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With its precise, sensitive, and nondestructive features, spectral unmixing-based fluorescence resonance energy transfer (FRET) microscopy has been widely applied to visualize intracellular biological events. In this report, we set up a spectral wide-field microscopic FRET imaging system by integrating a varispec liquid crystal tunable filter into a wide-field microscope for quantitative FRET measurement in living cells. We implemented a representative emission-spectral unmixing-based FRET measurement method on this platform to simultaneously acquire pixel-to-pixel images of both FRET efficiency (E) and acceptor-to-donor concentration ratio (RC) in living HepG2 cells expressing fusion proteins in the presence or absence of free donors and acceptors and obtained consistent results with other instruments and methods. This stable and low-cost spectral widefield microscopic FRET imaging system provides a new toolbox for imaging molecular events with high spatial resolution in living cells. [ABSTRACT FROM AUTHOR]

Several studies demonstrated that the cyclic adenosine monophosphate (cAMP), an important second messenger, is involved in the mechanism of low-level laser irradiation (LLLI) treatment. However, most of these studies obtained the cAMP level in cell culture extracts or supernatant. In this study, the cAMP level in living cells was measured with bioluminescence resonance energy transfer (BRET). The effect of LLLI on cAMP level in living cells with adenosine receptors blocked was explored to identify the role of adenosine receptors in LLLI. The results showed that LLLI increased the cAMP level. Moreover, the rise of cAMP level was light dose dependent but wavelength independent for 658-, 785-, and 830-nm laser light. The results also exhibited that the adenosine receptors, a class of G protein-coupled receptor (GPCR), modulated the increase of cAMP level induced by LLLI. The cAMP level increased more significantly when the A3 adenosine receptors (A3R) were blocked by A3R antagonist compared with A1 adenosine receptor or A2a adenosine receptor blocked in HEK293T cells after LLLI, which was in good agreement with the adenosine receptors' expressions. All these results suggested that measuring the cAMP level with BRET could be a useful technique to study the role of GPCRs in living cells under LLLI. [ABSTRACT FROM AUTHOR]

Optical spectroscopic techniques, including Raman spectroscopy, have shown promise for in vivo cancer diagnostics in a variety of organs. In this study, the potential use of a home-made Raman spectral system with a millimeter order excitation laser spot size combined with a multivariate statistical analysis for the rapid detection and discrimination of nasopharyngeal cancer from normal nasopharyngeal tissue was evaluated. Raman scattering signals were acquired from 16 normal and 32 nasopharyngeal carcinoma tissue samples. Linear discriminant analysis (LDA) based on principal component analysis (PCA) and partial least squares (PLS) were employed to generate diagnostic algorithms for the classification of different nasopharyngeal tissue types. Spectral differences in Raman spectra between the two types of tissues were revealed; the normalized intensities of Raman peaks at 1,001, 1,207 and 1,658 cm-1 were more intense for nasopharyngeal carcinoma tissue compared to normal tissue, while Raman bands at 848, 936 and 1,446 cm-1 were stronger in normal nasopharyngeal samples. The PCA-LDA algorithm together with leave-one-out cross validation yields a diagnostic sensitivity of 81% and a specificity of 87%, while the PLS method coupled with subwindow permutation analysis improves the diagnostic sensitivity and specificity to 85 and 88%, respectively. Therefore, Raman spectroscopy combined with PCA-LDA/PLS demonstrated good potential for improving the clinical diagnosis of nasopharyngeal cancers. [ABSTRACT FROM AUTHOR]

Since changes in the basement membranes are the critical indicators for differentiating normal, precancerous, and cancerous colonic tissues, direct visualization of these warning signs is essential for the early diagnosis and treatment of colonic cancer. Here, we present that second harmonic generation (SHG) microscopy can probe the changes of basement membranes in different colonic cancer stages. Our results also show the capability of using the quantitative analyses of images for quantifying these changes in different cancer stages. These results suggest that SHG microscopy has the potential in label-freely imaging the changes of basement membranes for effectively distinguishing between normal, precancerous, and cancerous colonic tissues. To our knowledge, this is the first demonstration of the dynamics of basement membrane changes in different colonic cancer stages using entirely intrinsic source of contrast. [ABSTRACT FROM AUTHOR]

