Photonics probing of pup brain tissue and molecular-specific nuclear nanostructure alterations due to fetal alcoholism via light scattering/localization approaches.

SIGNIFICANCE: Light is a good probe for studying the nanoscale-level structural or molecular-specific structural properties of brain cells/tissue due to stress, alcohol, or any other abnormalities. Chronic alcoholism during pregnancy, i.e., fetal alcoholism, being teratogenic, results in fetal alcohol syndrome, and other neurological disorders. Understanding the nano-to-submicron scale spatial structural properties of pup brain cells/tissues using light/photonic probes could provide a plethora of information in understanding the effects of fetal alcoholism. AIM: Using both light scattering and light localization techniques to probe alterations in nano- to-submicron scale mass density or refractive index fluctuations in brain cells/tissues of mice pups, exposed to fetal alcoholism. APPROACH: We use the mesoscopic physics-based dual spectroscopic imaging techniques, partial wave spectroscopy (PWS) and molecular-specific inverse participation ratio (IPR) using confocal imaging, to quantify structural alterations in brain tissues and chromatin/histone in brain cells, respectively, in 60 days postnatal mice pup brain, exposed to fetal alcoholism. RESULTS: The finer focusing PWS analysis on tissues shows an increase in the degree of structural disorder strength in the pup brain tissues. Furthermore, results of the molecular-specific light localization IPR technique show an increase in the degree of spatial molecular mass density structural disorder in DNA and a decrease in the degree in histone. CONCLUSIONS: In particular, we characterize the spatial pup brain structures from the molecular to tissue levels and address the plausible reasons for such as mass density fluctuations in fetal alcoholism.

Multi-component redox system for selective and potent antineoplastic activity towards ovarian cancer cells.

Ovarian cancer is the deadliest gynecological cancer which rarely causes symptoms, and goes undetected until reaching the advanced stage of drug-resistant metastases. The cationic porphyrin meso-tetra(4-N-methylpyridyl)porphine (TMPyP) is a well-known photosensitizer (PS) used in photodyamic therapy (PDT) for curing cancer due to its strong affinity for DNA and high yield of reactive oxygen species (ROS) upon light activation. The practicality to irradiate tumor cells alone in the physiological system being slim (due to the close proximity of healthy cells and tumors), we looked for a variation in the PDT using a mixture of TMPyP with 1,5-dihydroxynapthalene (DHN) and Fe(III) ions at a mole ratio of 1:20:17 (drug combo) respectively in aqueous solution. The drug combo needs no photoactivation in H2O2 rich environment (mimicking the microenvironment of cancer/tumor), where it generates È®H and juglone, the latter being a known potent anticancer agent. In vitro studies of the drug combo in drug resistant and sensitive ovarian cancer cell lines showed drastic growth inhibition and cell death compared to normal epithelial cells. The drug combo provides an effective and non-invasive alternative to conventional PDT, exploiting the cytosolic carcinogenic H2O2 to produce an efficient anticancer treatment. The unique action of cancer-specific cytotoxicity arises from the redox chemistry involving activation of Fe(III) as the oxidizing agent to generate juglone, which utilizes the cytosolic ROS in cancer cells against itself.

Photonic technique to study the effects of probiotics on chronic alcoholic brain cells by quantifying their molecular specific structural alterations via confocal imaging.

Molecular specific photonics localization method, the inverse participation ratio (IPR) technique, is a powerful procedure to probe the nano- to submicron scales structural alterations in cells/tissues in their abnormalities due to chronic alcoholism using confocal imaging. Chronic alcoholism introduces abnormalities in brain cells/tissue at the nanoscale level that results in behavioural and psychological disorders which are not well understood. On the other hand, probiotics such as Lactobacillus plantarum enhances brain functions in chronic alcoholism. Using the IPR technique, we probe the molecular specific spatial structural alterations in glial brain cells astrocytes and microglia, as well as in chromatins in the nuclei of cortex brain cells, with or without probiotic treatments in chronic alcoholism. The results show chronic alcoholism alone harms brain cells and the probiotic treatment in chronic alcoholism reverses alcoholic damage in the brain cells/tissues toward normalcy.

