Over the last week, I’ve been on safari. Not in Africa, or even a cheesy safari park, but in three drops of water on a microscope slide. I spent multiple hours looking at a number of slides with drops of river water on them from the Tama River, which runs just five minutes from our Tokyo apartment. The experience was, to a degree, relatively life-changing. I was amazed by not only the number and variety of organisms found in just a few drops but possibly more intriguing was their resilience.
Not only did they seem to be perfectly happy to live in a glass jar for around four days before I put them back in the river, but they also seemed to be mostly fine with being tapped off their root or scraped off of a piece of reed, and sandwiched between two pieces of glass for hours at a time. They just kept going about their business, rummaging around, eating, and crapping, regardless of where they were.
I actually emptied the four jars of water and various organisms back into the river last Sunday, and I’ve spent most of the last week creating a video, which I’ve embedded into this blog post below. I’d have completed this a day sooner, but DaVinci Resolve decided it would be a good idea to delete four days worth of editing while I had breakfast on Thursday, and the rest of Thursday was taken rebuilding what I’d lost, so this has taken longer than necessary, but I’m happy with the results. The video is the main content for this week, but I did want to share a few photos with you as well, and talk about some of the creatures that I met on my microscope slide.
I’d also like to warn you before you look at the video, that it may actually disturb some people to know what is in plain old river water. I found the majority of the organisms attached some plant life that was put into my sample jars, and the majority of what I saw was, in my opinion, as cute as can be. But while looking at some cute plankton, we were occasionally visited by what looked like, in comparison, I giant almost transparent anaconda snake, with lots of little four-toed legs.
I’ve put two warnings in the video, shortly before the anaconda enters the screen, and I mention how long it will be there as well if you want to overt your eyes. I personally found it fascinating, like the rest of the organisms, but I imagine some people will not be overall happy with the visit. In fact, even the plankton going about its business might disturb some people, so only watch if this sort of thing interests you. Anyway, here is the video, starting with an explanation of the project and a bit of footage of fetching the samples, followed by around 30 minutes of footage through the microscope. As you watch, keep in mind that there are two main types of plankton. Phytoplankton, which are plants, and zooplankton, which are animals. Pretty much everything in this video falls into these two groups of plankton.
I hope you enjoyed the video. Let’s also take a look at a few of my favorite photos from the project, and I’ll explain some of the challenges faced as well. First up, here is a light field photograph of a Nematode otherwise known as a roundworm. These are very common, and can exist in both water and in soil, and are also able to live in acidic substances such as vinegar. They can be parasitic, and some species will bore into the soles of human feet if you walk barefoot in a river or on soil with Nematodes in it. I’m including this shot mostly to start explaining the difficulties of photographing plankton.
Nematode
Because this was light field, with light being passed directly through the microscope slide, there was quite a lot of light to work with, especially as I’ve customized my microscope to add more light via an additional LED ring light around the original single LED light, but still, to try and freeze this Nematode, which tend to wriggle around all the time, I increased my shutter speed to a 1/500 of a second. With this shutter speed, because of the higher light levels, I was able to use an ISO of 6400, which is good compared to most of the following examples I’ll share.
I then switched to dark field microscopy, which, as I explain at the start of the video, is a technique that can be used by placing an opaque disk between the light source ant the subject, and adjusting the size of the disk, the distance of the light from the slide, and the diaphragm that controls the diameter of the light, so that the light spills onto the subject from the sides rather than passing directly through it, as in the previous image.
Rabbit Rotifer with Phytoplankton
What you are looking at here is a Lapadella, a type of Rotifer, which in my Japanese Plankton book translates as a Rabbit Rotifer, probably because the two-toed tail that you can just about see in this image looks like rabbit ears. You’ll see that better in the following images, but I wanted to include this to show how dark field microscopy illuminates the surrounding phytoplankton, and in my opinion makes for much more pleasing images. There is also less to clean up. With the previous light field image I had to clean up the background quite a lot, but this image is pretty much straight out of the camera, with just a simple tone curve applied to increase the contrast a little. Also, to try and freeze this Rotifer as it swam around, I tried working with 1/1000 of a second shutter speeds, which required ISO 20000 to get this exposure, so I was starting to push the limits a little here.
