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Articles

Near Real Time Confocal Microscopy of Cultured Amelanotic Cells: Sources of Signal, Contrast Agents and Limits of Contrast

[+] Author Affiliations
Colin Smithpeter, Andrew Dunn, Rebekah Drezek, Tom Collier, Rebecca Richards-Kortum

The University of Texas at Austin, Department of Electrical and Computer Engineering, Austin, Texas?78712

J. Biomed. Opt. 3(4), 429-436 (Oct 01, 1998). doi:10.1117/1.429853
History: Received Mar. 30, 1998; Revised July 23, 1998; Accepted Aug. 23, 1998
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Abstract

The use of high resolution, in vivo confocal imaging for noninvasive assessment of tissue pathology may offer a clinically important adjunct to standard histopathological techniques. To augment the present understanding of both the capabilities and limitations of in vivo confocal imaging, we investigated cellular sources of image contrast in amelanotic tissues, how contrast can be enhanced with external agents and how contrast is degraded by the scattering of overlying cells. A high-resolution reflected light confocal microscope was constructed and used to obtain images of various types of unstained amelanotic cells in suspension in real time before and after the addition of contrast agents. Reflectance images were compared to phase contrast images and electron micrographs to identify morphology visible with real time reflected light confocal microscopy. Mechanisms which decrease image contrast, including interference effects and scattering in overlying layers of cells, were considered. In amelanotic epithelial cells, fluctuations in the nuclear index of refraction provide signal which can be imaged even under several overlying cell layers. Acetic acid is an external contrast agent which can enhance this nuclear backscattering. Image contrast is degraded by the presence of multiple scattering in overlying cell layers. The degradation of image contrast by cell scattering depends on the scattering phase function; in vitro models which use polystyrene microspheres to approximate tissue underestimate the actual degradation caused by cell scattering. The loss in contrast can be explained using a finite difference time domain model of cellular scattering. We conclude that near real time reflected light confocal microscopy can be used to study cell morphology in vivo. Contrast degradation due to overlying tissue is a concern and cannot adequately be modeled using conventional tissue phantoms; however, acetic acid may be used to substantially increase intrinsic contrast, allowing imaging at significant depths despite distortion from overlying layers. © 1998 Society of Photo-Optical Instrumentation Engineers.

© 1998 Society of Photo-Optical Instrumentation Engineers

Citation

Colin Smithpeter ; Andrew Dunn ; Rebekah Drezek ; Tom Collier and Rebecca Richards-Kortum
"Near Real Time Confocal Microscopy of Cultured Amelanotic Cells: Sources of Signal, Contrast Agents and Limits of Contrast", J. Biomed. Opt. 3(4), 429-436 (Oct 01, 1998). ; http://dx.doi.org/10.1117/1.429853


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