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.