For this next image though, I changed to the 40X objective lens, to get a closer look at the Rabbit Rotifer, but with that, because of the lower light that the lens collects at this magnification, the ISO jumped up to 51200 at 1/1000 so I had to run this through On1’s NoNoise AI software to clean it up. That still produces a usable image, but definition drops slightly using these settings.
Rabbit Rotifer
I’m also fighting with the poor image quality that I get with my camera adapter, as well as the very shallow depth of field. I have been overcoming these restrictions by doing a lot of focus stacking, but for moving subjects, focus stacking isn’t possible. I got lucky with this next photo, as this creature, which I believe is a type of Copepod, stayed very still for a while as I shot a 39 frame stack. Shortly after I finished my stack he swam away at speed, so I was very lucky, but the stack also recorded trails in the debris that flowed past the organism as I made more frames, which I thought was quite effective as a photograph as well.
Copepod
Because it was stationary though, I was also able to reduce my shutter speed to 0.3 seconds and use ISO 400, so the image quality is greatly increased with this settings and the focus stack. This was one of just a few images that I was able to shoot like this though, with the majority of the rest being single shots, struggling with shutter speed and ISO settings trying to make the most of what I was being presented with.
Having learned from the first few days that I really needed to get my ISO down lower than it was going, I reduced my shutter speed to 1/250 of a second, and accepted a little more subjected movement over grain for this next image, in which I was able to get three Rabbit Rotifers in the frame together.
Three Rabbit Rotifers
They are providing a top, bottom and side view, so this is one of my favorite shots that really illustrates this beautiful life from.
In the next image we can see the outlined lorica which almost look like little wings, but it’s actually just the outer shell of the Rotifer being caught by the light and actually continues down to the base of the foot.
Rabbit Rotifer in Open Space
Still at 1/250 of a second shutter speed, my ISO was a 20000 for this shot, Once again here I ran this through On1 NoNoise AI to bring the grain caused by the large dark portion of this image under control.
This final shot is another of my favorites, shot with the 40X objective, the Rabbit Rotifer was stopping still for a few moments at a time, so I reduced my shutter speed to 1/125 of a second, and got a few shots where it was still, and relatively clear. There are also a couple of air bubbles which I was intrigued to find that the plankton has a very hard time with. The surface tension of the air is impenetrable despite seeing the Rotifer interact with the air a few times. It’s like a solid wall to them.
Rabbit Rotifer with Air Bubbles
I don’t really have anything that I’m all that happy with of the Mouse Rotifer, which appears frequently in the video. They are slightly smaller than the Rabbit Rotifer, but their behavior is just like little mice, scurrying around in their environment, eating the algae on the phytoplankton. Anyway, we’ll leave it there for now. Please do watch the video when you have time. If you are listening to this you can jump directly to the video on Vimeo with the short link https://mbp.ac/universe.
Following on from my previous post about the moral dilemma I’m struggling with regarding killing an insect for a photograph, we’re going to put that aside for this episode, and simply talk about how I went about the process. If you don’t agree with this and feel uncomfortable listening or reading, please turn this off or close your browser now. I won’t do a lot of this kind of post, but I do think that there will be at least a portion of the audience that will find this useful, so here goes.
I’ve been quietly preparing to photograph insects more over the last few weeks, based in part on the very informative videos from insect photographer guru Allan Walls, so a public shout-out and thank you to Allan for making that stuff available. You can actually see most of what I used in this photo, so let’s walk through this item by item, and I’ll fill in some gaps as we proceed. Firstly, you’ll see the jam jar that I caught the fly in, and then poured in 90% alcohol and 10% water which both kills and cleans the fly. Allan Walls also recommends using an ultrasonic jewelry cleaner to clean off stubborn dust that can form on insects, but so far I haven’t needed this. Maybe at some point, I’ll pick one up.