TEM study of chronic alcoholism effects on early carcinogenesis by probing the nanoscale structural alterations of cell nuclei.

Nanoscale structural alteration in the nuclei of cells with the progression of carcinogenesis is due to the rearrangements of the basic building blocks in the cell such as DNA, RNA, lipids, etc. Although epigenetic modifications underlie the development of cancer, exposure to carcinogenic chemicals such as alcohol also enhances the development of cancer. We report the effects of chronic alcoholism on early-carcinogenesis based on changes in the degree of nanoscale structural alterations (L d) in nuclei. For this, transmission electron microscopy (TEM) imaging of the nuclei of colonic cells is performed for the following four mouse models: control mice; chronic alcoholic mice treated with ethanol (i.e., EtOH mice); mice treated with colonic carcinogen azoxymethane (AOM) and dextran sulfate sodium (DSS) that induced colitis (i.e., AOM + DSS mice); and chronic alcoholic or EtOH treated mice, together with AOM and DSS treatment (i.e., AOM + DSS + EtOH mice). The disordered optical lattices are constructed from their respective TEM images of thin colonic cell nuclei and the L d values are calculated using the inverse participation ratio (IPR) technique from the spatially localized eigenfunctions of these lattices. Results show no significant difference in the average L d value of the colon cell nuclei of alcohol treated mice relative to its control [i.e., L d(C) ∼ L d(EtOH)]; however, an increase in the L d value of alcohol treated precancerous cells [i.e., L d(AOM + DSS + EtOH) > L d(AOM + DSS)], indicating that alcohol accelerates the early carcinogenic process.

Spectroscopic Study on Pseudomonas Aeruginosa Biofilm in the Presence of the Aptamer-DNA Scaffolded Silver Nanoclusters.

We report the effectiveness of silver nanocluster (Ag-NC) against the biofilm of Pseudomonas aeruginosa (PA). Two DNA aptamers specific for PA and part of their sequences were chosen as templates for growing the Ag-NC. While circular dichroism (CD) studies determined the presence of secondary structures, UV/Vis absorption, and fluorescence spectroscopic studies confirmed the formation of the fluorescent Ag-NC on the DNA templates. Furthermore, mesoscopic physics-based partial wave spectroscopy (PWS) was used to analyze the backscattered light signal that can detect the degree of nanoscale mass density/refractive index fluctuations to identify the biofilm formation, comparatively among the different aptamers with respect to the control sample. The importance of the secondary structure of the aptamer DNA in targeting, successfully binding with the cells and delivering the Ag-NC, is evidenced by the decrease in disorder strength (Ld) of the Ag-NC treated samples compared to the untreated PA cells, which showed the abundance of higher Ld in the PWS studies. The higher Ld value attributed to the higher mass density fluctuations and the formation of biofilm. We envision this study to open a new avenue in using a powerful optical microscopic technique like PWS in detection, and DNA aptamer enclosed silver nanoclusters to prevent biofilms for opportunist pathogens like Pseudomonas aeruginosa.

Studying nanoscale structural alterations in cancer cells to evaluate ovarian cancer drug treatment, using transmission electron microscopy imaging.

Understanding nanoscale structural changes can provide information about the physical state of cells/tissues. It has now been shown that increases in nanoscale structural alterations are associated with the progress of carcinogenesis in most cancer cases, including early carcinogenesis. Anti-cancerous therapies are designed to inhibit the growth of cancer cells; however, it is challenging to detect the efficacy of such drugs in the early stages of treatment. A unique method of assessing the impact of anti-cancerous drugs on cancerous cells/tissues is to probe the nanoscale structural alterations. In this paper, we study the effect of different anti-cancerous drugs on ovarian tumorigenic cells, using their nanoscale structural alterations as a biomarker. Transmission electron microscopy (TEM) imaging on thin cell sections is performed to obtain their nanoscale structures. The degree of nanoscale structural alterations of tumorigenic cells and anti-cancerous drug treated tumorigenic cells are quantified by using the recently developed inverse participation ratio (IPR) technique. Results show an increase in the degree of nanoscale fluctuations in tumorigenic cells relative to non-tumorigenic cells; then a near-reversal of the degree of fluctuation in tumorigenic cells to that in non-tumorigenic cells, following anti-cancerous drug treatment. These results support that the effect of anti-cancerous drugs in cancer treatment can be quantified by using the degree of nanoscale fluctuations in the cells via TEM imaging. Potential applications of the technique for cancer treatment are also discussed.

