Mohammad Zaffar

In the current paper, we represent intrinsic fluorescence anisotropies as four-dimensional normalized Stokes vectors defined by the maximum excitation and emission in the fluorescence process with respect to linear, linear-45 and circular polarizations of light. Depending upon the transition moments for absorption/excitation and emission of fluorophores, eight types of these Stokes vectors can be realized from the Mueller fluorescence matrix of the system. These Stokes vectors probe the orientational distribution of fluorophores and predict the nature of optically active biological proteins, whether laevorotatory or dextrorotatory. The orthogonality relation between the Stokes vectors corresponding to the excitation and emission processes of fluorescence connects the molecular ground and excited states of biological and non-biological systems.

Laser speckle contrast imaging (LSCI) methods are extensively used in assessment of blood flow to detect various pathological conditions in different parts of human body. In contrast to LSCI being deployed for larger region of interest (few centimeters), we present a laser speckle imaging at microscopic level. Rather than utilizing the conventional speckle contrast, we use intensity auto-correlation using recently developed Multi-step Volterra Integral method(MVIM) to quantify the micro-perfusion. The proposed laser speckle correlation microscopy (LSCM) system is validated using microfluidic flow phantom experiments.

We present a laser speckle contrast imaging (LSCI) device equipped with an image conduit to image microvascular blood flow in remote tissues like ear, nose, throat (ENT) and cervical region. The system is validated using a tissue mimicking microfluidic flow phantom with different widths and flow speeds. The proposed system is being developed as a point of care testing (POCT) device best suited for at-home self-monitoring in resource-limited areas as it is non-invasive, portable, affordable and real time.

A rigorous model has been proposed to qualify fluorescence scattering through the Mueller matrix viewpoint in terms of absorption/excitation, emission, and the process in between them. The process in between the excitation and emission processes of fluorescence, irrespective of the scattering directions, has been modeled as a depolarization process. The absorption/excitation of the fluorophore molecules gets revealed through the first-row elements while the emission of fluorescence has been observed through the first column elements of the fluorescence Mueller matrix. Information of the transitions between the molecular ground and excited states gets encoded into the diagonal elements following the photon selection rule. The other off-diagonal elements of the fluorescence Mueller matrix also exhibit very small nonzero values due to the anisotropic absorption and phase changes that the ground state of the fluorophore molecules imposes on the incident polarized beam while parallelly governing the emitted beam. The comparison of the current model with the earlier model has been discussed in a detailed way. The modeling of the in-between process as the depolarizing one enables us to qualify the fluorescence detected linear and circular dichroism and luminescence and very effectively overcomes the shortcomings in the earlier model.

Time-resolved Mueller matrix (MM) imaging polarimetry in transmission mode has been implemented in both epithelium and stromal regions of cervical tissues to explore the various polarization dynamics in connection with the diagnosis of cervical precancer. The picosecond-resolved intensity patterns of various MM elements, resulting from the various orders of scattering, at different time delays provide clear demarcation between the epithelium and stroma of cervical tissue. The time dependent depolarization and retardance maps are seen to differentiate the epithelium from stroma. The average values of time dependent linear, linear-45, and circular depolarization and linear, circular, and scalar retardance parameters in different regimes of scattering from the optically anisotropic stromal region identify the pre-malignancy in cervical tissue. As the disease evolves, time dependent linear depolarization varies to larger values as compared to time dependent circular depolarization. Interestingly, the chirality of the collagen network that rotates the plane of polarized light in either direction in normal samples is limited to only the clockwise direction during the progression of the disease. These results show potential in the early detection and understanding of the mechanisms of morphological changes in cervical cancer development.

The spatial autocorrelation and correlation map of amplitude and phase anisotropy along with depolarization parameter from the stroma of uterine cervix utilizing their Mueller matrix (MM) images have been reported for early diagnosis of cervical cancer and differentiation of precancerous stages. The comparative results of the evaluation of the spatial autocorrelation over MM images of optically anisotropic collagen structures from normal and various grades of cervical precancer reflect significant alterations which are correlated with the pathological changes. The spatially varying polarizance from different region of anisotropic stromal region gets correlated within a given spatial lag during the precancerous changes. The diattenuation governing elements M12, M13 and M14 clearly discriminate normal and various grades of precancerous cervical tissue through their autocorrelation profile and correlation map. Evaluation of autocorrelation of spatially varying linear birefringence and linear‐45 birefringence characterized by MM elements M34 and M43 and M24 and M42 are not found to differ between the precancer grades, indicating that these changes may be arising from highly directional collagen network while the changes displayed by MM elements M23 and M32 faithfully represent that the chirality of the stromal region is compromised as the cervical cancer evolves and only one type of nature dominates.