Preparing the Housefly to Photograph
I left the fly in the alcohol overnight, both because it was late when I caught it, and to ensure that any parasites that might have been inside the fly were also killed by the alcohol. Then, before I photographed it, I placed it in a Petri dish with some distilled water in it, to rehydrate the fly. I then selected a #2 insect pin which is the second to thinnest pin in that selection of pins you see in the white envelope on the right. You can also see the pin next to the Petri dish on that sheet of lens cleaning paper. I used a pair of very fine tweezers that you also see here to open up the wings of the fly a little, and also pull its legs away from its body a little too, then as it dried naturally in the air, I placed a tiny blob of superglue onto the insect pin and stuck the pin to the underside of the fly’s abdomen.
The fly was probably only 3 to 4mm in length, so I used the magnifying glass that you see on the left with the built-in USB-powered LED lights to see what I was doing. The paper is lens cleaning paper that I use to clean my microscope slides, and, of course, lenses, but reuse them like this when dried to guard against spilling alcohol, etc. on my work surface. Once they actually get dirty I throw them away. After preparing the fly, I use a soldering stand to hold the insect pin and position the fly under my Stereo microscope to start photographing it, as you see here.
The Housefly under a Stereo Microscope
I didn’t photograph every step of the shoot, because keeping the fly out of the alcohol or water gradually allows it to dry out, and the eyes start to collapse in, which doesn’t look good. For some of the images, I used a piece of black velvet below the fly to give it a black background. The microscope came with a black plate that replaces the semi-transparent circular plate in the above photo, but it’s so reflective that it appears almost white when illuminated from above, so I find that the black velvet soaks up the light much better. I am also using an LED ring light that attaches to the bottom of the microscope lens, providing really nice adjustable light for the subject.
Here is one of the first shots of the Housefly using my Stereo microscope and the 2X Barlow lens. My Stereo microscope has a zoom mechanism that provides 7-45X magnification with the 10X eyepieces, and the Barlow lens doubles that to a 14-90X magnification range. I haven’t yet calibrated my Stereo microscope to find the magnification of my camera adapter with this scope, but it’s probably around 2X, so we’re actually looking at around 24-180X magnification in the photo with the 2X Barlow lens in place.
Focus Rings in Eyes
I learned an important lesson with this first photograph though, and that is that you have to be very careful with the amount you adjust the focus when shooting images of insect eyes to focus stack. You can probably notice the four slightly darker rings around the top half of the eye that is caused by too large a gap between my focus stack images. To prevent this I turned on the “Focused” checkbox in Helicon Remote, which shows the area of the specimen that is in focus in blue, as you can see in this photograph of my computer screen as I worked.
Focused Zone in Helicon Remote
Once I’d identified the problem and the cause, I started to place my thumb on the back edge of the focused area on my screen and then adjusted focus for the next image with my finger marking the previous edge, and I only moved the focus halfway back from there, so it was half overlapping the previous image. When you shoot the eyes like this, you don’t get the nasty focus error rings that you see in the earlier image of the Housefly.
Extra “Hand”
At times like this, it helps to have an extra hand to trip the shutter, so I used the built-in Mac OS Voice Control feature, found under System Preferences Accessibility options. There were also times when I was photographing the fly with my compound microscope when I was holding a flashlight to illuminate the subject, and I also have to adjust the focus of the microscope between each frame for the stack, so I set up a voice command to enter the keyboard shortcut to take a photo in Helicon Focus every time I said “new photo”. I also configured a command to create a new stack every time I said, you guessed it, the words “new stack”.
Housefly Under a Compound Microscope
Scarab Torso (10X 40 Frames Stereo Microscope)
There were also times when although I was not holding a flashlight, or marking the focus zone with my thumb, but it just kept things more stable by not physically touching my keyboard. It helps reduce vibration on my work surface, so the voice control came in very handy.