Optical detection of the structural properties of tumor tissue generated by xenografting of drug-sensitive and drug-resistant cancer cells using partial wave spectroscopy (PWS).

A mesoscopic physics-based optical imaging technique, partial wave spectroscopy (PWS), has been used for the detection of cancer by probing nanoscale structural alterations in cells/tissue. The development of drug-resistant cancer cells/tissues during chemotherapy is a major challenge in cancer treatment. In this paper, using a mouse model and PWS, the structural properties of tumor tissue grown in 3D structures by xenografting drug-resistant and drug-sensitive human prostate cancer cells having 2D structures, are studied. The results show that the 3D xenografted tissues maintain a similar hierarchy of the degree of structural disorder properties as that of the 2D original drug-sensitive and drug-resistant cells.

Optical study of stress hormone-induced nanoscale structural alteration in brain using partial wave spectroscopic microscopy.

Chronic stress affects nano to microscale structures of the brain cells/tissues due to suppression of neural growths and reconnections, hence the neuronal activities. This results in depression, memory loss and even death of the brain cells. Our recently developed novel optical technique, partial wave spectroscopic microscopy has nanoscale sensitivity, and hence, can detect nanoscale changes in brain tissues due to stress. In this study, we applied this technique to quantify the stress related structural changes in the corticosterone-treated mouse model of stress. Our results show that brains from corticosterone-treated mice showed higher nanoscale structural disorder in the hippocampal region as compared to the brain from normal (vehicle) mice. The increase in structural alteration correlates with the duration of the stress. We further quantified the relative changes and the spatial localization of these changes in this mouse model and found out that the maximum changes occurred nearly symmetrically in both regions of the hippocampus. The mRNA for stress-related genes, brain-derived neurotrophic factor and tyrosine kinase-coupled receptor were also significantly reduced in the hippocampus of corticosterone-treated mice compared to that in control mice. These results indicate that chronic corticosterone treatment induces nanoscale structural alterations in mouse brain that corresponds to changes in stress-related gene expression.

Optical study of chemotherapy efficiency in cancer treatment via intracellular structural disorder analysis using partial wave spectroscopy.

As cancer progresses, macromolecules, such as DNA, RNA or lipids, inside cells undergo spatial structural rearrangements and alterations. Mesoscopic light transport-based optical partial wave spectroscopy (PWS) was recently introduced to quantify changes in the nanoscale structural disorder in biological cells. The PWS measurement is performed using a parameter termed as "disorder strength" (L d ), which represents the degree of nanoscale structural disorder inside the cells. It was shown that cancerous cells have higher disorder strength than normal cells. In this work, we first used the PWS to analyze the hierarchy of different types of prostate cancer cells, namely, C4-2, DU-145 and PC-3, by quantifying their average disorder strengths. Results expectedly showed that L d values increases in accordance with the increasing aggressiveness/tumorigenicity levels of these cells. Using the L d parameter, we then analyzed the chemoresistance properties of these prostate cancer cells to docetaxel drug compared to their chemosensitivity. Results show that chemoresistant cancer cells have increased L d values, that is, higher disorder strength, relative to chemosensitive cancer cells. Thus, use of the L d metric can be effective in determining the efficacy of particular chemotherapy.

Clinical significance of MUC13 in pancreatic ductal adenocarcinoma.

