Microscopic Photography

Microscopic Photography
Marc Brouwer

Microscopic Photography

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.

Phantom Midge Larva
Phantom Midge Larva

Camera

Olympus E-M1 mark II
Olympus E-M1 mark II

The best kind of camera to use is one which has an electronic shutter mode. Most mirrorless camera's have this option, but DSLR's usually don't. A mirrorless camera with electronic shutter has no mechanical components that can cause vibrations. This is very important when taking photos on a microscopic level. Some camera's are restricting in shutter speed when using the electronic shutter making it impossible to use a shutterspeed of, for example, 1 second. This does not have to be an issue if there is enough light on the object. Support for tethering can also be really helpful so that photos can be directly stored onto a computer rather than constantly emptying a memory card. This way, it is possible to process photos whilst more photos are still being made and it prevents disruption of the camera setup. I use the Olympus E-M1 mark II. This camera does not have any restrictions on the shutter speed of the electronic shutter and tethering is possible with the Olympus software.

Microscope objectives

A lot is already possible with a macro lens, especially if it has a magnification larger than 1:1 or if it is combined with extension tubes, converters or close-up lenses. As long as there are not too many components used, this is fine. However, by using too many components the quality decreases noticeably. To still work with an extreme magnification, a microscope objective can be used. There are two kinds of objectives, finite objectives and infinity objectives. The most popular fittings for both are RMS and M26.

Finite objectives are less popular than infinity objectives. In short, finite objectives require a fixed focal length and can be attached on a camera by using an adapter from the objective to extension tubes, where the distance in extension tubes matches the focal length of the objective. For the rest the same applies as for infinity objectives.

microscope objectives

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.

Olympus LMPLFLN 10x
Olympus LMPLFLN 10x
Objective specifications
  1. LMPlanFL N
  2. 10x / 0.25
  3. ∞ / - / FN26.5
  1. Objective name, letter indications for working and lenscoating
  2. Magnification / Numerical aperture
  3. Infinity objective (∞) or focal length / Thickness of cover glass or - when no cover glass is needed / Field number

The numerical aperture is sometimes followed by an immersion indication like Oil or Water. For the Olympus LMPLFLN objectives this is not needed and would not be handy for a long working distance/focal distance. Also the use of a cover glass is not possible for larger objects. Hence an objective that is used without a cover glass (indication -) is required for this purpose.

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.

Transmitted lighting
Transmitted lighting
Reflected lighting
Reflected lighting

Attachment

An infinity objective can be attached to a camera in two ways. The first way is to attach the microscope objective onto the front of a camera lens. To do so, an adapter ring is needed to convert the microscope objective fitting to the filter size of the camera lens. An example is a RMS to 62mm adapter ring, when the microscope objective has a RMS fitting and the filter size of the camera lens is 62mm. Next, the camera lens should be set to manual focus, with the focus on infinity (∞). Per camera it can differ which lens will lead to a sensor filling result, but mostly a telephoto lens is required. After setting it all up correctly, an object can be placed in front of the objective with a distance of a few millimetres to a few centimetres (depending on the microscope objective). Now everything is ready to start taking photos.

Infinity objective on telephoto lens

Option 2 may be slightly more complicated, but can lead to better results as less glass is used. Instead of using a camera lens, containing many glass elements, only 1 so called tube lens is used. This tube lens ensures that the light, like a camera lens, is projected correctly onto the sensor. Special tube lenses exist, but a good alternative is to use a Raynox close-up lens, like the Raynox DCR-250. Both the Raynox DCR-250 and the Raynox DCR-150 can be used, but the DCR-150 magnifies more than the DCR-250. This in contrast to their magnification for macro photography as then the DCR-250 has a larger magnification.

Components
  1. Extension tubes
  2. 52mm - 43mm adapter ring
  3. Raynox DCR-250 (tube lens)
  4. 49mm - RMS adapter ring
  5. Olympus LMPLFLN 10x (microscope objective)
Extension tubes, Raynox DCR-250, Olympus LMPLFLN 10x
  1. Extension tubes
  2. Raynox DCR-250 (tube lens)
  3. Olympus LMPLFLN 10x (microscope objective)

Attachment from extension tubes to a Raynox lens and then to a microscope objective is the same in both cases. An adapter is needed from the extension tubes to 43mm (Raynox diameter). In my case this is a 52mm to 43mm adapter ring, but this depends on the extension tubes. In some cases it may be useful to use a T2 or M42 adapter and then to use an adapter ring to 43mm. At the front the Raynox lens has a 49mm filter size and in combination with a RMS fitting for the microscope objective, this means a 49mm to RMS adapter ring is needed to attach the microscope objective onto the Raynox.

The distance of the extension tubes varies per camera mount and per Raynox lens. To make it a little easier, I have made a tool which computes the distance for you. Select your camera mount and which Raynox you want to use and the distance in mm for the extension tubes is computed. If it is setup correctly you will (almost) get a sharp photo when you only have the extension tubes and Raynox lens on your camera. This way it is possible to check that the Raynox is at the correct distance to the sensor. If it is not possible to set it up correctly using normal extension tubes, then there are also variable extension tubes which can be adjusted on millimetre level.

Calculate required extension tubes

- mm

Macro rail/setup

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.

macrorail
threaded shaft
LED lights

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.

Essential components:
  • Micro controller
  • Stepping motor
  • Threaded shaft
  • Camera trigger component
  • Linear axes
  • Fixing bars
Optional components:
  • LED lights
  • Bluetooth component
  • Quick release clamp
  • Quick release plates

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

Before Focus Stacking
After Focus Stacking

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

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.

stitch segment
stitch segment
stitch segment
stitch segment
stitched photo

Photographer

Name:Marc Brouwer
Age:25
Current Study:Master Computer Science and Engineering,
Eindhoven, University Of Technology
Work:Parttimer at CameraNU.nl
Owner of Theta 1 Software
More Photos:
Marc Brouwer

Contact

Do you have a question, comment, feedback or suggestions for additions or improvements? Feel free to let me know!

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