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Near Real Time Confocal Microscopy of Amelanotic Tissue: Dynamics of Aceto-Whitening Enable Nuclear Segmentation

Tom Collier, Peggy Shen, Benoit de Pradier, Kung-Bin Sung, Rebecca Richards-Kortum, Anais Malpica, and Michele Follen

Open Access Open Access

Abstract

High resolution, in vivo confocal imaging of amelanotic epithelial tissue may offer a clinically useful adjunct to standard histopathologic techniques. Application of acetic acid has been shown to enhance contrast in confocal images of these tissues. In this study, we record the time course of aceto-whitening at the cellular level and determine whether the contrast provided enables quantitative feature analysis. Confocal images and videos of cervical specimens were obtained throughout the epithelium before, during and post-acetic acid after the application of 6% acetic acid. Aceto-whitening occurs within seconds after the application. The confocal imaging system resolved sub-cellular detail throughout the entire epithelial thickness and provided sufficient contrast to enable quantitative feature analysis.

©2000 Optical Society of America

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Supplementary Material (3)

Media 1: MOV (3130 KB)     
Media 2: MOV (3098 KB)     
Media 3: MOV (2470 KB)     

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Figures (8)
Figure 1.
Figure 1. Set up of epi-illumination confocal microscope used to collect the images and video.

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Figure 2.
Figure 2. Images of cervical biopsy obtained prior to the application of acetic acid. a). Image taken with confocal microscope with the image plane parallel to the epithelial surface and the focus 20 microns below the surface. b). Same as (a), but with the focus 100 microns below the surface. c). Image of hemotoxylin and eosin stained transverse section using bright field microscopy. Contrast has been reversed in this black and white image to aid in comparing confocal and histologic images. Lines a and b indicate the approximate depth at which the confocal images in (a) and (b) were obtained.

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Figure 3.
Figure 3. Images of cervical biopsy obtained after the application of acetic acid. a). Image taken with confocal microscope with the image plane parallel to the epithelial surface and the focus 50 microns below the surface. b). Same as (a) but with the focus 100 microns beneath the surface. c). Same as (a) but with the focus 200 microns beneath the surface. d). Image of hemotoxylin and eosin stained transverse section using bright field microscopy. Contrast has been reversed in this black and white image to aid in comparing confocal and histologic images. Lines a, b and c indicate the approximate depth at which the confocal images in (a), (b) and (c) were obtained.

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Figure 4.
Figure 4. 3.1 MB Quicktime video of confocal images obtained as the image plane was translated from the epithelial surface to the basement membrane approximately 5 minutes after the application of acetic. The field of view is approximately 200 um.

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Figure 5.
Figure 5. 3.1 MB Quicktime video of confocal images from a fixed image plane approximately 50 um below the epithelial surface as acetic acid is applied. The field of view is approximately 150 um.

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Figure 6.
Figure 6. 2.4 MB Quicktime video of three dimensional rendering viewed from various angles. The rendering was created from 30 images taken at 1 um increments through 3 cell layers.

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Figure 7.
Figure 7. Segmentations of cell features with the feature detection algorithm on images taken without application of acetic acid. a). Unprocessed image of cervical biopsy. b). Same image as (a) with features outlined in yellow.

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Figure 8.
Figure 8. Segmentation of cell nuclei with our feature detection algorithm on images taken with the use of acetic acid. a). Unprocessed image of cervical biopsy. b). Same image as (a) with nuclei highlighted in yellow.

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