BACKGROUND: Poor prognosis of pancreatic cancer (PanCa) is associated with lack of an effective early diagnostic biomarker. This study elucidates significance of MUC13, as a diagnostic/prognostic marker of PanCa. METHODS: MUC13 was assessed in tissues using our in-house generated anti-MUC13 mouse monoclonal antibody and analyzed for clinical correlation by immunohistochemistry, immunoblotting, RT-PCR, computational and submicron scale mass-density fluctuation analyses, ROC and Kaplan Meir curve analyses. RESULTS: MUC13 expression was detected in 100% pancreatic intraepithelial neoplasia (PanIN) lesions (Mean composite score: MCS = 5.8; AUC >0.8, P < 0.0001), 94.6% of pancreatic ductal adenocarcinoma (PDAC) samples (MCS = 9.7, P < 0.0001) as compared to low expression in tumor adjacent tissues (MCS = 4, P < 0.001) along with faint or no expression in normal pancreatic tissues (MCS = 0.8; AUC >0.8; P < 0.0001). Nuclear MUC13 expression positively correlated with nodal metastasis (P < 0.05), invasion of cancer to peripheral tissues (P < 0.5) and poor patient survival (P < 0.05; prognostic AUC = 0.9). Submicron scale mass density and artificial intelligence based algorithm analyses also elucidated association of MUC13 with greater morphological disorder (P < 0.001) and nuclear MUC13 as strong predictor for cancer aggressiveness and poor patient survival. CONCLUSION: This study provides significant information regarding MUC13 expression/subcellular localization in PanCa samples and supporting the use anti-MUC13 MAb for the development of PanCa diagnostic/prognostic test.

Quantification of photonic localization properties of targeted nuclear mass density variations: Application in cancer-stage detection.

Light localization is a phenomenon which arises due to the interference effects of light waves inside a disordered optical medium. Quantification of degree light localization in optical media is widely used for characterizing degree of structural disorder in that media. Recently, this light localization approach was extended to analyze structural changes in biological cell like heterogeneous optical media, with potential application in cancer diagnostics. Confocal fluorescence microscopy was used to construct "optical lattices," which represents 2-dimensional refractive index map corresponding to the spatial mass density distribution of a selected molecule inside the cell. The structural disorder properties of the selected molecules were evaluated numerically using light localization strength in these optical lattices, in a single parameter called "disorder strength." The method showed a promising potential in differentiating cancerous and non-cancerous cells. In this paper, we show that by quantifying submicron scale disorder strength in the nuclear DNA mass density distribution, a wide range of control and cancerous breast and prostate cells at different hierarchy levels of tumorigenicity were correctly distinguished. We also discuss how this photonic technique can be used in examining tumorigenicity level in unknown prostate cancer cells, and potential to generalize the method to other cancer cells.

Light localization properties of weakly disordered optical media using confocal microscopy: application to cancer detection.

We have developed a novel technique to quantify submicron scale mass density fluctuations in weakly disordered heterogeneous optical media using confocal fluorescence microscopy. Our method is based on the numerical evaluation of the light localization properties of an 'optical lattice' constructed from the pixel intensity distributions of images obtained with confocal fluorescence microscopy. Here we demonstrate that the technique reveals differences in the mass density fluctuations of the fluorescently labeled molecules between normal and cancer cells, and that it has the potential to quantify the degree of malignancy of cancer cells. Potential applications of the technique to other disease situations or characterizing disordered samples are also discussed.

Quantitative analysis of nanoscale intranuclear structural alterations in hippocampal cells in chronic alcoholism via transmission electron microscopy imaging.

Chronic alcoholism is known to alter the morphology of the hippocampus, an important region of cognitive function in the brain. Therefore, to understand the effect of chronic alcoholism on hippocampal neural cells, we employed a mouse model of chronic alcoholism and quantified intranuclear nanoscale structural alterations in these cells. Transmission electron microscopy (TEM) images of hippocampal neurons were obtained, and the degree of structural alteration in terms of mass density fluctuation was determined using the light-localization properties of optical media generated from TEM imaging. The results, which were obtained at length scales ranging from ~30 to 200 nm, show that 10-12 week-old mice fed a Lieber-DeCarli liquid (alcoholic) diet had a higher degree of structural alteration than control mice fed a normal diet without alcohol. The degree of structural alteration became significantly distinguishable at a sample length of ~100 nm, which is the typical length scale of the building blocks of cells, such as DNA, RNA, proteins and lipids. Interestingly, different degrees of structural alteration at such length scales suggest possible structural rearrangement of chromatin inside the nuclei in chronic alcoholism.

