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DIC image reconstruction on large cell scans

The width of the emission spectrum of a common fluorophore allows only for a limited number of spectral distinct fluorescent markers in the visible spectrum, which is also the regime where CCD‐cameras are used in microscopy. For imaging of cells or tissues, it is required to obtain an image from which the morphology of the whole cell can be extracted. This is usually achieved by differential interference contrast (DIC) microscopy. These images have a pseudo‐3D appearance, easily interpreted by the human brain. In the age of high throughput and high content screening, manual image processing is not an option. Conventional algorithms for image processing often use threshold‐based criteria to identify objects of interest. These algorithms fail for DIC images as they have a range from dim to bright with an intermediate intensity equal to the background, so as to produce no clear object boundary. In this article we compare different reconstruction methods for up to 100 MB‐large DIC images and implement a new iterative reconstruction method based on the Hilbert Transform that enables identification of cell boundaries with standard threshold algorithms. Microsc. Res. Tech. 66:312–320, 2005. © 2005 Wiley‐Liss, Inc.