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Microscope photography

 

If you want to go up in magnification from normal macro photography, then a microscope is probably the best way. Some really weird stuff can be found by looking trough a microscope, like the examples below. Some more examples can be found here.

A Daphnia with two young ones.

 

A tardigrade.

 

Microscope set-up

 

My microscope is a 30+ year old Zeiss Jenamed 2, which I found online. It does not have the best optical quality of course since it is so old, but it works fine for my needs. On top of it is my camera, which is connected to a computer. When using a DSLR on a set-up like this, it is important to use either mirror delay or mirror lock-up, since the mirror of the camera will shake the whole set-up, giving blurry photos like the example below to the right.

 

My microscope set-up with a camera connected.

 

The difference mirror delay can make => [mirror delay on] [mirror delay off]

 

Illumination techniques

 

There are a whole bunch of different illumination techniques around, but below are the ones I use. For comparison of subjects taken with different illumination techniques you can go to the microscope part, where many pictures are shown with several different illumination techniques.

 

Illumination techniques - brightfield

 

Brightfield illumination is the simplest technique, with the light source sending light through the subject directly to the camera.

A desmid, brightfield.

 

A tardigrade, brightfield.

 

Illumination techniques - darkfield

 

In this case a filter is used to block any direct light from the light source to the camera, and the only light reaching the subject comes at an angle. If this incident light is scattered by the subject, then it will show up against a dark background.

An insect larva, darkfield.

 

A desmid, darkfield.

 

Illumination techniques - phase contrast

 

This is a complicated technique which makes use of the fact that the subject has a slightly different refractive index than the surrounding water. With some smart optical design, this difference is translated to an image. This technique is especially useful for thin specimens that are hardly visible in brightfield.

A rotifer, phase contrast.

 

A diatom, phase contrast.

 

Illumination techniques - cross-polarized light

 

In this case, a polarizer is placed below the subject, and a second one is placed above the subject, rotated at an angle of 90 degrees compared to the first polarizer. If the subject does not alter the polarization of the light, then the image will be black (or at least very dark). But if the subject does alter the polarization (that is, if it is birefringent), then an image is shown against a dark background. More information about birefringence can be found here.

 

Usually when using fully cross-polarized light, the background will turn very dark or even black. If you don't want a completely black background, then you can turn one of the polarizers slightly (so that they are no longer perpendicular) to obtain partially polarized light which will give a lighter background. Below on the right is an example of this.

 

A desmid, cross-polarized light.

 

Examples of different degrees of polarization =>
[fully] [partial 1] [partial 2] [partial 3]

 

 

Illumination techniques - cross-polarized light with cellophane retardation

 

Another way to change the background with cross-polarized light is to add a retarder, which will change the direction of the polarized light coming from the first polarizer. There are dedicated retarders for sale, but it can also be done in a cheaper way with a sheet of cellophane.

 

By playing with the directions of both the cellophane and the polarizers you can get a very wide range of colors, as you can see in the examples to the left. You can also play with amount of layers of cellophane to get different results.

 

 

 

Illumination techniques - UVIVF

 

UVIVF (ultraviolet induced visible fluorescence) is the same principle as the light painting using a UV-lamp described here and here. The principle behind this technique is that some substances get excited by the energy of the UV light, after which they return to their ground state by sending out visible light.

 

There are dedicated microscopes for this technique, but I use the Convoy S2+ to expose the samples to UV light, and this works reasonably well. But long exposure times are needed, so your subject can't move around during the exposure.

A desmid, UVIVF.

 

A desmid, UVIVF.

 

Illumination techniques - Rheinberg filters

 

Some Rheinberg filters that I made from colored sheets of plastic.

Rheinberg filters are a special way to illuminate your subject. They can be bought, or made yourself using colored sheets of plastic. On the right are some examples of filters that I made for my microscope. The first two rows are in fact darkfield filters, but with the incident light gaining colors.

The size of the inner part of these Rheinberg filters depends on your microscope and the magnification you are using. So you have to make a series of filters for each magnification that you want to use.

 

• The first row will send different colors of light from each side of the subject.

• In the case of the second row, the colors will be directed perpendicular to eachother, giving the possibity to get some directional information from using these filters (see the second pair of examples below).

• The third row will result in a background color from the center part of the filter, whereas the colored edge of the filter will illuminate the subject with a different color. In this way, you will get a color contrast between the subject and the background.

 

Below are some examples of how the use of Rheinberg filters will look. In each case, the Rheinberg filter I used is shown in the lower right corner.

 

Examples of Rheinberg illumination =>
[brightfield] [darkfield] [Rheinberg 1] [Rheinberg 2]
[Rheinberg 3] [Rheinberg 4] [Rheinberg 5]

 

Examples of Rheinberg illumination =>
[brightfield] [darkfield] [Rheinberg 1] [Rheinberg 2]
[Rheinberg 3] [Rheinberg 4] [Rheinberg 5] [Rheinberg 6]

Examples of Rheinberg illumination => [situation 1] [situation 2]

 

Examples of Rheinberg illumination => [situation 1] [situation 2]

 

Focus stacking

 

Just like conventional photography, it is perfectly possible to make focus stacks using a microscope. The biggest problem is usually that the subject moves around during the focus stack.

Example of a focus stack => [focus stack] [single photo]

 

Example of a focus stack => [focus stack] [single photo]

 

Stitching

 

Example of a stitch of eight photos.

Stitching together several photos to a larger photo is also possible, but it needs a bit of tweaking to find the optimal settings for the stitching software. The properties of your microscope objectives (for example distortion and field curvature) also play an important role in how well the stitching works. A uniformly illuminated background is also very important, and, unfortunately, quite a challenge on my old microscope.

 

On the left is an example of a mosquito larva, made of eight separate photos stitched together. There are one or two errors in the stitching, which I think could have been prevented by taking smaller steps between the photos, but I haven't tested this.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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