Introduction

It has always been possible to combine photographic images but the advent of digital imaging has made the process far easier, although it has also created pitfalls. 

My particular interest in image stacking comes from the desire to produce photomicrographs of butterfly and moth scales, pollen grains and other minute plant structures with far more depth of field than I could obtain with film. When I used to show Kodachrome 25 transparencies of butterfly or moth wing scales photographed at x100 through the microscope, audiences were usually wowed, but because the wings themselves are not flat, depth of field was limited. The impact is far greater when the image is sharp overall. 

As stated above, butterfly and moth wings are not flat and there can be millimetres of difference between the highest and lowest areas of a wing under the microscope. This is far too much for edge-to-edge sharpness at x100. Using a cover slip to flatten the wing is useful for transmitted light images, but isn’t really an option when photographing by reflected light. 

I have never had access to electron microscopy or had the technique, time and patience which some darkroom workers have used to produce multiple image film photomicrographs; so when I heard that software could be used to combine digital images it seemed to be worth investigating. 

As I’m primarily a Mac user I began by downloading a free trial version of the only reasonably-priced compatible software I could find – Helicon Focus – which seemed simple and straightforward. I began by scanning some old film photomicrographs and tried to combine them, but this failed because they didn’t register properly. 

I then tried using our Canon digital Ixus 500 compact, held over the microscope eyepiece (we didn’t have a microscope adaptor for this camera at the time) but although the individual images were very good indeed, registration was once again a problem. 

At about this time Pentax brought out the K10D which is compatible with all my old Pentax film gear including the microscope adaptor. At last it was possible to take images which were accurately registered and suitable for blending with Helicon Focus. An added bonus was the gift of an old Nikon photographic microscope which was ‘broken’ – it actually needed nothing more than a new mains plug! Obviously this has superb optics. It also has a numbered scale on the fine focus knob and a fixed mark on the coarse focus, which makes selection of the necessary focus range, and of the vertical interval between images, very straightforward. 

Reflected light images

I obtain my butterfly and moth wings from road casualties, spiders’ webs and by scrounging from butterfly houses, so no live creatures are harmed in the process!

 

Illustration 1: Fifteen Images of Orange-tip 'green' scales (Images 1 and 15 at top) are combined using Helicon Focus to produce the Final Image.

For this example I chose the underside of the hind wing of an Orange-tip butterfly. It has what appear to be green patches, but these in fact are made up of a mixture of yellow and black scales. 

Firstly I cut out a piece of the wing and attached it as flat as I could to a microscope using Scotch Magic tape. This was then placed on the microscope stage and a suitable area selected by looking through the microscope. The camera was then mounted and the lighting set up; this was a cold swan-neck (optic fibre) lamp, with a silver foil reflector placed so as to reduce hard shadows. The lamp was turned to full power for initial focusing purposes – a live view facility would have made this far easier, but the K10D doesn’t have this! 

I found the highest focus point of the image and noted the reading on the scale (20), then the lowest focus point (35). A series of 15 images were then taken, using the same manual exposure (varying the exposure can confuse the software) and ensuring precise registration, focusing down one scale mark at a time. I quickly found that it was important to stand absolutely still during each exposure. 

It would have been easiest to take JPEG images as this shortens processing times; but I used RAW as I may one day wish to produce prints rather than files for projection. Processing for blending and eventual projection involved opening the RAW file and making any necessary adjustments – these must be identical for each file. Each file was then saved as an uncompressed TIFF. 

The files for blending were then selected from within the Helicon Focus programme, using default settings (I still haven’t explored the programme properly) and I hit the ‘run’ button. Eventually the software produced a blended file, which was saved as a TIFF, then resized for projection and sharpened appropriately. 

Transmitted light

Transmitted light images are produced using the built-in microscope lamp (help – does anyone know of a source of old Nikon microscope bulbs? - please click here to contact the Editor if you do) with the camera white balance set to tungsten – no more need for all those blue filters which I had accumulated. The same procedure as for a reflected light image is followed, finding the highest and lowest focus points and taking a series of images. 

The biggest problem I found was in getting absolute cleanliness of optics and microscope slides – a disadvantage of living in a dusty old house! However, Photoshop was used to ‘spot out’ the final image. 

I particularly wanted, for a talk on ‘Fruits and Seed Dispersal’, to produce an image of the barbs which are found in Burdock seed heads – the ‘itching powder’ which makes dogs scratch when they get burdock heads in their fur. Interestingly it takes a day or two for the barbs to reach the skin, so the dog will probably be some distance from the plant when it scratches, so aiding seed dispersal. 

These barbs – at the most 5mm long in real life – are beautifully sculptured, as can be seen from the blended image.

 

Illustration 2: Six Images of Burdock barbs (Shots 1 and 6 at top) are combined using Helicon Focus to produce the Final Image.

Conclusions about Helicon Focus

Although I can’t make comparisons, it’s a reasonably cheap bit of software which works well for me. I have tried the sort of thing which John McCormack describes in his masterclass, both with flowers and with moths (the best moth image has 4.5cm depth of field with 40 images taken at f5.6 combined), but I mainly use it for photomicrography. I would say that it is user-friendly and effective.