The aim of this study is to develop a light-emitting diode- (LED-) based illumination system that can be used as an alternative light source for in vitro photodynamic therapy (PDT). This illumination system includes a red LED array composed of 70 LEDs centered at 643 nm, an air-cooling unit, and a specific-designed case. The irradiance as a function of the irradiation distance between the LED array and the sample, the homogeneity and stability of irradiation, and the effect of long-time irradiation on culture medium temperature were characterized. Furthermore, the survival rate of the CNE1 cells that sensitized with 5-aminolevulinic acid after PDT treatment was evaluated to demonstrate the efficiency of the new LED-based illumination system. The obtained results show that the LED-based illumination system is a promising light source for in vitro PDT that performed in standard multiwell plate. [ABSTRACT FROM AUTHOR]

We demonstrate the application of multiphoton microscopy (MPM) based on two-photon excited fluorescence and second harmonic generation signals in imaging two-layered structures of cervical tissue in mice. It was found that MPM is effective for visualizing two-layered cervical microstructures, monitoring the metabolic activity and the density (nuclear-to-cytoplasmic) of epithelial cells, measuring the size of epithelial cell nuclei and the thickness of the epithelial layer, and quantifying collagen in the stroma. These results suggest that, with the advent of clinical portability of typical multiphoton endoscopes, the MPM technique has the potential to be applied in vivo to the clinical diagnosis and monitoring of cervical cancer. [ABSTRACT FROM AUTHOR]

A new numerical method for solving the generalized regularized long wave equation is devised and analyzed. By using a reproducing kernel function, the numerical solution at each discrete time step is obtained by an explicit integral expression even though the scheme is truly implicit, and, hence, the computation is fully parallel. The error estimates are given and some numerical results are presented. [ABSTRACT FROM AUTHOR]

Background/purpose: The purpose of this work was to demonstrate the use of the nonlinear spectral imaging technique with the image-guided spectral analysis function and the extracting channels function in visualizing the extracellular maxtrix (ECM) structures and sensing fibroblasts metabolism within fresh and untreated human dermis. Methods: A Zeiss LSM 510 META laser scanning microscopy and a femtosecond Ti: sapphire laser was used to obtain the nonlinear spectral images of human dermis. Results: The nonlinear spectral imaging technique was useful in obtaining the biomorphology and biochemistry information from human dermis. By combining the image-guided spectral analysis function, intrinsic components were identified and their corresponding emission spectra can be analyzed. By integrating the extracting channels function, the information of ultrastructure of the ECM can be quantitatively obtained. Conclusion: The nonlinear spectral imaging technique has been demonstrated to be an effective technique for a detailed analysis and qualitative visualization of the ECM structure, and sensing of cell metabolism within fresh and untreated human dermis. Specifically, the ultrastructure of the ECM can be revealed, and collagen fibril spacing, elastic fiber diameters, the ration of collagen content over elastin can be quantitatively determined. It was found that the nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) signals excited at 830 nm provide the information for a good estimation of cellular redox ratio and a parameter (NF), the ration of NADH over FAD fluorescence signal, is used for sensing fibroblasts metabolism. With the advent of the clinical portability of typical multiphoton microscopy, the technique has the potential to be applied for in vivo studies and clinical examinations. [ABSTRACT FROM AUTHOR]

This work reports on the measurement of optical properties from nine normal and cancerous human esophageal stroma pairs using reflectance-based confocal microscopy. It was found that the scattering coefficient of cancerous stroma is significantly lower than that of normal stroma. The results suggest that the decreased scattering in cancerous stroma may provide a possible indicator for differentiating normal and cancerous stroma. [ABSTRACT FROM AUTHOR]

Collagen alteration is critical for epithelial tumor initiation and progression. Quantitatively linking collagen alteration and epithelial tumor progression is essential for developing an optical endoscopy to evaluate epithelial tumor progression. In this work, we established a quantitative link between collagen alteration and epithelial tumor progression using second harmonic generation (SHG) microscopy. It was found that SHG microscopy can provide quantitative features to effectively evaluate epithelial tumor progression, and to locate tumor and determine the margin of tumor regions. These results suggest that SHG microscopy has the potential in offering a noninvasive in vivo imaging tool to quantify epithelial tumor progression. [ABSTRACT FROM AUTHOR]

