Tom Collier

Plasmon resonance coupling of metal nanoparticles for molecular imaging of carcinogenesis in vivo

An effective cancer control strategy requires improved early detection methods, patient-specific drug selection, and the ability to assess response to targeted therapeutics. Recently, plasmon resonance coupling between closely spaced metal nanoparticles has been used to develop ultrasensitive bioanalytical assays in vitro. We demonstrate the first in vivo application of plasmon coupling for molecular imaging of carcinogenesis. We describe molecular-specific gold bioconjugates to image epidermal growth factor receptor (EGFR); these conjugates can be delivered topically and imaged noninvasively in real time. We show that labeling with gold bioconjugates gives information on the overexpression and nanoscale spatial relationship of EGF receptors in cell membranes, both of which are altered in neoplasia. EGFR-mediated aggregation of gold nanoparticles in neoplastic cells results in more than a 100-nm color shift and a contrast ratio of more than tenfold in images of normal and precancerous epithelium in vivo, dramatically increasing contrast beyond values reported previously for antibody-targeted fluorescent dyes.

Near real-time confocal microscopy of amelanotic tissue: detection of dysplasia in ex vivo cervical tissue.

RATIONALE AND OBJECTIVES The authors performed this study to determine whether images of ex vivo tissue obtained with a near real-time confocal microscope can be used to differentiate between normal and dysplastic tissue. MATERIALS AND METHODS Biopsy specimens of colposcopically normal and abnormal cervical tissue were obtained from 19 patients and imaged at various depths with a confocal microscope. Nuclear morphologic features were extracted from the confocal images; in addition, a group of reviewers examined the images and attempted to identify whether the specimen contained high-grade dysplasia. Results of both analyses were compared with the histopathologic findings of the same specimens provided by a board-certified pathologist with expertise in gynecologic pathology. RESULTS The morphologic feature measurements compared well with the findings at pathologic examination. The use of the nuclear-cytoplasmic ratio to determine the presence of dysplasia resulted in a sensitivity of 100% and a specificity of 91%. The untrained reviewers had an average sensitivity of 95% and an average specificity of 69% in the determination of dysplasia. CONCLUSION The results indicate the clinical potential of in vivo confocal imaging in the detection of dysplasia.

Determination of epithelial tissue scattering coefficient using confocal microscopy

Most models of light propagation through tissue assume the scattering properties of the various tissue layers are the same. The authors present evidence that the scattering coefficient of normal cervical epithelium is significantly lower than values previously reported for bulk epithelial tissue. They estimated the scattering coefficient of normal and precancerous cervical epithelium using measurements of the reflectance as a function of depth from confocal images. Reflectance measurements were taken from ex vivo cervical biopsies and fit to an exponential function based upon Beers law attenuation. The mean scattering coefficients derived were 22 cm/sup -1/ for normal tissue and 69 cm/sup -1/ for precancerous tissue. These values are significantly lower than previously reported for bulk epithelial tissues and suggest that scattering of bulk tissue is dominated by the stroma. They also suggest that computational models to describe light propagation in epithelial tissue must incorporate different scattering coefficients for the epithelium and stroma. Further, the lower scattering of the epithelium suggests greater probing depths for fiber optic probes used by optical diagnostic devices which measure reflectance and fluorescence in epithelial tissue. The difference in scattering between normal and precancerous tissue is attributed to increased nuclear size, optical density, and chromatin texture. The scattering coefficients measured here are consistent with predictions of numerical solutions to Maxwells equations for epithelial cell scattering.

Fiber-optic confocal reflectance microscope with miniature objective for in vivo imaging of human tissues

We have built a fiber-optic confocal reflectance microscope capable of imaging human tissues in near real time. Miniaturization of the objective lens and the mechanical components for positioning and axially scanning the objective enables the device to be used in inner organs of the human body. The lateral resolution is 2 micrometers and axial resolution is 10 micrometers. Confocal images of fixed tissue biopsies and the human lip in vivo have been obtained at 15 frames/s without any fluorescent stains. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope.

Sources of scattering in cervical tissue: determination of the scattering coefficient by confocal microscopy

Most models of light propagation through tissue assume that the scattering properties of various tissue layers are the same. We present evidence that the scattering coefficient of cervical epithelium varies by a factor of 3 within the epithelium owing to variations in nuclear density and to the presence of keratin. We estimated the scattering coefficient from regions of normal and precancerous cervical epithelium by fitting reflectance measurements from confocal images to an exponential function of depth based on Beers law of attenuation. The results suggest that the normal cervix is characterized by highly variable scattering in the superficial epithelium, low scattering in the intermediate epithelium, and high scattering in the basal and stromal regions. In high-grade dysplasia, high scattering from high-density nuclei is observed throughout the entire epithelium.

