The Nikon 2020 story, or: I’d trade a kingdom for clownfish embryos ...

The first attempt is not always successful. But it's worth sticking with it, whether in coral reef aquaristics or in photomicrography

Two clownfish embryos on the eighth day of their development, shortly before hatching from the egg (focus stacking setup, Mitutoyo M Plan Apo 10x, three system flash units)

It all started with a photo of a clownfish embryo that I saw in a scientific publication. This embryo, about two millimetres in size, was still in its egg and had been photographed in such a way that some details could be seen quite well – with all the blurring that such a close-up inevitably entails. What a fascinating picture idea: nascent life waiting in the transparent egg shell to be allowed out into the world and already looking at its surroundings with large, silvery eyes.

This spontaneously awakened in me the desire to make it at least as good, ideally even better. So that you could see anatomical details in the photo – in the center the yellow, bulging yolk sac, which should provide the little animal with food during its first days after hatching.

However, I saw the whole thing as a challenge and didn't want to take the easiest route and choose the common, widespread clownfish Amphiprion ocellaris, which is very easy to obtain from captive breeding. Instead, I planned to use the very similar, but somewhat prettier colored Amphiprion percula, which was the model for the TV movie “Finding Nemo”.

It took five years to mate two of the rare percula clownfish

Finally, the first clutch of eggs was available for the focus stacking project

However, this made things much more difficult, as it is very rare to come across a breeder who works with exactly this species, especially as the two species are extremely difficult to tell apart. There was a further complication: two juveniles that have hatched from a clutch of eggs can hardly be mated, because the pairing of these protandric fish is a socially controlled sex change; initially the two juveniles are male, and the stronger of the two suppresses the other in continuous fights, with the result that he himself develops into the dominant female, while this is suppressed in the other and he becomes the subdominant male of the pair. This is the prerequisite for pair formation, and littermates are usually the same age and of similar strength, so they wear each other out in endless scuffles.

Clownfish pair formation

It took me a whole five years to succeed in pairing two healthy offspring perculas in the coral reef aquarium. They lived in their host anemone, a magnificent red Entacmaea quadricolor, which also came from aquarium propagation. The two clownfish snuggled permanently between the anemone tentacles and, after some waiting time, produced their first clutch of around two hundred eggs.

I was already present with the camera during the fertilization process. The smaller male attached numerous tiny sperm sacs to the eggs, which the stronger and larger female was sticking to the rock substrate, each of which stuck to the transparent egg membrane, which was around two millimeters long. Inside the eggs there was nothing but a yellow globule in which numerous tiny droplets of fat could be seen.

The 10x microscope objective from Mitutoyo can use the focus stacking technique to make the small sperm sac visible on the 2 mm long egg, and even individual sperm cells can be recognized!

The first series of pictures

Every day I stole some of the eggs to put them on a glass slide with a concave depression in the middle, which was filled with a drop of seawater and covered with a cover glass. Under the microscope, the development inside the eggs was very easy to observe. I used my Leitz Orthoplan microscope, which was one of the most popular research microscopes in clinics and universities worldwide from the 1960s to the late 1980s. I had equipped it with transmitted light objectives and used a 10x Planapo. However, I needed incident light for an aesthetically pleasing image, and this was provided by a two-headed LED light that shone onto the egg from both sides. A series of images was created with gentle rotations of the fine adjustment knob and then compiled into one photo.

Every morning and every evening I took three series of 100 individual pictures in this way, for eight days, until the embryos had hatched and passed into the larval stage. The picture results were phenomenal – at least I thought so at the time. I looked at the pictures documenting the entire embryonic development with great pride. It was easy to make out the U-shaped folded body in the transparent egg membrane, with swim bladder, eyes and numerous anatomical details. But somehow the colors were pale and unattractive, the light distribution was horrible. There had to be a better way ...

The first series of experiments: Orthoplan microscope with transmitted light objectives, but photographed with incident light

The second series of images

I also used the Orthoplan microscope for the next clutch of eggs produced by my still quite young pair of clownfish, but I used different objectives, namely a set of HD Planachromats from Zeiss Jena. These East Germany objectives, almost half a century old, were able to supply themselves with light: the halogen light from the microscope was directed downwards through a ring-shaped light channel on the outside of the objective lenses and shone through a ring mirror onto the object, either in bright field or in dark field, i.e. with a white or black background. This seemed to me to be better suited to achieving a good and even light distribution throughout the clownfish egg.

So I took the series of pictures again and once more observed the entire development of the embryos, after the first cell divisions the gastrulation, during which the embryonic layers form and the main embryonic axis is created, then the segmentation, during which the typical fish body is formed and the chorda dorsalis, a precursor of the central nervous system, forms inside. This is followed by the late stages with the development of the organs and finally the hatching from the transparent egg membrane.

The typical fish body is formed during segmentation, and the large eye primordium with iris and pupil can already be seen in the head. The heart, which can soon be seen beating under the microscope, is developing in the bulging membrane in front of the head.

The second series of images, which again portrayed the entire embryonic development of the clownfish, was significantly better than the first. The ring lighting around the objective lenses had produced vibrant colors, in contrast to the pale yellow of the first series. I also found all the details inside the eggs more sharply contoured and easier to perceive. But the only way to get better is to always be dissatisfied with your work – not to the point of frustration, but to the point where it's hard to sit back and relax. So I decided to be dissatisfied.