Analysis of spatial frequency of Mueller matrix (MM) images in the Fourier domain yields quantifying parameters of anisotropy in the stromal region in normal and precancerous tissue sections of human uterine cervix. The spatial frequencies of MM elements reveal reliable information of microscopic structural organization arising from the different orientations of collagen fibers in the connective tissue and their randomization with disease progression. Specifically, the local disorder generated in the normal periodic and regular structure of collagen during the growth of the cervical cancer finds characteristic manifestation in the Fourier spectrum of the selected Mueller matrix elements encoding the anisotropy effects through retardance and birefringence. In contrast, Fourier spectra of differential polarization gated images are limited to only one orientation of collagen. Fourier spectra of first row elements M11, M12, M13, and M14 and first column elements M11, M21, M31, and M41 discriminates cervical inter-epithelial neoplasia (CIN)-I from normal cervical tissue samples with 95%–100% sensitivity and specificity. FFT spectra of first and fourth row elements classify CIN-I and CIN-II grades of cervical cancerous tissues with 90%–100% sensitivity and 87%–100% specificity. Normal and CIN-II grade samples are successfully discriminated through Fourier spectra of every MM element while that of M31 element arises as the key classifier among normal, CIN-I, and CIN-II grades of cervical cancer with 100% sensitivity and specificity. These results demonstrate the promise of spatial frequency analysis of Mueller matrix images as a novel, to the best of our knowledge, approach for cancer/precancer detection.

The mapping of diattenuation, polarizance and retardance vector (normalized Stokes vector) on Poincare sphere, evaluated from Mueller matrix of optically anisotropic stromal region of cervical tissues, is presented for cervical precancer detection and its staging. This reveals that the changes in the polarization states shown by these normalized Stokes vectors corresponds to the degradation of linearly arranged collagen fibers, breakage of the collagen cross links in the stromal region and change in the density of scattering sites when cervical cancer evolves. The distinct nature of real and imaginary parts of the refractive index for linear, linear‐45 and circular polarization from the optically anisotropic stromal region underscore the various polarization structures of the connective tissue region which get modified during the pathological changes. It has been found that versatility of these vectors for normal and precancerous cervical tissue of various grades may be utilized as a key distinction for qualitative staging of cervical precancer tissue. Quantitative classification of precancerous stages of cervical precancer has been determined with 95%–100% sensitivity and 93%–100% specificity through the evaluation of linear and circular diattenuation, linear polarizance and linear birefringence from the components of the respective vectors.

An autocorrelation study has been conducted on Mueller matrix images of stromal region of cervical tissue sections for early cancer detection. Changes in multiple scattering and deterioration of stromal architecture through their spatial autocorrelation maps are observed among healthy and various grades of cervical precancerous tissues. Autocorrelation maps of diagonal elements reflects increasing depolarization property while the disease progresses. These maps of Mueller elements governing polariance, di-attenuation and birefringence properties qualitatively establish a demarcation of different grades of cancer from normal tissue. Moreover, spatial autocorrelation on optical parameters like circular di-attenuation, liner and linear-45 di-attenuation along with circular birefringence is decreasing with the evolution of cervical cancer. These preliminary results about the stromal biology are very promising in the early detection of cervical cancer.

A systematic study has been conducted on application of wavelet based multifractal de-trended fluctuation analysis (MFDFA) on Mueller matrix (MM) images of cervical tissue sections for early cancer detection. Changes in multiple scattering and orientation of fibers are observed by utilizing a discrete wavelet transform (Daubechies) which identifies fluctuations over polynomial trends. Fluctuation profiles, after 9th level decomposition, for all elements of MM qualitatively establish a demarcation of different grades of cancer from normal tissue. Moreover, applying MFDFA on MM images, Hurst exponent profiles for images of MM qualitatively are seen to display differences. In addition, the values of Hurst exponent increase for the diagonal elements of MM with increasing grades of the cervical cancer, while the value for the elements which correspond to linear polarizance decrease. However, for circular polarizance the value increases with increasing grades. These fluctuation profiles reveal the trend of local variation of refractive -indices and along with Hurst exponent profile, may serve as a useful biological metric in the early detection of cervical cancer. The quantitative measurements of Hurst exponent for diagonal and first column (polarizance governing elements) elements which reflect changes in multiple scattering and structural anisotropy in stroma, may be sensitive indicators of pre-cancer.