The ability to image and quantitate fluorescently labeled markers in vivo has generally been limited by autofluorescence of the tissue. Skin, in particular, has a strong autofluorescence signal, particularly when excited in the blue or green wavelengths. Fluorescence labels with emission wavelengths in the near-infrared are more amenable to deep-tissue imaging, because both scattering and autofluorescence are reduced as wavelengths are increased, but even in these spectral regions, autofluorescence can still limit sensitivity. Multispectral imaging (MSI), however, can remove the signal degradation caused by autofluorescence while adding enhanced multiplexing capabilities. While the availability of spectral "libraries" makes multispectral analysis routine for well-characterized samples, new software tools have been developed that greatly simplify the application of MSI to novel specimens.
We present a method for selective detection of size-dependent scattering characteristics of epithelial cells in vivo based on polarized illumination and polarization sensitive detection of scattered light. We illustrate the method using phantoms designed to simulate squamous epithelial tissue and progressing to epithelial tissue in vitro and in vivo. Elastic light scattering spectroscopy with polarized illumination/detection dramatically reduces background signals due to both diffuse stromal scattering and hemoglobin absorption. Resulting spectra can be described as a linear combination of forward and backscattering components determined from Mie theory. Nuclear sizes and refractive indices extracted by fitting experimental spectra to this model agree well with previous measurements. Reflectance spectroscopy with polarized light can provide quantitative morphological information which could potentially be used for non-invasive detection of neoplastic changes.
Optical coherence tomography (OCT) acquires cross-sectional images of tissue by measuring back-reflected light. Images from in vivo OCT systems typically have a resolution of 10 to 15 mm, and are thus best suited for visualizing structures in the range of tens to hundreds of microns, such as tissue layers or glands. Many normal and abnormal tissues lack visible structures in this size range, so it may appear that OCT is unsuitable for identification of these tissues. However, examination of structure-poor OCT images reveals that they frequently display a characteristic texture that is due to speckle. We evaluated the application of statistical and spectral texture analysis techniques for differentiating tissue types based on the structural and speckle content in OCT images. Excellent correct classification rates were obtained when images had slight visual differences (mouse skin and fat, correct classification rates of 98.5 and 97.3%, respectively), and reasonable rates were obtained with nearly identical-appearing images (normal versus abnormal mouse lung, correct classification rates of 64.0 and 88.6%, respectively). This study shows that texture analysis of OCT images may be capable of differentiating tissue types without reliance on visible structures.
There is no satisfactory mechanism to detect premalignant lesions in the upper aero‐digestive tract. Fluorescence spectroscopy has potential to bridge the gap between clinical examination and invasive biopsy; however, optimal excitation wavelengths have not yet been determined. The goals of this study were to determine optimal excitation–emission wavelength combinations to discriminate normal and precancerous/cancerous tissue, and estimate the performance of algorithms based on fluorescence. Fluorescence excitation–emission matrices (EEM) were measured in vivo from 62 sites in nine normal volunteers and 11 patients with a known or suspected premalignant or malignant oral cavity lesion. Using these data as a training set, algorithms were developed based on combinations of emission spectra at various excitation wavelengths to determine which excitation wavelengths contained the most diagnostic information. A second validation set of fluorescence EEM was measured in vivo from 281 sites in 56 normal volunteers and three patients with a known or suspected premalignant or malignant oral cavity lesion. Algorithms developed in the training set were applied without change to data from the validation set to obtain an unbiased estimate of algorithm performance. Optimal excitation wavelengths for detection of oral neoplasia were 350, 380 and 400 nm. Using only a single emission wavelength of 472 nm, and 350 and 400 nm excitation, algorithm performance in the training set was 90% sensitivity and 88% specificity and in the validation set was 100% sensitivity, 98% specificity. These results suggest that fluorescence spectroscopy can provide a simple, objective tool to improve in vivo identification of oral cavity neoplasia.
Actinic Keratosis (AK) arises from sun-damaged skin and is the first clinical manifestation in the multistep process of skin carcinogenesis to invasive squamous cell carcinoma. Thus, it is an ideal target for chemopreventive efforts. Noninvasive measures of AK severity are needed to assess the efficacy of chemoprevention agents. We performed a pilot study on 20 participants to investigate the OCT appearance of sun-protected skin of the upper inner arm as well as sun-damaged skin and early AKs of the dorsal forearms, and to determine if features or quantitative measures in Optical Coherence Tomography (OCT) images could be used to reliably differentiate between these categories. OCT images of upper inner arm (normal appearing skin) showed skin layers and features (stratum corneum, epidermis, dermis, blood vessels) seen in previous studies; additionally in this participant group the subcutaneous fat layer was usually identified. Sun-damaged skin was characterized by increased signal in the epidermis and rapid attenuation of light. AKs were diverse in appearance but frequently characterized by high surface reflection, the presence of a low-signal band in the stratum corneum, and heterogeneous appearance in the epidermis/dermis. Significant differences were found between skin categories using measures of stratum corneum and epidermal/dermal depths and intensities. The presence of a dark band in the stratum corneum was 79% sensitive and 100% specific for AK. This study indicates that OCT holds promise as a useful technique for identifying and characterizing AKs and monitoring their response to chemoprevention agents.
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