The second series of experiments: Orthoplan microscope with reflected light dark field objectives; the light reached the object through an annular mirror

The second series of images of embryonic development was ready, but also failed to meet expectations

The third series of images

So: Next attempt with a new clutch a few weeks later. This time, however, I chose a completely different approach, as I had meanwhile completed my third focus stacking setup and put it into operation. The objective: Mitutoyo M Plan Apo 10x – a legend! Once again, an egg with embryo was placed on the concave ground slide. This time, however, the glass plate was mounted vertically on the focus stacking setup using a specially made holder. Behind it was black felt to make the background pitch black. The lighting was provided by two system flashes, as I had not yet achieved good results with LED lighting.

For the third series of experiments, a focus stacking setup with microscope objective was used and a height-adjustable vertical holder for the microscope slide was constructed

First discovery

I also discovered that the embryo's state of stress is reflected in the scattering of pigments in its body: If it is relaxed, the blackish and the yellow pigments are evenly distributed in the respective pigment cell, so that a homogeneous yellow coloration occurs on the body, and the yellow yolk sac is overlaid by the dispersed melanin pigments, which make it appear brownish. If, on the other hand, the embryo comes under stress, the pigments concentrate in the center of the pigment cells and you only see yellow and black dots, which radically changes the color effect of the entire embryo. I was therefore able to read the state of stress of the two-millimetre embryo in the egg membrane from its body coloration – that was new.

The pigment cells are controlled by the sympathetic nervous system. If the embryo is relaxed, the pigments are distributed over a very large area within the pigment cells, as can be seen on the yolk sac on the left. Under stress, however, they contract so that the pigments can only be seen as roundish, black spots (yolk sac on the right).

With my usual routine, I took three of the eggs each morning and evening to make a series of 100 pictures of each. At a certain stage of development, I also noticed a complete body rotation that each of the embryos performed: Initially, the head with the large eye primordium was developing at the bottom, but the following day this head was on top! I had already seen this change in maturing eggs in the aquarium without realizing what was happening. In order to document this, I took a series of 100 individual pictures of a particular embryo every hour for a whole day to show the course of this rotational movement.

Around the 3rd day, the embryo performs a rotation that brings the head, which was initially at the bottom, all the way to the top. The two pictures show a partial rotation with a time interval of several hours. Here, too, the pigment changes clearly show that the embryo in the right-hand picture is more stressed than the one on the left, presumably due to a lack of oxygen.

Further findings

And at some point, the third series of images was in the box and could be mounted. I found the result much better than the second series, especially because the light distribution inside the eggs was phenomenally even – exactly what I had imagined, made with a light diffuser recommended to me by Robert O'Toole, who had sadly died much too young early in 2024.

On the day of hatching, another observation was made, actually two: while in the morning the bodies of the small embryos were still homogeneously colored yellow, with evenly distributed xanthine pigments – according to my hypothesis they were therefore stress-free – in the evening, a few hours before nightfall, i.e. the approaching time of hatching, I saw a radical change of color in all of them: the yellow and black pigments distributed over a large area had concentrated into small, orange or black dots in all of the embryos. In my layman's terms, this was the result of a hormone release, e.g. serotonin, which was supposed to prepare them for strong muscle contractions to burst the egg membrane.

On the morning of hatching, the pigment distribution of the embryos within the pigment cells is still very extensive, which should indicate a relaxed state (left). In the evening, however, they all become stressed (right), possibly due to a hormone release in preparation for the hatching process.

The second observation was in the behavior immediately before the egg shell burst for hatching: With the U-shaped body position of the embryos, which is necessary to be able to grow to around four millimeters in length in an egg shell only two millimeters in size, many lack the strength to burst the shell by stretching their body like an opening pocket knife. The solution to the problem is simple and probably unknown to science: The embryo moves into an S-position so that it can now exert more force on the egg membrane by stretching its body and burst it – a success for the embryos and a gain in knowledge for me.

During the hatching process itself, some of the embryos move into an S-position in order to be able to exert more force on the egg shell by stretching their bodies and bursting it

The photo competition

Months later, I came across the Nikon Small World Award photo competition on the Internet for the first time. Wouldn't that also be something for my clownfish embryos? But then again, I had never entered any competition before, let alone a photo competition! And this was the world's largest and most important microscope photography competition! Every year around 2000 entries from around 90 countries around the world! The crème de la crème of microphotographers from all over the world met there for their annual rendezvous, mostly experts from university institutes, who took exciting fluorescence images in all colors of the rainbow every day with highly complex, sinfully expensive confocal microscopes, razor-sharp calculations by high-performance computers – that's how I saw it back then. And I, a complete beginner, was supposed to mingle with them?

No matter – taking part is everything. I produced a special short version of the embryonic development, which was suitable as a competition entry in terms of format, and submitted it to the Nikon Small World Award. And nothing happened for months.

Done: The microscopic image documentation of the embryonic development of Amphiprion percula is finished and photographed in such detail that it could easily be enlarged to an edge length of two meters

In October 2020, I almost fell off my chair when I found out that my entry had come second – second! And I got a bit of a stomach ache when I realized that I had accidentally sent a test file to the competition, which I had built in advance to try the whole thing out. The final competition file, which was much nicer, has still not been seen by anyone. It might have moved up one place – there was one before the second.

Nikon Small World Award 2020: An excerpt from the image series takes second place, and the embryos make the rounds of the international press

In the following weeks, I was interviewed by numerous editors abroad who reported on the competition, and my embryos were printed in several scientific reference books. The German magazine “Bild der Wissenschaft” also featured them on two pages at the beginning of the magazine. And I was finally reconciled to my mixed-up files when I heard by chance from the USA that the editors of the scientific journal “Nature” had chosen my competition picture as “Science Photo of the Year 2020” alongside a few others – what an honor for my little embryos!

The editors of the US science journal “Nature” choose the clownfish embryos as the Science Photo of the Year 2020

Daniel Knop