A simple and stable autofocusing protocol for long multidimensional live cell microscopy
Focus maintenance is a challenging problem in multidimensional wide-field microscopy. Most automated microscopes use software algorithms, which are applied to z-sections of the object, to select for the plane with the best signal to noise ratio. When applied automatically in multidimensional imaging applications, autofocus routines significantly increase light exposure and can become cytotoxic if applied too frequently. In addition, automated focusing procedures can readily focus on unwanted high contrast objects. By labelling a defined position with a fluorescent marker, we were able to separate the focusing procedure from the actual image acquisition positions and therefore overcome some of the major drawbacks of routine autofocus procedures. To implement this method in a multidimensional acquisition experiment, we created a visual basic-based program, which is run prior to each image acquisition. This technique allows tight control of focus whilst keeping light toxicity in live cell imaging experiments to a minimum.