The calcium ion (Ca2+) is a ubiquitous second messenger that is responsible for regulating a wide range of cellular and biological processes, including cell proliferation and cell death, gene expression, muscle contraction and neurotransmission. Here, we demonstrate the change of intracellular Ca2+ release in dorsal root ganglion (DRG) neurons in vitro by femtosecond laser stimulation using confocal microscopy combined with special fluorescence probe Fluo-3/AM. The results showed that relative fluorescence intensity increased significantly after the axons of DRG neurons was performed with photostimulation. It demonstrated that femtosecond laser stimulation could affect the intracellular Ca2+ release in the sensory neurons and might be used to treat pain by modulating intracellular Ca2+ release in DRG neurons. [ABSTRACT FROM AUTHOR]

The purpose of this study is to illustrate experimentally the optical coherence tomography (OCT) signal slope and diffuse reflectance (DR) spectra of 30% and 80% glycerol combined with electroporation (EP) diffusion in normal, benign and cancerous human gastric tissues in vitro. The results of OCT showed that the permeability coefficients of 80% and 30% glycerol (both with and without EP) have the following trend: human cancerous gastric tissue  >  human benign gastric tissue  >  human normal gastric tissue under the same conditions. The permeability coefficient of the 30% glycerol group is larger than that of the 80% glycerol group under the same circumstances; the permeability coefficient of glycerol combined with the EP group is larger than that without the EP group under the same conditions. The permeability coefficient and the reduction of the DR spectra have perfect linear correlation (R2  =  0.9745). The research results suggest that OCT and the DR spectra combined with an optical clearing agent (glycerol) and the EP method can potentially become a powerful tool for the early diagnosis and monitoring of human gastric cancer. [ABSTRACT FROM AUTHOR]

Lipid rafts have been extensively studied and shown to be involved in many cancers, including breast cancer. However, the exact role of lipid rafts in the migration of breast cancer cells remains unclear. This study was designed to examine lipid rafts (cholesterol) in the plasma membrane of breast cancer cells (MDA-MB-231 and MCF-7) and normal breast epithelial cells (MCF-10A) through generalized polarization values, and further investigate the role of cholesterol-rich lipid rafts in the migration of breast cancer cells. The results showed that the plasma membrane in breast cancer cells was more orderly than that in normal epithelial cells; this was correlated with expression changes of matrix metallopeptidase 9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR), the markers of cancer cell migration. Moreover, the breast cancer cells were more sensitive to the reagent that induced cholesterol depletion than the normal breast epithelial cells, while the capacity of cancer cells to migrate decreased significantly according to changes in MMP-9 and uPAR expression. To our best knowledge, this is the first demonstration of the relationship between cholesterol-rich lipid rafts and the migration of breast cancer cells; it could be useful for the prevention of breast cancer and early treatment through reduction of the level of cholesterol in the plasma membrane of the cells. [ABSTRACT FROM AUTHOR]

The new time high-order energy-preserving schemes are proposed for the nonlocal Benjamin-Ono equation. We get the Hamiltonian system to the nonlocal model, and it is then discretized by a Fourier pseudospectral method in space and the Hamiltonian boundary value method (HBVM) in time. This approach has high order of convergence in time and conserves the total mass and energy in discrete forms. We further develop a time second-order energy-preserving scheme and a time fourth-order energy-preserving scheme for the nonlocal Benjamin-Ono equation. Numerical experiments test the proposed schemes with a single solitary wave and the interaction of two solitary waves. Results confirm the accuracy and conservation properties of the schemes. [ABSTRACT FROM AUTHOR]

Combining atomic force microscopy (AFM) with Raman spectroscopy (RS), three different subtypes of breast cell lines, including metastatic cancer cells (MDA-MB-231), non-malignant cancer cells (MCF-7) and benign cells (MCF-10A), were studied to compare their differences in nano-mechanical and biochemical properties. Based on AFM measurements, two cancerous cells were found to have a close elasticity modulus, but were significantly softer than that of their benign counterparts. Raman spectral analysis revealed that the data points for two cancerous cells were distinct with completely separated clusters. The results demonstrate that combined AFM and RS techniques could obtain information about the biomechanical and biochemical properties necessary to distinguish different subtypes of breast cancer cells. This will hold great promise for cancer detection at the single cell level. [ABSTRACT FROM AUTHOR]

