Improvement in focusing accuracy of DNA sequencing microscope with multi-position laser differential confocal autofocus method.

High focusing accuracy in microscopes could improve the imaging quality to reduce the error rate in DNA sequencing. We propose a new feedback method to improve the focusing condition to a very high accuracy. A reference laser reflected by the sample is detected by two or more sensors around the confocal point. After acquiring the signals from the out-of-focus positions, online data processing is implemented to provide feedbacks for real-time focus-plane locking on the sample surface. This method provides an accuracy better than 1/10 of the objective depth-of-focus. To balance optical aberrations, a specific optical feedback system should be designed, with athermal design considerations to adapt DNA sequencing work to temperature fluctuations.