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

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.

Near real time in vivo fibre optic confocal microscopy: Sub-cellular structure resolved

We have built a fibre optic confocal reflectance microscope capable of imaging biological tissue in near real time. The measured lateral resolution is 3 µm and axial resolution is 6 µm. Images of epithelial cells, excised tissue biopsies, and the human lip in vivo have been obtained at 15 frames s−1. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope. This device has the potential to enable reflected light confocal imaging of internal organs for in situ detection of pathology.

Real-time reflectance confocal microscopy: comparison of two-dimensional images and three-dimensional image stacks for detection of cervical precancer

Confocal microscopy can provide real-time, 2-D and 3-D images of the cellular morphology and tissue architecture features that pathologists use to detect precancerous lesions without the need for tissue removal, sectioning, and staining. The utility of 3-D confocal image stacks of epithelial tissue for detecting dysplasia has not yet been explored. We aim to extract morphometry and tissue architecture information from 2-D confocal reflectance images and 3-D image stacks from fresh, unstained cervical biopsies and compare their potential for detecting dysplasia. Nine biopsies are obtained from eight patients; confocal images are acquired pre- and postacetic acid at multiple epithelial depths in 1.5 mum-intervals. Postacetic acid images are processed to segment cell nuclei; after segmentation, 2-D images taken at 50 mum below the tissue surface, and the entire 3-D image stacks are processed to extract morphological and architectural features. Data are analyzed to determine which features gave the best separation between normal and high-grade cervical precancer. Most significant differences are obtained from parameters extracted from the 3-D image stacks. However, in all cases where the 2-D features were multiplicatively scaled by the depth of acquisition divided by the epithelial thickness or scaled by the scattering coefficient, the significance level is equal to or greater than the comparable feature extracted from the 3-D image stacks. A linear discriminant function previously developed to separate 19 samples of normal tissue and high-grade cervical precancer based on the nuclear-to-cytoplasm (N/C) ratio and epithelial scattering coefficient is prospectively applied to the nine biopsies examined to determine the accuracy with which it could separate normal tissue from cervical intra epithelial neoplasia (CIN) 23. For the entire data set of 28 biopsies, a sensitivity and specificity of 100% is produced using this discriminant function; the scattering coefficient provides more discriminative capacity than the N/C ratio. The success of the scaled 2-D image features has important implications for using confocal microscopy to detect precancer in the clinic. Acquisition of the epithelial thickness or scattering coefficient requires less time than 3-D image sets and little additional effort is required to gain the added information compared to 2-D images alone.

Multispectral digital microscopy for in vivo monitoring of oral neoplasia in the hamster cheek pouch model of carcinogenesis

In this study we use a multi-spectral digital microscope (MDM) to measure multi-spectral auto-fluorescence and reflectance images of the hamster cheek pouch model of DMBA (dimethylbenz[alpha]anthracene)- induced oral carcinogenesis. The multi-spectral images are analyzed both in the RGB (red, green, blue) color space as well as in the YCbCr (luminance, chromatic minus blue, chromatic minus red) color space. Mean image intensity, standard deviation, skewness, and kurtosis are selected as features to design a classification algorithm to discriminate normal mucosa from neoplastic tissue. The best diagnostic performance is achieved using features extracted from the YCbCr space, indicating the importance of chromatic information for classification. A sensitivity of 96% and a specificity of 84% were achieved in separating normal from abnormal cheek pouch lesions. The results of this study suggest that a simple and inexpensive MDM has the potential to provide a cost-effective and accurate alternative to standard white light endoscopy.

Near real-time in vivo confocal imaging of mouse mammary tumors.

The goal of this study was to evaluate the ability of near real-time reflectance confocal microscopy to image tumor metastasis in vivo in an animal model. We used an epi-illumination confocal microscope to capture images of mouse mammary tumors in nude immunodeficient and Balb/C immunocompetent mice. In vivo confocal images and videos of normal and neoplastic areas were obtained before and after the application of a 6% acetic acid solution, with a lateral resolution of 0.8 microns and an axial resolution of 2-3 microns. Average imaging depths ranged from 150 microns to greater than 300 microns. We were able to differentiate between normal and abnormal tissue areas within the mammary gland, including areas of adipose tissue, fibroblasts and connective tissue, neoplastic tissue, and blood flow within blood vessels. Intravital imaging with reflectance confocal microscopy appears to be a useful tool to study tumor metastasis in vivo.