Parallax Shift
There is one limiting factor working with a Stereo-microscope for photography, in that, because they are designed to provide a stereoscopic view of the specimen, there is a parallax shift as you adjust focus when redirecting the image from a single lens to the camera port. This means that as I adjust the focus the subject gradually drifts across the screen, and will eventually move so far that there is nothing left to stack, so I am still limited to a certain depth of field. In this photograph of a scarab beetle, which I luckily found dead outside of my apartment recently, I initially tried to stack enough images to get the entire beetle in focus, but the parallax shift caused the beetle to move so far over to the right that I ended up with only half a beetle after stacking. To avoid that, I stopped my stack around halfway down the beetle, and I actually like the out-of-focus abdomen anyway, so all was well. This still took 40 frames to create this image, with around half of the scarab beetle in focus.
Anyway, having figured out how to photograph the eyes on the housefly without the nasty focus rings, I also shot a few stacks that resulted in some images that I am relatively happy with, such as this one, off slightly to the left of the face. I darkened down the body in this and a few of the images, as there are areas that don’t look great, but also there were a few stacking issues that left a few too many remnants, but I actually am mostly interested in the face of the Housefly.
To get this look relatively naturally I used an adjustment brush in Capture One Pro and brushed out the background in a number of different layers, probably up to five or six per image, and allowed some of the layers to overlap with the back of the eyes a little so that it looked like the fly was poking into a spotlight rather than being more fully illuminated from the start. I reduced the Exposure and sometimes used a tone curve adjustment on the layers to gradually darken the background.
Common Housefly (60X Stereo Microscope)
Common Housefly (60X Stereo Microscope)
To the right, above, I’ve also included a shot where I didn’t totally darken down the body, and again, without the focus rings in the eyes, so I’m pretty happy with this. Both of these images were shot at around 60X magnification through the eyepieces of the microscope. It’s a little bit scary to first look into the eyepieces at this magnification, but seeing all of the detail in this creature is incredibly fascinating.
Here are two final images to finish with. The first was shot with my Compound microscope, illuminating the Housefly with an LED flashlight, at 100X magnification. The second and final image is from the Stereo microscope again, and I had started to run out of time as the eyes were gradually losing their luster and starting to concave very slightly in a few areas.
Common Housefly (100X Darkfield Microscope)
Common Housefly (50X Stereo Microscope)
As I mentioned in my previous episode about the moral aspect of doing this, this fly was pretty much doomed the moment it came into our apartment, but I don’t feel 100% happy with killing it for these photographs. I’m struggling with the decision a little bit, although I do find it fascinating. For larger insects, I’m pretty much going to rely on finding them dead after they’ve lived their lives, and see how much I can make of that. As I mentioned earlier, I’m not going to do a lot of this kind of episode, so if you don’t like it, stay tuned anyway, as we’ll be back to my regular work more often than not as we move forward.
Exactly two weeks after my Compound Microscope arrived, today I’m going to pull together another status update, to let you know how I’m getting along and share some of my new work, which I will tell you upfront, I’m very happy with. I also took possession of my Stereo Microscope a week ago, but some of you, I know, will be pleased to hear that I’ve decided to keep both microscopes. I was a little hesitant as to whether or not I would keep the Compound Microscope, mostly due to the total financial outlay involved in buying both together, and partially due to the fact that I was initially finding the learning curve relatively steep.
Not wanting to let it get the better of me though, I continued to study various techniques, bought a few more supporting pieces of equipment, and I am now having so much fun, I doubt that either microscope will be leaving my possession in the foreseeable future. As I mentioned in my first post on this topic, a large part of me diving down this rabbit hole was inspired by my friend Don Komarechka who’s just released a new book on Macro Photography and it has a section on using Microscope optics to get photos and covers the procedure for making crystals out of citric acid mixed with water and or alcohol, and I also got a lot of great information from the Canadian Nature Photographer website.
Both resources made it sound relatively easy, although I initially tried simply mixing the citric acid with water just by stirring it in a plastic Petri dish and found that the surface tension of the resulting liquid was so great that it wouldn’t spread out on the slide, so I ended up with little mountains of crystals which although could be photographed, weren’t really satisfying me artisticly. The mounds of liquid were also so thick that they took around 12 hours to dry.
