A new approach to extended focus for high-speed high-resolution biological microscopy

Sara Abrahamsson; Satoru Usawa; Mats Gustafsson

doi:10.1117/12.647022

23 February 2006 A new approach to extended focus for high-speed high-resolution biological microscopy

Sara Abrahamsson, Satoru Usawa, Mats Gustafsson

Proceedings Volume 6090, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII; 60900N (2006) https://doi.org/10.1117/12.647022
Event: SPIE BiOS, 2006, San Jose, California, United States

Abstract

Microscopic study of rapid biological processes often requires both high resolution and high acquisition speed. When the speed requirement precludes acquiring a full 3D focal series at each time point, it can be attractive to sacrifice all axial information and instead record a single, 2D image per time point. This can be done at very high frame rates. High-resolution objectives, however, have a very short depth of focus. There are several established methods to achieve extended depth of focus, including annular pupil masks; mechanical sweeping of the focus and wavefront coding, which uses a pupil-plane optical device to introduce geometric aberrations. We have developed a new pupil plane approach where the light is manipulated chromatically rather than geometrically. A phase mask with circularly symmetric stair steps divides the pupil plane into a series of annular zones. The stair steps are large compared to the coherence length of the observation light, so that images from different zones form independently and combine incoherently into a final image. Each zone carries only a fraction of the objective's axial resolution, but the larger zones still carry the full lateral resolution of the objective. The incoherent addition of the different single-zone images results in a smooth and circularly symmetric point spread function with a depth of focus that is extended by a factor approximately equal to the number of zones in the mask. The method has been demonstrated both on bead samples and on whole cells with a performance that is well in accordance with the theoretical predictions.

Citation Download Citation

Sara Abrahamsson, Satoru Usawa, and Mats Gustafsson "A new approach to extended focus for high-speed high-resolution biological microscopy", Proc. SPIE 6090, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII, 60900N (23 February 2006); https://doi.org/10.1117/12.647022

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