ×25 is ideal.Best to minimise them by having a clean sensor to start with. Dust spots on the camera sensor create irritating trails during the operation of the stacking software, which appear on the final output and can give some tedious and tricky cleanup tasks in post processing if they are numerous. Make sure your camera sensor is as clean as possible before you start.Failure to allow enough overlap causes banding to appear in the stacked output. ![]() This is important for the stacking software to work properly. Allow plenty of overlap between steps the idea is to move smoothly though the depth of the subject just as though you were focusing normally.If you have good parfocality between your visual eyepieces and your camera, and your trinocular head permits simultaneous viewing and photography, then it is possible to judge steps by eye, otherwise use the tick marks on your fine focus and be careful not to loose concentration and forget which direction you are turning the focus wheel!.Check visually that the size of focus step you have chosen is appropriate to the subject and magnification.Stacking works best with a series of approximately equal focus steps between top and bottom settings.bottom → top or top → bottom, not randomly. Stacking software needs images to be acquired in a logical sequence, i.e.Note the settings of the fine focus at the top and bottom focal plane of the subject. Make sure your fine focus operates smoothly.Start with a nice simple prepared slide, choosing a subject with significant depth. Practise your stacking technique on subjects that don’t move! Live subjects like water fleas and mosquito larvae make attractive subjects for stacking, but you need to learn how to confine or anaesthetise them first.Stacking inevitably has its limitations, and there are a few general rules that will help you to make better stacked images. What if we took a new picture each time we made an adjustment to the fine focus, and then combined each of these ‘slices’ together to reconstruct the full depth of the subject? In fact we can do exactly this and use software to select the sharply-focused parts of each slice and combine them into a single composite image showing greatly extended apparent depth of field. That way, we get a much better impression of the 3D structure we are seeing. To appreciate them, we use the fine-focus control to bring various parts into sharp focus in sequence. ![]() Many of the subjects that we enjoy examining through our microscope have significant depth. Stacking is the process of combining a series of digital images of the same subject area, taken at different focal planes, into a single image with great depth of field, using a computer program. Home | Resources | Stacking & stitching | Stacking Stacking Enhancing depth of field by combining image stacks Introduction Bulletin of the Quekett Microscopical Club.
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