As you can see in the image on the left, I attach either my Canon 40D or Canon 5D to a beautiful L3000BHTG research trinocular microscope from GX microscopes to take full (real) colour images of the Micro-World. In addition I have the most remarkable software called “Helicon Focus” which allows me to create focus-stacked images of microscopic objects at very high magnification. What this means in practice is that I can focus at the bottom of the object, take a picture, then move the focus up a tiny amount and take another picture, finishing with an image of the very top of the object in sharp focus. Helicon Focus then does its magic on this set of “focus stacked” images and gives a final image which is in good focus from top to bottom which looks like the sort of image you would normally get from an SEM (scanning electron microscope) – that is it creates an image with an enormous “depth of focus”. The Helicon Focus approach has one major advantage over the SEM however – the final image is in full (real) colour!! A good quality DSLR together with a high quality research microscope and the Helicon Focus software can create some amazing images in the right hands. In this section I will describe in detail how you can take impressive photomicrographs.
I hadn’t a clue how to set up a DSLR to take pictures through a microscope when I first started. I knew I needed a good quality trinocular microscope and a DSLR and that was about it. I had seen some great digital photomicroscopy on the web and the setup these guys used had me stumped (at the time). The DSLR was often fixed on a precision Z-axis manipulator and connected to a bellows – what was that all about? When I finally put my own system together I could see what was being done – but not before 🙂
Looking again at my setup you can the beautiful research trinocular microscope L3000BHTG from GX microscopes, and on the third optical port there is the GX microscopes digital camera adapter (you need to buy this as well as the microscope) with a Canon T-adapter (bought separately) and a Canon 40D sitting on the top. The digital camera adapter comes with a low power eyepiece which sits in the adapter tube. You can now take pictures down your microscope with this system.
So why did these other guys have precision Z-stages and bellows. It is to make their microscopy imaging system parfocal – in other words if the object is in focus through the eyepieces then it is also in precise focus on the digital camera. You need to have a degree of movement in the camera height (Z-axis) to achieve this. My system IS NOT parfocal, when I have looked at my specimen in good focus through the stereo eyepieces, if I don’t change the focus at all, it is out of focus (not by much) in the digital camera. This doesn’t actually bother me at all, in fact I much prefer working this way as focusing closely using the “Liveview” feature and x10 magnification on the 40D or 5D guarantees I am going to get a good in focus image – every time!
The only other thing that might not be familiar to you in the image is the fibre light guide illuminator (also from GX microscopes) which is the black box at the back with the two stainless steel “shower hose” attachments coming to the front. This light guide illuminator provides flexible top illumination for any opaque specimens I want to photomicrograph.
So that’s my hardware setup, and Helicon Focus is my software setup (together with Photoshop) and together they give me a system that satisfies all my photomicrography requirements.
The blue coloured organism in this image is Spirogyra (in conjugation) and it was taken with the setup just described. It is a single frame, focus stacked image of a prepared slide.
But. with just a little imagination, we can of course go much further than this. We could take overlapping frames of a much large specimen to form a mosaic, and then also focus stack each individual frame as well. This is pretty work intensive stuff both on the microscope, and on the computer – but the end result can be well worth the effort. After creating each focus-stacked frame, stitch all the overlapping frames together using software such as PTGui and you’ll end up with something like I’ve managed to do with this Fruit Fly 🙂