Robust autofocusing in microscopy.

Background: A critical step in automatic microscopy is focusing. This report describes a robust and fast autofocus approach useful for a wide range of microscopic modalities and preparations.

Methods: The focus curve is measured over the complete focal range, reducing the chance that the best focus position is determined by dust or optical artifacts. Convolution with the derivative of a Gaussian smoothing function reduces the effect of noise on the focus curve. The influence of mechanical tolerance is accounted for.

Results: The method is shown to be robust in fluorescence, bright-field and phase contrast microscopy, in fixed and living cells, as well as in fixed tissue. The algorithm was able to focus accurately within 2 or 3 s, even under extremely noisy and low contrast imaging conditions.

Conclusions: The proposed method is generally applicable in light microscopy, whenever the image information content is sufficient. The reliability of the autofocus method allows for unattended operation on a large scale.