Then I noticed in an article by Robert Berdan of the Canadian Nature Photographer website that he used a Vortex Mixer to mix the ingredients, so another $60, and two days later I tried that method and created ten slides with just IPA (Isopropyl Alcohol) and citric acid and another ten slides with half water and half IPA. As you can see from the center image below, there is some citric acid settled at the bottom of the test tube. This is because the liquid became saturated before all of the citric acid had melted, so I simply used a pipette to take just the solution from above the settled acid crystals and applied a few drops to each slide. This time it spread out very quickly, pretty much over the entire face of the slide, and both solutions dried very quickly, providing very similar results.
INTLLAB Vortex Mixer
Citric Acid with 1:1 Water and IPA
Makeshift Polarization Setup (Not Ideal)
The way the patterns in the crystals formed was very similar with IPA alone and when I also mixed it with water, but simply by the nature of this experiment, the crystals that form are unique, not only from slide to slide but from millimeter to millimeter of each slide. I created a short video which is embedded at the bottom of this post to show you through the microscope as I surveyed just a small area of one slide.
Note too, that to turn these crystals into beautiful abstract art, the light has to be adjusted in a specific way by two separate polarizer filters. As you can see in the photo (above right) there is one polarizer filter between the light source and the subject, and a second between the subject and the viewing lenses. Apparently, this happens when the subject is what’s known as anisotropic or birefringent, which means it has double refraction.
The makeshift setup I’ve used so far is not ideal because the thickness of the filter prevents me from using the 40X objective lens, which would give me 400X magnification. Over the last week, I’ve been restricted to using my 4X and 10X objective lenses for magnifications of 40X and 100X through the ocular lens and on my camera. My Camera is actually magnified by around 2.5X magnification because of the adapter, so in real terms, we’re probably talking between 100 and 250X magnification in the images, but I’ve not accurately measured that yet.
Because my first filter setup was not ideal, yesterday, I managed to find some inexpensive cylindrical diamond cutters that contained sizes that would enable me to use the piece of polarizer from the inside of the cut to fit into the filter holder below the diaphragm of my microscope and a second in the space between the objective lens turret and the rotating head that houses the two eyepieces and camera port. The filter above the turret will be more difficult to rotate, but the bottom filter above the LED light is more accessible, and so far my tests have shown that it doesn’t really matter which of the two filters you rotate. It’s more about the degree of rotation between the two filters, so I think this new setup will be better and allow higher magnifications to be used. Here are two photos of the new, somewhat roughly cut filters that I made, in place.
Polarizer Filter in Diaphragm Filter Holder
Second Polarizer Between Objective Lens Turret and Head
Before we take a look at a few of the resulting images, here too is a photo of my work table as I got started photographing the slides while they dried. For the first few minutes, I was actually watching the crystals form through the microscope, which I also found fascinating. Since this first evening using my laptop as you see in the photo, I moved my microscope to my main desk and connected my camera to my iMac instead, so that I could use the large screen, and the more powerful computer to deal with the focus stacking using Helicon Focus, which I’ve started using to process the images that I’m making.
Photographing Polarized Citric Acid Crystals
I was happy to get my camera connected up using the adapter that I bought, although the image quality is about as good as I’d expected, it’s not as good as I’d hoped for. At some point, I think I’m going to take the plunge and go for the LM Microscope adapter, but spending almost one and a half times more than the cost of both microscopes just for the adapter is not a decision I’m really able to make at this point, especially as I’m getting good final results, mostly down to processing.
My First Results I’m Happy With
Anyway, let’s look at some of my favorite example photos from the last week, starting with one of the first ‘scenes’ that I was presented with as the slides I prepared dried. As you can see, the variation of the shapes and textures of the crystals varies greatly. The blue and brown colors that you see, as well as the rainbow colors visible in some small areas, come from the polarization. When you rotate one of the two polarizing filters the colors change dramatically.
