| Popis: |
Differential Interference Contrast (DIC) and phase contrast light microscopy are the two most popular methods of obtaining contrast when acquiring images of unstained cells and tissues. DIC images lack the halo effect found in phase contrast images and can provide optically sectioned images especially for high-resolution (high spatial frequency) cellular features. A DIC image is essentially the derivative of the optical path length, nt, defined as the index of refraction times the specimen thickness. This derivative is obtained by splitting polarized light into two closely spaced beams with a Wollaston prism, allowing both beams to interact with the specimen and recombining the beams with a second Wollaston prism and analyzer. The shear is the distance between the beams which are oriented along the shear axis of the DIC optics (usually 45° from horizontal). DIC imaging produces positive and negative peaks at the edges of cell structures while unchanging structure results in a gray background intensity similar to that found outside the cell. Since much of the internal structure of a cell has the same intensity as the image background, standard image segmentation methods are not very effective. Therefore some preprocessing step should be taken to reverse the effects of differentiation. Many methods such as low-pass filtering, line-integration along the shear direction, Hilbert Transformation, Weiner filtering and deconvolution have been suggested to prepare DIC images for further image processing or display. The Hilbert Transform has been shown to reverse the effects of differentiation without introducing high frequency noise into the image. A combination of Hilbert Transformation followed by deconvolution with a bright-field point spread function (psf) produces images that are highly amenable to further processing and display. An ImageJ macro routine has been written to automatically process a series of images using this algorithm so they are ready for further analysis. |
