My images of Stigonema highlight one of the biggest problems in photomicroscopy: the very shallow depth of field that is inevitable at high magnifications means that any object that is distinctly three-dimensional will only ever be partly in focus. In this case, I managed to get the main branch in crisp focus but the side branches are blurry and out-of-focus. After I had taken those photographs, however, I made my first experiments with a technique called “stacking”.
One “slice” of the “stack” of images of Stigonema mamillosum, collected from the Atma River in July 2013. Note how parts of the image are out of focus whilst others are crisply focussed.
The idea is that you take a series of photographs of the same object, adjusting the focus slightly each time so you end up with a series of images, each showing a different “level” of the object in crisp focus. I then downloaded a software package called Helicon Focus that takes this “stack” of images and selects the sharply-focused parts of each which it then combines into a single composite image that appears to have much greater depth of field than any of the originals.
The composite image of Stigonema mamillosum produced from a “stack” of seven images, including the earlier image in this post. Note how the depth of field now appears to be much deeper in the composite image. Scale bar: 50 micrometres (1/20th millimetre).
Helicon Focus is not the cheapest of the packages that create composite stacked images, but a quick trawl of the internet (in particular, the Quekett Microscopical Club’s useful web pages) suggested that this was the easiest and most intuitive to use. And it did prove fairly straightforward: probably no more than thirty minutes elapsed between downloading the software and producing my first “stacked” image from a batch I had taken the day before. I’ll be trying this again before too long…