Spatial temperature gradients guide axonal outgrowth.

Formation of neural networks during development and regeneration after injury depends on accuracy of axonal pathfinding, which is primarily believed to be influenced by chemical cues. Recently, there is growing evidence that physical cues can play crucial role in axonal guidance. However, detailed mechanism involved in such guidance cues is lacking. By using weakly-focused near-infrared continuous wave (CW) laser microbeam in the path of an advancing axon, we discovered that the beam acts as a repulsive guidance cue. Here, we report that this highly-effective at-a-distance guidance is the result of a temperature field produced by the near-infrared laser light absorption. Since light absorption by extracellular medium increases when the laser wavelength was red shifted, the threshold laser power for reliable guidance was significantly lower in the near-infrared as compared to the visible spectrum. The spatial temperature gradient caused by the near-infrared laser beam at-a-distance was found to activate temperature-sensitive membrane receptors, resulting in an influx of calcium. The repulsive guidance effect was significantly reduced when extracellular calcium was depleted or in the presence of TRPV1-antagonist. Further, direct heating using micro-heater confirmed that the axonal guidance is caused by shallow temperature-gradient, eliminating the role of any non-photothermal effects.

Enhancing the sensitivity of mesoscopic light reflection statistics in weakly disordered media by interface reflections.

Reflection statistics have not been well studied for optical random media whose mean refractive indices do not match with the refractive indices of their surrounding media. Here, we theoretically study how this refractive index mismatch between a one-dimensional (1D) optical sample and its surrounding medium affects the reflection statistics in the weak disorder limit, when the fluctuation part of the refractive index (Δn) is much smaller than the mismatch as well as the mean refractive index of the sample (Δn ≪ 〈n〉). In the theoretical derivation, we perform a detailed calculation that results in the analytical forms of the mean and standard deviation (STD) of the reflection coefficient in terms of disorder parameters ( [Formula: see text] and its correlation length lc ) in an index mismatched backscattering system. Particularly, the orders of disorder parameters in STD of the reflection coefficient for index mismatched systems are shown to be lower (~(〈Δn2〉lc )1/2) than that of the matched systems (~〈Δn2〉lc ). By comparing STDs of the reflection coefficient values of index matched and mismatched systems, we show that reflection coefficient at the sample boundaries in index mismatched systems can enhance the signal of the STD to the "disorder parameters" of the reflection coefficient. In terms of biophotonics applications, this result can lead to potential techniques that effectively extract the sample disorder parameters by manipulating the index mismatched conditions. Potential applications of the technique for enhancement in sensitivity of cancer detection at the single cell level are also discussed.

Open source software for electric field Monte Carlo simulation of coherent backscattering in biological media containing birefringence.

ABSTRACT. We present an open source electric field tracking Monte Carlo program to model backscattering in biological media containing birefringence, with computation of the coherent backscattering phenomenon as an example. These simulations enable the modeling of tissue scattering as a statistically homogeneous continuous random media under the Whittle-Matérn model, which includes the Henyey-Greenstein phase function as a special case, or as a composition of discrete spherical scatterers under Mie theory. The calculation of the amplitude scattering matrix for the above two cases as well as the implementation of birefringence using the Jones N-matrix formalism is presented. For ease of operator use and data processing, our simulation incorporates a graphical user interface written in MATLAB to interact with the underlying C code. Additionally, an increase in computational speed is achieved through implementation of message passing interface and the semi-analytical approach. Finally, we provide demonstrations of the results of our simulation for purely scattering media and scattering media containing linear birefringence.

Nanocytology of rectal colonocytes to assess risk of colon cancer based on field cancerization.

