Microscopic photography
Marc Brouwer

Photography on a microscopic level can lead to very special photos. Due to the extreme magnification, it is possible to capture details which are invisible to the human eye. There are countless unique patterns and structures which can be discovered and also insects and arthropods can be captured with much more detail than with a standard macro lens. On this website I don't just want to show photos, but I also want to explain how these photos can be taken and what is required to do so.

Infinity objectives can be found in many variations. In price, quality, lighting technique, magnification and focus distance they can largely differ. The required magnification strongly depends on what you want to take photos of. If a little more detail is required than possible with a macro lens then 5x magnification is sufficient. For a more significant increase of magnification, 10x or 20x is much better suited. This allows you to capture much more detail. 50x or more is also possible, but the extreme magnification results in a shallower sharpness depth which does not make it easier to take photos. The actual magnification is not only dependent on the objective, but also on how it is attached to the camera.

Focus distance is important when taking photos of larger and deeper objects. In most cases the focus distance is only a few millimetres, making it impossible to capture, for example, the inside of a flower without pulling it apart first. Also for insects and other larger objects a focus distance of a few millimetres is not sufficient. Luckily there are also objectives with a long working distance, in other words, a larger focus distance. Depending on the magnification, this can be a few centimetres. This allows you to capture larger objects without the risk of damaging them.

There are many lighting techniques within microscopy and for different techniques different microscope objectives are used. So when choosing a microscope objective, it is important that it is suitable for the desired lighting technique. An example is the use of electrons as illumination. There are special objectives suited for this purpose, but are unnecessary when only the visible light spectrum is used. When using the visible light spectrum, the objective must be suited for brightfield illumination. In the case of brightfield illumination there are also different techniques of which reflected lighting and transmitted lighting are most common and can also be combined. For reflected lighting the light source is above the object, that is, on the same side as the camera. The light shines on the object and hence passes through the lens as reflected light. In the case of transmitted lighting, the light source is behind the object, that is, the opposite side of the camera, and shines through the object into the lens. Reflected lighting is almost always possible, whereas transmitted is not since the light cannot always pass through an object. The get a black background when using reflected lighting, the object must be placed on a microscope slide and light must be blocked so there is no light underneath the slide.

Microscope objectives only have a tiny depth of field (it is in terms of a few micron...). In order to get an object sharp from front to back, the object must be captured in slices. Each slice has a different and small part of the object in focus. All these slides will be combined into one overall sharp result. This technique is called focus stacking, of which I will explain more further on, but first, how all the slices can be taken.

Depending on the size of the object that will be captured, a few hundreds to several thousands of photos may be needed to capture all the previously mentioned slices. The displacement in focus between two photos is usually only a few micron. Due to the large number of photos and the extremely small displacement it is no longer possible to do this manually. For this an automated macro rail is necessary, which is capable of shifting the camera a few micron each time and triggers the camera to take a photo. Automated macro rails are for sale, but it is also possible to build one yourself. The macro rail I built consists of Makeblock components with a micro controller base on the Arduino UNO.

The combination of the stepping motor and the threaded shaft determine how accurate the camera can be shifted. The threaded shaft determines the distance per rotation of 360°, that is, the distance by rotating the shaft one complete round. For my threaded shaft this is 2mm per 360°. The step size in degrees of the stepping motor determines how much the threaded shaft can be rotated. My stepping motor has a step size of 1.8° per full step, but it is also possible to have 1/16th of the step size each time. This leads to the minimum rotation of 1.8°/16 = 0.1125° per step of this stepping motor. For a full rotation of 360° with a step size of 0.1125° means there are 360°/0.1125° = 3200 steps needed for a 360° rotation. The displacement per step is now 2mm/3200 = 0.000625mm = 0.625µm. Hence the accuracy of my macro rail is 0.625µm per step.

For the rest, the process of the macro rail is simple. From the starting point till the end point, the camera is continiously shifted, LED lights are switched on, a photo is taken, LED lights are switched off, again and again. After shifting the camera it can be good to wait shortly before taking a photo to prevent vibrations. Switching the LED light off after each photo is not always necessary.

Focus stacking is a technique to get more sharpness than is possible with one photo. This is done by combining multiple photos. Each photo has a small segment of an object in focus and by combining all these photos it is possible to get it all sharp. How these photos can be taken is described in section macro rail/setup. The best program I have seen so far is Zerene Stacker. This program has multiple stacking methods and aligns the photos when they are slightly shifted. Only for deep stacks of a thousand photos or more there is a noticeable decrease of sharpness. In the future I would like to write my own stacking software, which I have started, but it requires a lot of time so it may take a while. Important for focus stacking is that the object itself should not move, as this cannot be corrected. Also the in focus segments should overlap to prevent unsharp segments.

Stitching can be seen as a kind of panorama. When an object cannot be completely captured within one frame, it must be taken in segments. Here each segment is a photo to which focus stacking is applied first to get the object sharp from front to back. After all segments are stacked, they can be combined into one photo like a puzzle, this is called stitching. When there is enough overlap, a program like Photoshop can combine the segments using the panorama function. If this does not work, the segments must be combined manually. For manually aligning the segments it is easiest to use layers and make the top layer half transparent. Then the segments can be aligned such that the difference is minimal. Afterwards it is possible to use a mask (or eraser) to remove the hard edges between two layers and then the layers can be smoothly combined together.

One of my most remarkable projects was photographing miniscule creatures for Koppert's new packaging. The goal was to capture these tiny creatures with microscopic details. In order to make the photos more attractive (but therefore also extra difficult) some of the photos had to be taken with a graduation of colour in the background.

Some of the bugs were only a few millimetres in size, while others were only a fraction of that. The En-Strip, Ercal, Aphipar-M, and Enermix are minuscule flies. The Swirski-Mite and Thripex are predatory mites. Both the flies and the predatory mites were very active, so capturing them well was quite a challenge. With a microscopic lens on the camera it is difficult enough to get an object in focus, even when it doesn't move. Additionally, the point of focus had to be at the right place, the composition had to be good and the background had to be as desired. Fortunately, I had a very good assistant who could help me because doing it alone would have been impossible.

All these creatures are intended as natural pest controllers and are used, among other places, in greenhouse horticulture. If you want to know more about this, you should definitely visit Koppert's website. There you can also find more photos that I have taken. Below is just a small selection.