Openlab 3 - Auto Focus now supported for motorised focus drives

Technical Note: 164
Reads: 6664
Creation Date: 29/01/2001
Modification Date: 12/02/2004

Auto Focus operates on motorised focus drives only, that is, in Openlab supported electronic microscopes, and Ludl and Orbit hardware.

Auto Focus is based on analysis of the image for contrast. Focus is a subjective process when carried out by any microscopist, one operator may prefer a different focal point to another. Users may well disagree with the decision of the Auto Focus algorithm as they would with a colleague.

A perfectly focused sample should show the highest contrast within the range of movement of the Auto Focus, but the techniques of DIC and phase contrast can generate highest contrast at a plane differing from the best focus for the sample. Auto Focus is therefore not recommended for these techniques.

The sample must be illuminated for it to function. Samples which bleach rapidly or have limited tolerance to light will not be appropriate for Auto Focus. Low light levels will not work as well as images which use a greater portion of the dynamic range of the camera. Saturated pixels within the image will, however, interfere with Auto Focus and should therefore be avoided. Using Auto Exposure in conjunction with Auto Focus may help. The microscope and camera must be set up in such a way that the camera forms the best quality image possible.

Auto Focus searches within a maximum range of +/- 50 microns from its starting point. This is to keep the process within a reasonable time frame.

Dirt, debris and other artefacts of microscopy typically show far better contrast than any sample and therefore will attract the attention of the Auto Focus. It is vital that all optics, including the objective and camera glass are clean and dust free. Also consider that if you cannot focus the sample yourself, because, for example, the coverslip is the wrong thickness for the lens, or old immersion oil mixing with new on the coverslip, no Auto Focus algorithm will be able to achieve what you could not.

Auto Focus is more effective and reproducible at higher magnifications and it works better on objectives with a smaller depth of field. In general high-power objectives have small depths of field and low-power objectives have large depths of field, therefore presenting more possible focus points.

Auto Focus will operate on both brightfield and fluorescent illumination, however the greater the contrast the more effective Auto Focus will be. Fluorescent samples with background detectable by the camera may not be effectively focussed. During Auto Focus the software searches for the best contrast within the focus range. The speed of this process is directly related to the integration time, overall speed of camera and speed of drive. Longer integration times such as those often required for fluorescent imaging will prolong this process, which may be to the detriment of the sample.

To improve speed, the camera could be set up with increased binning and/or gain (depending on the capabilities of the camera), allowing shorter exposure and faster imaging whilst using the Auto Focus, and then returned to high quality imaging for actual capture. The State Saver could be used with many cameras to make this switch easier.