Developing a minimally invasive and cost-effective prescreening strategy for colon cancer is critical because of the impossibility of conducting colonoscopy on the entire at-risk population. The concept of field carcinogenesis, in which normal-appearing tissue away from a tumor has molecular and, consequently, nano-architectural abnormalities, offers one attractive approach to identify high-risk patients. In this study, we investigated whether the novel imaging technique partial wave spectroscopic (PWS) microscopy could risk-stratify patients harboring precancerous lesions of the colon, using an optically measured biomarker (L(d)) obtained from microscopically normal but nanoscopically altered cells. Rectal epithelial cells were examined from 146 patients, including 72 control patients, 14 patients with diminutive adenomas, 20 patients with nondiminutive/nonadvanced adenomas, 15 patients with advanced adenomas/high-grade dysplasia, 12 patients with genetic mutation leading to Lynch syndrome, and 13 patients with cancer. We found that the L(d) obtained from rectal colonocytes was well correlated with colon tumorigenicity in our patient cohort and in an independent validation set of 39 additional patients. Therefore, our findings suggest that PWS-measured L(d) is an accurate marker of field carcinogenesis. This approach provides a potential prescreening strategy for risk stratification before colonoscopy.

Alternate formulation of enhanced backscattering as phase conjugation and diffraction: derivation and experimental observation.

Enhanced backscattering (EBS), also known as weak localization of light, is derived using the Huygens-Fresnel principle and backscattering is generally shown to be the sum of an incoherent baseline and a phase conjugated portion of the incident wave that forms EBS. The phase conjugated portion is truncated by an effective aperture described by the probability function P(s) of coherent path-pair separations. P(s) is determined by the scattering properties of the medium and so characterization of EBS can be used for metrology of scattering materials. A three dimensional intensity peak is predicted in free space at a point conjugate to the source and is experimentally observed.

Quantification of nanoscale density fluctuations by electron microscopy: probing cellular alterations in early carcinogenesis.

Most cancers are curable if they are diagnosed and treated at an early stage. Recent studies suggest that nanoarchitectural changes occur within cells during early carcinogenesis and that such changes precede microscopically evident tissue alterations. It follows that the ability to comprehensively interrogate cell nanoarchitecture (e.g., macromolecular complexes, DNA, RNA, proteins and lipid membranes) could be critical to the diagnosis of early carcinogenesis. We present a study of the nanoscale mass-density fluctuations of biological tissues by quantifying their degree of disorder at the nanoscale. Transmission electron microscopy images of human tissues are used to construct corresponding effective disordered optical lattices. The properties of nanoscale disorder are then studied by statistical analysis of the inverse participation ratio (IPR) of the spatially localized eigenfunctions of these optical lattices at the nanoscale. Our results show an increase in the disorder of human colonic epithelial cells in subjects harboring early stages of colon neoplasia. Furthermore, our findings strongly suggest that increased nanoscale disorder correlates with the degree of tumorigenicity. Therefore, the IPR technique provides a practicable tool for the detection of nanoarchitectural alterations in the earliest stages of carcinogenesis. Potential applications of the technique for early cancer screening and detection are also discussed.

The influence of chromosome density variations on the increase in nuclear disorder strength in carcinogenesis.

Microscopic structural changes have long been observed in cancer cells and used as a marker in cancer diagnosis. Recent development of an optical technique, partial-wave spectroscopy (PWS), enabled more sensitive detection of nanoscale structural changes in early carcinogenesis in terms of the disorder strength related to density variations. These nanoscale alterations precede the well-known microscopic morphological changes. We investigate the influence of nuclear density variations due to chromosome condensation on changes of disorder strength by computer simulations of model chromosomes. Nuclear configurations with different degrees of chromosome condensation are realized from simulations of decondensing chromosomes and the disorder strength is calculated for these nuclear configurations. We found that the disorder strength increases significantly for configurations with slightly more condensed chromosomes. Coupled with PWS measurements, the simulation results suggest that the chromosome condensation and the resulting spatial density inhomogeneity may represent one of the earliest events in carcinogenesis.