It’s not always easy to see stomata, the pores on the surfaces of leaves that allow plants to breathe. I tried the leaves of pepper and tomato plants with a dissecting microscope to no avail. However, compared to these, the stomata on basil leaves were enormous. They were actually visible on the lowest magnification (6x), but the higher magnification is necessary to make out the detail.
For these pictures, I was lucky enough to have gotten to try out one of Leica’s research grade, digital microscopes (the DMS1000b). Given that I only had a hour or so, I did not take the time to experiment with all the optimization options, however, the pictures turned out remarkably well, none-the-less. In particular, you might note the highest magnification images, labeled 48(d)x, are just digital enlargements from the 48x magnification pictures.
Having the built in camera makes it an awful lot easier to put images into the online microscope app, and I suspect will make focus stacking a lot easier as well. Since this scope is a bit out of the range of my small high-school budget, I really need to figure out a good, solid way of mounting my point-and-shoot cameras on the microscopes I have.
Now that I have a new set of microscopes I didn’t think I would actually need to have an online, simulated microscope to show samples. However, I thought having a series of picture that I could scroll through would be useful to illustrate microscopy concepts such as depth-of-field when I talk about them to the whole class. Once I’d created the depth-of-field simulation, I figured it would not be too much extra trouble to put in a few different magnification levels. Now I have this embeddable online microscope simulator.
It’s started off with a single fly wing as a sample, but I’ll be adding to it as I take more pictures.
At higher magnifications, microscope lenses will only be able to focus on layers within your specimen. You could take a series of images with different focal planes and stack them together, but without a camera mounted on the microscope, getting images to line up right for focus stacking is quite the challenge. The alternative is focusing in and out until you get a feeling for the three dimensional shape of the specimen.
Since I don’t have a camera mount I’ve created an html5/javascript page that simulates focusing in and out of a sample. It’s embedded above, but a direct link is here.
You can use the knobs to the right of the image to adjust the focal plane. You should be able to see hairs on the top and bottom of the transparent wing.
Close up of a fly’s smaller, rear wing 1 hour after being mounted in nail polish. Image taken using my compound microscope at 100x magnification.
Now that I have a few new microscopes, I’d like students to be able to make their own, permanent, slide collections. Walter Dioni has some superbly detailed pages on how to mount samples for microscopy. I could not find a good, up-front, index, so for the record, here are his pages on mounting slides:
Most of these methods use chemicals that are safe to work with (all are non-toxic), but using nail polish appears to be the easiest — it’s a mount and a sealant in one — so that’s the one I tried first, using a bottle of Strengthener, Nail Hardener. The Karo syrup, and glycerin methods also seem reasonably easy, and it may preserve some of the organic colors better so I may try those later when I have the time.
The fly’s rear wing one hour after being mounted in nail polish. Image taken using my compound microscope — 100x magnification.
While walking through the woods to recover the skeleton the other day, I picked up, or rather was boarded by, a few ticks. So when I got back to school I plopped them under the stereoscopes to try to identify them.
Lone star tick (Amblyomma americanum) (adult female?) from the woods behind Maggie’s house (Missouri). Magnification ~20x; dorsal view.
They were both lone star ticks (Amblyomma americanum): one adult and one juvenile.
Lone star tick nymph (magnification 45x; dorsal view).
Under the microscope, they were quite pretty with their very interesting red and black patterns.
Much greater detail can be found in the Tick Gross Anatomy Ontology, but the best reference I’ve found so far is the USDA’s Handbook (485): Ticks of Veterinary Importance (pdf). There you can find great anatomy diagrams and interesting biological and ecological information. One curious piece of information is that ticks can survive a long time (three years in one case) without a blood meal. It also includes some excellent diagrams:
Diagrams of lone star tick external anatomy from USDA Handbook 485: Ticks of Veterinary Importance.External anatomy of hard bodied ticks. From the USDA Handbook 485: Ticks of Veterinary Importance.
There’s a tradeoff involved when you try to focus on things under a microscope. The higher the magnification the less you’re able to focus on at a time. Images 1, 2, and 3 in Figure 1 show a catalpa pollen grain under 400x magnification. In each image the microscope is focused slightly differently to bring a different level of the pollen grain into focus.
Three images (image 1, image 2, and image 3) are stacked together to create a final, focus-stacked image that is in better focus (has a larger depth of field). The images are of catalpa pollen grains at 400x magnification, stacked using Hugin Tools.
I tried two methods for doing the image stacking. The first was with the command-line programs in Hugin Tools, while the second was by hand using GIMP.
Hugin Tools
The method for focus stacking with Hugin Tools is described here by Patrick David and here by Edu Perez. It requires two commands, one to align the images, because focus-stacking requires very well aligned images, and another to stack them together.
Align the images using align_image_stack:
align_image_stack -m -a als_ catalpa-c[123].tif
This command takes three image files (catalpa-c1.tif, catalpa-c2.tif, and catalpa-c3.tif) and produces three aligned images prefixed with “als_” (als_0000.tif, als_0001.tif, and als_0002.tif).
The images are stacked together using enfuse (details here).
which produces an output file called catalpa-pollen-out-b.tif.
Focus-stacked image of a catalpa pollen grain using Hugin Tools. (Magnification 400x).
GIMP
Focus-stacking with GIMP requires opening all the files as layers and adding transparency masks to the layers to leave behind just the areas that are in focus. The general method is shown in this GIMP tutorial.
Aligning images by hand and then selecting the areas to cut out can be quite tedious.
400x magnified, catalpa, pollen grain that was focus-stacked by hand with GIMP.
Notes
Two key things to keep in mind are:
the better aligned images are to start with the easier they are to focus-stack. Aligning images is tedious even with align_focus_stack;
the quality of the camera matters a lot. The images above were taken with a Moticam 2 megapixel camera attached to the microscope to reduce misalignment. The image below, however, was taken with a point-and-click, 6 megapixel camera down the eyepiece. You can see a lot more detail.
Catalpa pollen grain taken with a higher resolution camera.Higher resolution, focus-stacked image of catapa pollen grain. (magnification 400x)
More testing of the higher powered stereo-microscope with this flower specimen from a catalpa tree on campus.
Anthers with pollen grains (~25x).
The catalpa tree leaf and flower for reference.
Large catalpa leaf and two flowers.Detail of longitudinally bisected flower (~7x).Closeup of petal (~35x).The colors on the petals come from cells having different colors (~90x).
