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Method for observing phase objects without halos and directional shadows

A new microscopy method for observing phase objects without halos and directional shadows is proposed. The key optical element is an annular aperture at the front focal plane of a condenser with a larger diameter than those used in standard phase contrast microscopy. The light flux passing through the annular aperture is changed by the specimen's surface profile and then passes through an objective and contributes to image formation. This paper presents essential conditions for realizing the method. In this paper, images of colonies formed by induced pluripotent stem (iPS) cells using this method are compared with the conventional phase contrast method and the bright-field method when the NA of the illumination is small to identify differences among these techniques. The outlines of the iPS cells are clearly visible with this method, whereas they are not clearly visible due to halos when using the phase contrast method or due to weak contrast when using the bright-field method. Other images using this method are also presented to demonstrate a capacity of this method: a mouse ovum and superimposition of several different images of mouse iPS cells.