Agarwal, Nitin Biancardi, Alberto M. Patten, Florence W. Reeves, Anthony P. Seibel, Eric J.
Affiliations A University of Washington, Human Photonics Laboratory, Department of Bioengineering, 204 Fluke Hall, Seattle, Washington 98195 b Cornell University, Vision & Image Analysis Group, School of Electrical and Computer Engineering, 392 Rhodes Hall, Ithaca, New York 14850 c VisionGate Inc., 275 N. Gateway Drive, Phoenix, Arizona 85034 d University of Washington, Human Photonics Laboratory, Department of Mechanical Engineering, P.O. Box 352600, Seattle, Washington 98195.
General Fluorescence Microscopy
Aneuploidy is typically assessed by flow cytometry (FCM) and image cytometry (ICM). We used optical projection tomographic microscopy (OPTM) for assessing cellular DNA content using absorption and fluorescence stains. OPTM combines some of the attributes of both FCM and ICM and generates isometric high- resolution three-dimensional (3-D) images of single cells. Although the depth of field of the microscope objective was in the submicron range, it was extended by scanning the objective’s focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. These projections were later reconstructed using computed tomography methods to form a 3-D image. We also present an automated method for 3-D nuclear segmentation. Nuclei of chicken, trout, and triploid trout erythrocyte were used to calibrate OPTM. Ratios of integrated optical densities extracted from 50 images of each standard were compared to ratios of DNA indices from FCM. A comparison of mean square errors with Thionin, Hematoxylin, Feulgen, and SYTOX green was done. Feulgen technique was preferred as it showed highest stoichiometry, least variance, and preserved nuclear morphology in 3-D. The addition of this quantitative biomarker could further strengthen existing classifiers and improve early diagnosis of cancer using 3-D microscopy.
… “an epi-fluorescence setup was used, in which light from the light-emitting diodes (pE100, CoolLED, River Way, Andover, United Kingdom) was filtered by an excitation filter and then reflected to an objective lens by a dichromic mirror.” …
Product Associated Features
pE-100: A range of compact, simple-to-use, single wavelength illumination systems for screening fluorescence.
Journal of Medical Imaging
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