Crab spider on Fleabane flower, 5x life-size (in camera)
Depth of Field (DOF) (the plane that is sharp) can be extremely limited in macrophotography - as magnification increases, the DOF decreases. And as you enlarge the image, DOF decreases even more. Let me use this image as an example.
I brought this young Crab spider on the Fleabane flower into my studio for this image - or more accurately, this series of images to make this 8 image composite. I used Canon's fantastic MP-E 65mm/2.8 lens, which allows you to shoot from 1x to 5x life-size without adding extension tubes or bellows. I set the lens for full magnification (5x); my plan was to get all the "face and head" into focus. And the spider cooperated, allowing me to take all 8 images before it moved. Here are the two "book-end" images, showing the small depth of field and the front and back of my "sharp" area:
At 5x magnification with a 65mm lens using an aperture of f/27 (and planning on printing to 8x print magnification), the DOF is a meager 0.02mm - that's 2/100 of a millimeter! If you plan on using the image "printed" smaller - for example, like a 4"x6" image, the DOF increases to 0.05mm - still pretty small!
Now, there are a lot of assumptions that go into determining what's acceptably sharp, for deciding what your depth of field - or depth of "what is acceptably sharp to you" is. One is how large you intend to enlarge the final image, like I mentioned above. Another is how well you can see - the arguable standard is 5 to 6 line pairs per millimeter. For my formulas, I've settled on 10 lp/mm - just because I am quite near-sighted and can make out 10 lines per mm without my glasses. I could probably double the 0.02mm to 0.04 to be practical - and then double that again for showing it at this magnification on-line - especially with the post-process sharpening we can now do in digital images.
For proper photo-stacking, you need to overlap the sharp areas of each image by about 50% (overlapping 25% front and back). That means that the combined DOF for this image, as seen in this posts, is (for all practical purposes) about 1 millimeter.
How much bigger than life is the image you're seeing? Here's how to figure that out.
The image was taken at 5x life-size with a "full-size" 35mm digital camera, so the height of the chip is about 0.9 inch. To determine the magnification you are seeing, measure the height of the larger image above on your computer and multiply it by 4.5 (which you would get by multiplying 0.9 by 5). For example, on my computer the larger composite image measures 4.5 inch high. So, 4.5 x 4.5 (or doing it the long way, 4.5 x 0.9 x 5) = 20.25; or about 20x the spider's actual size.