Influence of the phase effect on gradient-based and statistics-based focus measures in bright field microscopy

Autofocusing is essential to high throughput microscopy and live cell imaging and requires reliable focus measures. Phase objects such as separated single Chinese hamster ovary cells are almost invisible at the optical focus position in bright field microscopy images. Because of the phase effect, defocused images of phase objects have more contrast. In this paper, we show that widely used focus measures exhibit an untypical behaviour for such images. In the case of homogeneous cells, that is, when most cells tend to lie in the same focal plane, both gradient-based and statistics-based focus measures tend to have a local minimum instead of a global maximum at the optical focus position. On the other hand, if images show inhomogeneous cells, gradient-based focus measures tend to yield typical focus curves, whereas statistics-based focus measures deliver curves similar to the case of homogeneous cells. These results were interpreted using the equation describing the phase effect and patch-wise analysis of the focus curves. Bioprocess engineering experts are also influenced by the phase effect. Forty-four focus positions selected by them led to the conclusion that they prefer to look at defocused images instead of those at the optical focus.