Photographing the Fluorescent Bones of Amphibians using “X-Ray Vision”

“How did you make that photo look like an x-ray?” It’s one of the questions I get asked most frequently, usually referring to one of the images below. The answer is that I get this x-ray effect by using fluorescent lighting while photographing some species of amphibian! Here’s a quick behind-the-scenes look into my process of shooting fluorescent, “x-ray” photos in the field.

What Is Fluorescence?

Before we go any further, let me take a moment to explain what fluorescence – and, in this case, biofluorescence – is.

First of all, biofluorescence is not to be confused with bioluminescence. Bioluminescence is what most people are more familiar with: organisms producing their own light. For example, fireflies, angler fish, and glow worms are well known examples.

In contrast, organisms with biofluorescence do not emit their own light. They look normal under everyday lighting conditions. The fluorescence of these organisms is only apparent to the human eye when activated by light of a specific wavelength. When that light hits certain pigments, they reflect the light back at a lower energy level, which is what gives the fluorescent appearance.

A popular example of biofluorescence is in scorpions. When an otherwise bland scorpion is lit by a black light (aka UV light), it will appear very bright and look like it’s glowing. In reality, it is not glowing; it is just fluorescent.

Fluorescence in Bones

As it turns out, the collagen in bones is fluorescent. This is why teeth glow eerily under black light! You may recall seeing this in haunted houses around Halloween.

Teeth aren’t technically bones, but bones do the same thing – it’s just that we don’t see it because, well, they are in our bodies. But some amphibians have such thin and nearly translucent skin that their bones are actually visible with the naked eye.

One More Thing to Know About Fluorescence

As nice as it would be, not all types of fluorescence are visible by simply shining the right light on the critter. With many organisms, an additional filter is necessary to block out all the light that is not reflected as fluorescence. This way, only the right wavelengths of light reach your camera lens (or your eye when wearing filtered glasses) and the activation light does not overwhelm the viewing experience.

So, to be clear, in order to observe some cases of biofluorescence, the light from the activation light must travel to the critter, get absorbed and re-emitted by the pigment, travel through the filter, and hit your eye or camera lens! The activation light and filter must be carefully tailored, too, to the wavelengths of light that cause fluorescence in your subject. It also looks a bit funny when you’re wearing the observation goggles, especially when a frog jumps on your face!

Behind the Scenes

Capturing the following image of the hand of an arboreal salamander (Aneides lugubris) was no easy task! It was a team effort with a friend, made more challenging by the fact it was raining when I pulled it off. Luckily the salamander was kind enough to stay put for the camera.

The Arboreal Salamander is a species of climbing salamander that can be found crawling on trees during rainy nights in California. Their excellence in climbing is likely part of the reason their hands are eerily human-like, especially the bone structure!

Let me go into a bit more detail about how I captured it in the field.

1. The Gear

To pull off that shot, I used a kit from Nightsea, specifically a bluelight and yellow longpass filter. The bluelight is simply a flashlight that shines bright, blue light. The filters that came in the package were simply a pair of glasses and a small square of the filter material.

For photographic purposes, I had to shoot through either of these. (Apparently there are some screw-on yellow filters that can also work, but I didn’t have my hands on these.) As you can see, haphazardly dangling the filter or glasses in front of the lens was not an easy task, especially while illuminating the subject with the flashlight to capture the fluorescence. But it wasn’t impossible.

2. The Settings

Although the blue light itself is quite strong, once it is passed through the filter, the fluorescence is not very bright. For this reason, I had to use a long exposure and a tripod. I also wanted a reasonably large depth of field to keep the salamander’s entire hand in focus, so I stopped down my aperture to f/11. Lastly, because I shoot Micro Four Thirds, I needed to keep my ISO on the low side (in this case, ISO 160). Combined, all of this necessitated a long shutter speed of 10 seconds.

While 10 seconds is usually way too long for wildlife photography, the tripod held my camera steady and the salamander thankfully stayed relatively still. The result is the nice, crisp shot that you saw a moment ago.

3. The Process

While I took the photo, my friend dangled the filter glasses in front of my 60mm macro lens. I fired the shutter and started light painting the salamander’s foot with the bluelight, coming from several angles to avoid any harsh shadows.

It took a few attempts because the salamander would occasionally move, or something in the light painting process would go wrong. Plus it was raining. I recall it being a challenge, but not one of the fun ones! I was close to my wit’s end, but I’m glad I kept trying, because the final shot is quite otherworldly.

Skeleton Frog

Glass frogs are awesome little frogs that live in the rainforests of Central and South America. They are called glass frogs for their extremely thin skin. You can often see many of their organs and bones. Their bones also fluoresce, though not quite as brightly as the salamander’s, as you can see below:

The process behind this shot of a Reticulated Glass Frog, Hyalinobatrachium valerioi, was more or less the same as the image of the salamander foot. However, I used a faster shutter speed at the cost of a wider aperture and less depth of field. I really wanted the whole frog in focus, though, so I did a focus stack of three images.

I want to bring your attention to one more aspect of these shots, which is that the bones are not the only things in the shot exhibiting fluorescence! The red areas of the photos are the result of chlorophyll in nearby plant material. Chlorophyll are the pigments in plants responsible for photosynthesis, and they’re what make leaves green. The collagen in the amphibian bones fluoresces bright green, but the chlorophyll fluoresces bright red.

Viewing your surroundings under fluorescent conditions truly paints the world in a new light!

Final Thoughts

Ultimately, I think the fluorescing bones of these amphibians simply look cool, as opposed to having some significance to the species’ natural history. Most likely, many instances of biofluorescence are simply a random side-effect of the pigments, in the same way that our teeth fluoresce under UV light. But perhaps there are some reasons for these traits that scientists will one day uncover. For instance, some deep-sea fish are capable of seeing fluorescence (again, this is different from their bioluminescent capabilities), so it is not an entirely “hidden” aspect of these creatures.

There is plenty to speculate about the purpose, or lack thereof, of biofluorescence. But one thing is certain – it looks pretty rad.