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Video-enhanced contrast optical microscopy (VECOM) has emerged in response to the need for high-contrast intracellular imaging (Allen 1985). It is a combination of optical microscopic techniques with video and digital image processing (Inoué and Spring 1997) that has permitted dramatic improvements in the quality of the optical microscopy images (Shotton 1988). VECOM has successfully addressed the problem of illumination background across the field of view (background mottle) that obscured fine features in images of biological assays (Weiss et al 1989). The background mottle is primarily caused by non-uniformity of the sample illumination, as well as by the non-uniform photoresponsivity of an imaging detector and light scattering in the microscope optical train.
In a typical implementation of VECOM, two images of the object, e.g. glass slide, were recorded using a differential interference contrast (DIC) microscope equipped with a video camera: one image contained the specimen, whereas the other image was specimen-free. Digital subtraction of the specimen-free image resulted to a mottle free specimen image featuring low-contrast details. The DIC microscope can now be operated in an alternative regime taking full advantage of the mottle subtraction concept. Indeed, in conventional interference contrast microscopes, the phase difference between two beams was set to a small value minimizing the unwanted illumination background. Since that background can be efficiently subtracted in VECOM, the phase difference can be enlarged, leading to a considerable enhancement of the image details (Fig. 1).
The application of this seemingly simple concept has resulted in unprecedented visualisation of various sub-cellular structures, including mitochondria, microtubules (see also, Waterman-Storer 1998), and vesicles, as reviewed by Shotton (1988). With this technique, even weakly scattering details, as microtubules, and also gold immunolabels as small as 5 nm were easily detected (De Brabander et al 1985).
| CITATION: Zvyagin A. V., Plakhotnik T. 2006. Video-enhanced contrast optical microscopy (www.tpdsci.com/Tpc/MicVECO.php). In: Top. Part. Disp. Sci. (www.tpdsci.com). |
HISTORY: Published: 24-Nov-2006 Modified: 21-Nov-2006 Peer-reviewed: PENDING |
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