This was a 14 frame stack initially shot with Capture One Pro tethered to my Canon EOS R5, but Capture One froze on me as I created my stacking sets, and I lost around 40 of my last images, so I turned to Helicon Remote which is built for shooting sets of images for stacking, and there is no looking back. I’m going to dedicate the next post on this subject to Helicon Focus and Helicon Remote, so stay tuned for that if you are interested in focus stacking.
This next image has quickly become a firm favorite and caused a bit of a stir when I shared it on the social networks last week. To me, this looks like an alien flower head that has been discarded, although you may see something completely different. Notice the small line in the center of the disc, which will be a bit of fiber or dust that fell onto the slide and is responsible for the disc of crystals forming at that location. Both of these images were shot at 100X magnification.
Alien Flower Head (Polarized Citric Acid Crystals 100X)
Another type of image that I’ve been getting is this kind of electric rainbow-colored image, with very rich colors. Again the intensity can be changed by the degree of rotation between the two polarizer filters. I chose this section to photograph because to me it looks like a Hokusai print of a blue Mount Fuji here in Japan.
Blue Fuji (Citric Acid Crystals 100X 7 Frames)
Almost in complete contrast, this image looks like frail feathers or flowers to me. Watching these crystals form under the microscope was one of the most fascinating things I’ve ever done. I mentioned a few weeks ago that I was scratching a long term itch by starting this project, but at the time I had no idea that it was going to feel this good.
Once again, in contrast, here is my electric alien fox with his golden grasses and whacky alien flowers, running along the edge of a magical lake. I actually placed a glass cover on a couple of the slides to see how the cover interfered with the crystals as they formed. I was happy to see that the crystals reacted by forming along the seem as you can see here.
Foxy Alien (Polarized Citric Acid Crystals 40X)
Here is another very colorful shot that seems like an explosion in a Japanese anime movie. Again, the variations that can be found are astounding!
Here We Go! (Citric Acid Crystals 100X 12 Frames)
This next shot is another favorite. It reminds me of a wreath made from feathers. This is actually two separate 28 image stacks stitched together to form a square image. I purposefully rotated the polarizer filter so that the colors become more subdued for this shot, as I thought the cool blues would suit the subject better. There were richer colors initially, but it didn’t quite work for me.
Space Fairy with Pet (Citric Acid Crystals 100X 17 Frames)
The image to the right above looks to me like a fluffy alien fairy sitting in tall grasses or even the feathers of a peacock’s tail. The little alien has a canary pet in its arms. I wonder if you can see which part of the image I’m referring to? Don’t forget to click on the images to open them up in the Lightbox for a better view.
This next image to me is almost a complete miracle. Nature has created an almost perfect mountain range with a glacier and even the light of the moon shining through thin cloud coverage. This is almost untouched, with the only changed being a little bit of cloning in the corners to fill in the slight vignette that I’m getting from the camera adapter.
Mountain Range and Moon (Citric Acid Crystals 40X 20 Frames)
OK, so one final image, that has become pretty much my favorite of all the images I’ve shot over the last week. I see something different in this image every time I look at it, and just find the overall feel of the image very appealing.
Convergence (Citric Acid Crystals 100X 8 Frames)
Crystal Micrographs Portfolio
There are a few more things that I’d like to relay as we start to wrap this up. Firstly, I’ve pulled together a portfolio of my best Crystal Microphotographs so if you want to see more, please check that out.
Wall Art Now Available!
Also, as I think many of these photos would make good wall art, I’ve added a number of them to my wall art store on the Art Storefronts Website, which you can check out here.
Surveying a Slide and Polarization Effects Video
Finally, here is a video showing some footage of me surveying my first slide and also showing the effect of rotating one of the two polarizing filters. It’s under two minutes, really just a glimpse so that you can see what this is like.
As I mentioned earlier, in the next post on this topic I’ll walk you through how I’m using Helicon Remote to shoot my sets of images and then Helicon Focus to stack my images together. I hope you are finding this interesting. If you are, note that I have also added a Micrography page under the Posts menu, and will pull together everything about this new endeavor on that page, so bookmark that if you are interested in following my Micrography antics.
