Introduction

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It has always been possible to combine photographic images but theadvent of digital imaging has made the process far easier, although ithas also created pitfalls.
My particular interest in image stacking comes from the desire toproduce photomicrographs of butterfly and moth scales, pollen grainsand other minute plant structures with far more depth of field than Icould obtain with film. When I used to show Kodachrome 25transparencies of butterfly or moth wing scales photographed at x100through the microscope, audiences were usually wowed, but because thewings themselves are not flat, depth of field was limited. The impactis far greater when the image is sharp overall.
As stated above, butterfly and moth wings are not flat and there can bemillimetres of difference between the highest and lowest areas of awing under the microscope. This is far too much for edge-to-edgesharpness at x100. Using a cover slip to flatten the wing is useful fortransmitted light images, but isn’t really an option when photographingby reflected light.
I have never had access to electron microscopy or had the technique,time and patience which some darkroom workers have used to producemultiple image film photomicrographs; so when I heard that softwarecould be used to combine digital images it seemed to be worthinvestigating.
As I’m primarily a Mac user I began by downloading a free trial versionof the only reasonably-priced compatible software I could find –Helicon Focus – which seemed simple and straightforward. I began byscanning some old film photomicrographs and tried to combine them, butthis failed because they didn’t register properly.
I then tried using our Canon digital Ixus 500 compact, held over themicroscope eyepiece (we didn’t have a microscope adaptor for thiscamera at the time) but although the individual images were very goodindeed, registration was once again a problem.

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At about this time Pentax brought out the K10D which is compatible withall my old Pentax film gear including the microscope adaptor. At lastit was possible to take images which were accurately registered andsuitable for blending with Helicon Focus. An added bonus was the giftof an old Nikon photographic microscope which was ‘broken’ – itactually needed nothing more than a new mains plug! Obviously this hassuperb optics. It also has a numbered scale on the fine focus knob anda fixed mark on the coarse focus, which makes selection of thenecessary 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 areharmed in the process!

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

Forthis example I chose the underside of the hind wing of an Orange-tipbutterfly. It has what appear to be green patches, but these in factare made up of a mixture of yellow and black scales.

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

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I found the highest focus point of the image and noted the reading onthe scale (20), then the lowest focus point (35). A series of 15 imageswere then taken, using the same manual exposure (varying the exposurecan confuse the software) and ensuring precise registration, focusingdown one scale mark at a time. I quickly found that it was important tostand absolutely still during each exposure.
It would have been easiest to take JPEG images as this shortensprocessing times; but I used RAW as I may one day wish to produceprints rather than files for projection. Processing for blending andeventual projection involved opening the RAW file and making anynecessary adjustments – these must be identical for each file. Eachfile was then saved as an uncompressed TIFF.
The files for blending were then selected from within the Helicon Focusprogramme, using default settings (I still haven’t explored theprogramme properly) and I hit the ‘run’ button. Eventually the softwareproduced a blended file, which was saved as a TIFF, then resized forprojection and sharpened appropriately.
Transmitted light
Transmitted light images are produced using the built-in microscopelamp with the camera white balance set to tungsten – no more need forall those blue filters which I had accumulated. The same procedure asfor a reflected light image is followed, finding the highest and lowestfocus points and taking a series of images.
The biggest problem I found was in getting absolute cleanliness ofoptics and microscope slides – a disadvantage of living in a dusty oldhouse! However, Photoshop was used to ‘spot out’ the final image.
I particularly wanted, for a talk on ‘Fruits and Seed Dispersal’, toproduce an image of the barbs which are found in Burdock seed heads –the ‘itching powder’ which makes dogs scratch when they get burdockheads in their fur. Interestingly it takes a day or two for the barbsto reach the skin, so the dog will probably be some distance from theplant 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 Imagesof Burdock barbs (Shots 1 and 6 at top) are combined usingHelicon Focus to produce the Final Image.

Conclusionsabout Helicon Focus

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Although I can’t make comparisons, it’s areasonably cheap bit of software which works well for me. I have triedthe sort of thing which John McCormack describes in his

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masterclass

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, both with flowers andwithmoths (the best moth image has 4.5cm depth of field with 40 imagestaken at f5.6 combined), but I mainly use it for photomicrography. Iwould say that it is user-friendly and effective.