The track length calibration procedure depends on the equipment used. There are various calibration steps which are necessary, but each of these only needs to be carried out once. The essential tool (which is supplied with every full system sold by Autoscan Systems) is a "stage micrometer", a microscope slide engraved with very accurately positioned fine lines.
a) it is necessary to calibrate the optical train for each "objective/Optovar/camera/camera mount lens" combination used. As a minimum, this means that a calibration process must be carried out for every objective (if the other components stay the same). This process, which is part of our Trakscan program, involves placing the stage micrometer under the microscope and entering, via the keyboard, the number of microns which are visible across the screen, both horizontally and vertically. This creates a "micron per pixel" calibration constant for that particular objective etc. combination. This means that when the user clicks a distance on the screen during the course of the program (which the program can only sense as a screen pixel distance), that distance is immediately converted into real-life microns. This procedure needs to be carried out for each objective etc. during installation.
b) in our older systems, the length measurements were carried out using a drawing tube (now no longer available for Zeiss microscopes) and a digitiser tablet. The digitiser tablet cursor (or puck) was fitted by us with a special fine-point LED. The light from that LED was projected through the drawing tube over the top of the image of the track in the eyepiece and camera. To make a length measurement, the user clicked on both ends of the track, but using the digitiser puck instead of the mouse. A calibration step, using the stage micrometer and clicking on a known distance, allowed the generation of a calibration coefficient which related digitiser pixels or "lines", also sometimes called "mickeys" to micron lengths. Our Trakscan software allowed use of either the mouse or digitiser, as per the user's preference. Again, this procedure needs to be carried out for each objective etc. during installation.
c) the reason it is necessary to recalibrate when any of the elements are changed is that (for instance) different models of objectives have slightly different magnifications, different cameras have different pixel sizes, and different camera C-mounts are available with various magnifying or reducing lenses. Optovars, or magnification changers, add a whole new dimension, where the objective magnifications can be varied by a number of multplying factors, thus creating a whole range of possibilities, each of which will need to be calibrated. (It should be added that overall magnifications in excess of 1000 x will only produce something known as "empty magnification". That is to say, they do not add any extra information, as a physical limit of image resolution called the Raleigh limit has already been reached. The image simply becomes blurrier as it becomes larger. However, such magnifications are popular with some users, as they confer psychological comfort.