In this work, the potential use of nanoparticles as contrast agents by using spectral domain optical coherence tomography (SD-OCT) in liver tissue was demonstrated. Gold nanoparticles (average size of 25 and 70 nm), were studied in human normal and cancerous liver tissues in vitro, respectively. Each sample was monitored with SD-OCT functional imaging for 240 min. Continuous OCT monitoring showed that, after application of gold nanoparticles, the OCT signal intensities of normal liver and cancerous liver tissue both increase with time, and the larger nanoparticles tend to produce a greater signal enhancement in the same type of tissue. The results show that the values of attenuation coefficients have significant differences between normal liver tissue and cancerous liver tissue. In addition, 25 nm gold nanoparticles allow higher penetration depth than 70 nm gold nanoparticles in liver tissues. [ABSTRACT FROM AUTHOR]

Direct measurement of near-infrared (NIR) luminescence around 1270 nm is the golden standard of singlet oxygen (1O2) identification. In this study, the influence of pulse-height discrimination threshold on measurement accuracy of the 1O2 luminescence that is generated from the photoirradiation of meso-tetra (N-methyl-4-pyridyl) morphine tetra-tosylate (TMPyP) in aqueous solution was investigated by using our custom-developed detection system. Our results indicate that the discrimination threshold has a significant influence on the absolute 1O2 luminescence counts, and the optimal threshold for our detection system is found to be about [?] 41.2 mV for signal discrimination. After optimization, the derived triplet-state and 1O2 lifetimes of TMPyP in aqueous solution are found to be 1.73 +- 0.03 and 3.70 +- 0.04 us, respectively, and the accuracy of measurement was further independently demonstrated using the laser flash photolysis technique. [ABSTRACT FROM AUTHOR]

Intrinsic two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) signals are shown to differentiate between normal and neoplastic human esophageal stroma. It was found that TPEF and SHG signals from normal and neoplastic stroma exhibit different organization features, providing quantitative information about the biomorphology and biochemistry of tissue. By comparing normal with neoplastic stroma, there were significant differences in collagen-related changes, elastin-related changes, and alteration in proportions of matrix molecules, giving insight into the stromal changes associated with cancer progression and providing substantial potential to be applied in vivoto the clinical diagnosis of epithelial precancers and cancers. [ABSTRACT FROM AUTHOR]

Because of frequent exposure to carcinogens, the bronchus is prone to early pathologic alterations. The assessment of these early changes is of key significance in physiological studies and disease diagnosis of the bronchus. We utilize nonlinear optical microscopy (NLOM) to image mouse bronchial tissue based on intrinsic nonlinear optical contrast. Our results show that NLOM is effective for imaging the bronchial intact microstructural components, providing quantitative information about the biomorphology and biochemistry of tissue. Our findings also display that NLOM can provide a two-photon ratiometric redox fluorometry, based on mitochondrial signals and reduced pyridine nucleotide (NADH and NADPH) and oxidized flavoproteins (Fp) signals, to assess the metabolic state of the epithelial cells and chondrocytes. It was found that NLOM can offer a sensitive tool, based on the second-harmonic signal depth-dependent decay, to obtain quantitative information on the optical property of the stroma associated with normal and diseased tissue states. Our results suggest that with the advent of the clinical portability of typical nonlinear optical endoscopy, the NLOM technique has the potential to be applied in vivo to the clinical diagnosis and monitoring of bronchial disease. [ABSTRACT FROM AUTHOR]

Mast cell (MC) degranulation is an important step in the healing process. In this study, silver-nanoparticles-based surface-enhanced Raman spectroscopy (SERS) was used to investigate the spectral characteristics of degranulation of MCs activated by low-intensity laser. The significant spectral changes, such as Raman peak intensities, suggested the concentration variation of some degranulated substances. The Raman intensity ratio of 799-554 cm-1 could be used as a potential internal indicator for the degranulation degree of MCs. Principal component analysis (PCA) was employed to reduce the high dimension of spectra into a few principal components (PCs) while retaining the most diagnostically significant information for sample differentiation. Using the diagnostically significant PC scores (P < 0.05), linear discriminate analysis (LDA) was applied to identify different cell degranulation groups with high sensitivity, specificity and accuracy. This exploratory work demonstrates that SERS technique combined with a PCA-LDA algorithm possesses great potential for developing a label-free, comprehensive, non-invasive and accurate method for measuring MC degranulation. [ABSTRACT FROM AUTHOR]