Reflectance spectroscopy contains information of scatterers and absorbers present inside biological tissues and has been successfully used to diagnose disease. Success of any diagnostic tool depends upon the potential of statistical algorithm to extract appropriate diagnostic features from the measured optical data. In our recent study, we have used the potential of the classification algorithm, Nonlinear Maximum Representation and Discrimination Features (NMRDF) to extract important diagnostic features from reflectance spectra of normal and dysplastic human cervical tissue. This NMRDF algorithm uses the higher order correlation information in the input data, which helps to represent the asymmetrically distributed data and provides the closed form solution of the nonlinear transform for maximum discrimination. We have recorded unpolarized, co and cross-polarized reflectance spectra from 350nm to 650nm, illuminating the human cervical tissue epithelium with white light source. A total of 139 samples were divided into training and validation data sets. The input parameters were optimized using training data sets to extract the appropriate nonlinear features from the input reflectance spectra. These extracted nonlinear features are used as input for nearest mean classifier to calculate the sensitivity and specificity for both training as well as validation data sets. We have observed that co-polarized components provide maximum sensitivity and specificity compared to cross-polarized components and unpolarized data. This is expected since co-polarized light provides subsurface information while cross-polarized and unpolarized data mask the vital epithelial information through high diffuse scattering.
Intrinsic fluorescence spectra of the human normal, cervical intraepithelial neoplasia 1 (CIN1), CIN2, and cervical cancer tissue have been extracted by effectively combining the measured polarized fluorescence and polarized elastic scattering spectra. The efficacy of principal component analysis (PCA) to disentangle the collective behavior from smaller correlated clusters in a dimensionally reduced space in conjunction with the intrinsic fluorescence is examined. This combination unambiguously reveals the biochemical changes occurring with the progression of the disease. The differing activities of the dominant fluorophores, collagen, nicotinamide adenine dinucleotide, flavins, and porphyrin of different grades of precancers are clearly identified through a careful examination of the sectorial behavior of the dominant eigenvectors of PCA. To further classify the different grades, the Mahalanobis distance has been calculated using the scores of selected principal components.
The two highest principal components of fluorescence spectra in visible region obtained, using Xenon lamp as an
excitation source of normal and dysplastic human cervical tissues are analyzed using scatter plots and probability
density functions. These yield significant differences between the tissue types.
Fluorescence spectroscopy has been demonstrated as a viable tool for noting subtle biochemical changes that occur
during early-stage cervical cancer progression. Due to multiple fluorophore contributions, the individual fluorophore
activities often get masked due to overlapping spectra of neighboring fluorophores. Recently synchronous fluorescence
spectroscopy has been demonstrated as an efficient technique for investigation of such non-dominant fluorophores. With
synchronous fluorescence spectroscopy individual fluorophore responses are highlighted as sharp peaks by choosing
appropriate offsets during signal acquisition. Such peaks may, however be missed due to absorption effects. By
correcting the measured synchronous fluorescence spectrum with elastic scattering data, it was observed that the masked
fluorophores are highlighted while the broader bands are sharpened. Interestingly, fluorophore activities of
protoporphyrin, collagen, NADH, FAD and porphyrin can now be studied using this technique, as compared to only
collagen and NADH seen earlier. The results have been verified using tissue phantoms with known fluorophores and
scatterers. Use of normalized synchronous spectra has led to enhancement of several fluorophore responses. It was also
observed that among the different offsets, the lower ones show sharper features, whereas the larger offsets show a
broadband response. Among the different offsets 120nm is found optimal for further investigation.
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