Tuesday, October 15, 2013

Photographing of insect specimens using focus stacking techniques

This is the first of a series of posts describing the methods I have used to photograph specimens of flies to illustrate "Britain's Hoverflies" and various identification keys using focus stacking.

Ceratinostoma ostiorum (Diptera, Scathophagidae), male. Focus stack of 25 shots taken in July 2013 at approx. 1.5x life size using Canon 60D with MP65 macro lens, processed using Zerene Stacker.

Why is focus stacking necessary?

When you look at an insect specimen through a microscope, you focus up and down and move the specimen around and what you "see" is a 3D model formed by your visual system - the combination of eye and brain. If you take a photograph of the same specimen at the same sort of magnification the resulting image is often rather disappointing! It fails to match your perceptions because, due to the limited depth of field, it is not all in focus. It is possible to increase the depth of field of a photo by decreasing the aperture of the lens (selecting a higher F-number) but, as the magnification you are trying to achieve increases, the depth of field available is simply not sufficient. It is not possible to decrease the aperture, and hence gain more depth of field, beyond certain limits because diffraction leads to unacceptable image degradation.

The way out is "focus stacking" - taking a series of photos (the "stack") at different focal points and merging the most focussed parts into a single, composite image that is in focus throughout. The result, like the one shown above, satisfyingly matches our perceptions of what the specimen ought to look like!

Whilst this technique has been known for a long time, the availability of computer hardware and image processing software that will do a good job in a reasonable amount of time and at a cost affordable to an amateur enthusiast is relatively recent development. Sufficiently powerful home-computers with enough memory have only become available and affordable over the past decade or so (say, since 2000).

My experiences of focus stacking

I have been interested in the technique for a long time. I first encountered it when I was a PhD student at Newcastle University (1975-78). At that time, the camera microscope and image capture setup cost something in the high tens to low hundreds of thousands of pounds and the processing software needed the resources of a university mainframe computer! In Dec 2002 I bought a Nikon Coolpix 4500 and used that, mounted on a binocular microscope, to capture focus stacks which were processed using software I wrote myself (called "DeepFocus") written with Delphi 7.

Sphaerophoria scripta (Diptera, Syrphidae). Focus stack of 14 images taken in April 2004 with Coolpix 4500 mounted on binocular microscope and processed using self-written "DeepFocus" software.

More recently, Roger Morris and I produced about 180 images of  hoverflies for the WildGuide "Britain's Hoverflies". Producing this number of images at the sort of quality required for publication in a book, was only really possible because I had developed better and faster techniques.

Melanostoma scalare (Diptera, Syrphidae), female head. Focus stack of 15 shots taken in April 2011 at approx 3x life size using Canon 60D with MP65 macro lens. Processed using Helicon Focus.
My latest projects are an update my key to the British Scathophagidae and a Field Studies Council fold-out identification card on Garden Hoverflies (still in preparation). For these, I have been shooting a series of whole-animal images.

In the next post I will describe the setup I have developed to acquire the stack of images.