|Setup for taking focus stacks ofa specimen|
|The jig. The insets show the vertical slider without the specimen carrier in place (bottom left) and the way in which the three flash brackets, used for mounting the lights, are attached (top right).|
|Underside of base|
|Specimen carrier||Mounting pin|
The actual specimen is mounted on a plastazote stage on a long pin to get it well in front of the background so that stays completely out of focus. The plastazote stage into which the specimen's micro pin is mounted has been coloured blue using a standard highlighter pen. This makes the mount easy to mask out of the finished image because this florescent blue is not a colour that is likely to occur naturally! (I will talk about post processing the stacked images in a later post.)
The second sheet of 6mm plastazote pinned to the bottom of the specimen carrier simply acts as a reflector. It throws some light upwards and tends to fill in any shadows underneath the specimen arising from the fact that there is no light below the specimen in my setup (although there is no real reason why you could not mount a fourth light on the base of the jig, below the specimen, if required, to get really flat, all round illumination).
|Stackshot focussing rail|
The focussing rail is driven by a stepping motor which is controlled by the programmable controller. There are many options, but I use the one where you set the start and end point (by driving the rail forward and backwards using the "Fwd" and "Back" buttons on the controller) and step size. I set the step size using a Depth of Field table for the MP-E 65mm macro lens. I choose a step size that is 30% less than the Depth of Field for the F-stop and reproduction ratio I have set on the lens. This gives some overlap of the parts that are in focus between shots which helps the stacking software to merge the shots successfully. So, if the chart says that the Depth of Field is 0.5mm for my chosen lens settings, I would set a step size of 0.35mm (0.7 * 0.5). The controller works out how many steps of that size are needed to travel from the selected start to end points. When you tell it to start shooting the stack, the controller moves the rail to the preset start position, fires the shutter, moves by one step of the selected size, fires the shutter, etc. until it arives at the preset end position.
There are many options to control how fast the rail moves and the length of time between operations. For example, you can set how long to pause between the rail arriving at the next position for a photo and the shutter being triggered (i.e. how long to allow for vibrations caused by the rail's movement to damp down). It can be very useful to modify these settings, for example, to make the time between shots longer to allow a flash to recharge (if you are using flash to expose the shots). It also has an option to trigger the shutter twice for each shot so that you can use mirror lock up (as I normally do).
In the next post I will consider the lighting and compare flash and continuous LED lights.