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Posttranslational modification (PTM) of proteins is likely to be the most common mechanism of altering the expression of genetic information. It is essential to characterize PTMs to establish a complete understanding of the activities of proteins. Here, we present a sensitive detection method using surface-enhanced Raman spectroscopy (SERS) that can detect PTMs from as little as zeptomoles of peptide. We demonstrate, using model peptides, the ability of SERS to detect a variety of protein modifications, such as acetylation, trimethylation, phosphorylation, and ubiquitination. In addition, we show the capability to obtain positional information for modifications such as trimethylation and phosphorylation using SERS and wavelet decomposition data analysis techniques. We further show that it is possible to apply SERS to detect PTMs from biological samples such as histones. We envision that this detection method might be a valuable technique that is complementary to mass spectrometry in obtaining orthogonal chemical and modification-specific information from biological samples at sensitive levels.

For in vivo optical diagnostic technologies to be distributed to the developed and developing worlds, optical imaging systems must be constructed of inexpensive components. We present a fiber-optic confocal reflectance microscope with a cost-effective injection-molded plastic miniature objective lens for in vivo imaging of human tissues in near real time. The measured lateral resolution is less than 2.2 [micro]m, and the measured axial resolution is 10 [micro]m. Confocal images of ex vivo cervical tissue biopsies and in vivo human lip taken at 15 frames/s demonstrate the microscope's capability of imaging cell morphology and tissue architecture. OCIS codes: 170.1610, 170.1790, 170.2150, 170.3880, 170.4440, 170.4730.

Brain functions have been proved to be affected by external stimuli. Low-Level-Light Therapy (LLLT) using near-infrared photons is one of the effective ways to modulate the hemodynamic activities in the brain. However, the biphasic hormetic dose-response where bioenergetics are stimulated at a low dose and inhibited at a high dose is well observed in all photon stimulations. The amount of photon energy delivered to the brain are affected by the wavelength as well as the multilayered head structure with variations of optical parameters (OPs). A real 3D volume head model is built for each participant in this study, and the boundary conditions of each OP in each layer is considered. The Monte Carlo simulation with wavelengths ranging from 650 nm to 1064 nm is implemented to investigate the energy delivered to the brain under different radiation profiles. Results show that 1064-nm photons penetrate deeper than 810-nm photons except for scalp absorption at the lower bound due to low melanin content. Collimated-beam radiation is better than diverging-beam due to a more uniform intensity distribution at the scalp surface. Further research to optimize LLLT dosage for each individual is imperative due to the high inter-person variability in structure and OPs. [ABSTRACT FROM AUTHOR]

Significance: Quantifying subject-specific optical properties (OPs) including absorption and transport scattering coefficients of tissues in the human head could improve the modeling of photon propagation for the analysis of functional near-infrared spectroscopy (fNIRS) data and dosage quantification in therapeutic applications. Current methods employ diffuse approximation, which excludes a low-scattering cerebrospinal fluid compartment and causes errors. Aim: This work aims to quantify OPs of the scalp, skull, and gray matter in vivo based on accurate Monte Carlo (MC) modeling. Approach: Iterative curve fitting was applied to quantify tissue OPs from multidistance continuous-wave NIR reflectance spectra. An artificial neural network (ANN) was trained using MC-simulated reflectance values based on subject-specific voxel-based tissue models to replace MC simulations as the forward model in curve fitting. To efficiently generate sufficient data for training the ANN, the efficiency of MC simulations was greatly improved by white MC simulations, increasing the detectors' acceptance angle, and building a lookup table for interpolation. Results: The trained ANN was six orders of magnitude faster than the original MC simulations. OPs of the three tissue compartments were quantified from NIR reflectance spectra measured at the forehead of five healthy subjects and their uncertainties were estimated. Conclusions: This work demonstrated an MC-based iterative curve fitting method to quantify subject-specific tissue OPs in-vivo, with all OPs except for scattering coefficients of scalp within the ranges reported in the literature, which could aid the modeling of photon propagation in human heads. [ABSTRACT FROM AUTHOR]

Microenvironment stiffening plays a crucial role in tumorigenesis. While filopodia are generally thought to be one of the cellular mechanosensors for probing environmental stiffness, the effects of environmental stiffness on filopodial activities of cancer cells remain unclear. In this work, we investigated the filopodial activities of human lung adenocarcinoma cells CL1-5 cultured on substrates of tunable stiffness using a novel platform. The platform consists of an optical system called structured illumination nano-profilometry, which allows time-lapsed visualization of filopodial activities without fluorescence labeling. The culturing substrates were composed of polyvinyl chloride mixed with an environmentally friendly plasticizer to yield Young's modulus ranging from 20 to 60 kPa. Cell viability studies showed that the viability of cells cultured on the substrates was similar to those cultured on commonly used elastomers such as polydimethylsiloxane. Time-lapsed live cell images were acquired and the filopodial activities in response to substrates with varying degrees of stiffness were analyzed. Statistical analyses revealed that lung cancer cells cultured on softer substrates appeared to have longer filopodia, higher filopodial densities with respect to the cellular perimeter, and slower filopodial retraction rates. Nonetheless, the temporal analysis of filopodial activities revealed that whether a filopodium decides to extend or retract is purely a stochastic process without dependency on substrate stiffness. The discrepancy of the filopodial activities between lung cancer cells cultured on substrates with different degrees of stiffness vanished when the myosin II activities were inhibited by treating the cells with blebbistatin, which suggests that the filopodial activities are closely modulated by the adhesion strength of the cells. Our data quantitatively relate filopodial activities of lung cancer cells with environmental stiffness and should shed light on the understanding and treatment of cancer progression and metastasis. [ABSTRACT FROM AUTHOR]

Significance: Investigating cell death dynamics at the single-cell level plays an essential role in biological research. Quantitative phase imaging (QPI), a label-free method without adverse effects of exogenous labels, has been widely used to image many types of cells under various conditions. However, the dynamics of QPI features during cell death have not been thoroughly characterized. Aim: We aim to develop a label-free technique to quantitatively characterize single-cell dynamics of cellular morphology and intracellular mass distribution of cells undergoing apoptosis and necrosis. Approach: QPI was used to capture time-lapse phase images of apoptotic, necrotic, and normal cells. The dynamics of morphological and QPI features during cell death were fitted by a sigmoid function to quantify both the extent and rate of changes. Results: The two types of cell death mainly differed from normal cells in the lower phase of the central region and differed from each other in the sharp nuclear boundary shown in apoptotic cells. Conclusions: The proposed method characterizes the dynamics of cellular morphology and intracellular mass distributions, which could be applied to studying cells undergoing state transition such as drug response. [ABSTRACT FROM AUTHOR]

Quantification of three-dimensional (3D) refractive index (RI) with sub-cellular resolution is achieved by digital holographic microtomography (DHμT) using quantitative phase images measured at multiple illumination angles. The DHμT system achieves sensitive and fast phase measurements based on iterative phase extraction algorithm and asynchronous phase shifting interferometry without any phase monitoring or active control mechanism. A reconstruction algorithm, optical diffraction tomography with projection on convex sets and total variation minimization, is implemented to substantially reduce the number of angular scattered fields needed for reconstruction without sacrificing the accuracy and quality of the reconstructed 3D RI distribution. Tomogram of a living CA9-22 cell is presented to demonstrate the performance of the method. Further, a statistical analysis of the average RI of the nucleoli, the nucleus excluding the nucleoli and the cytoplasm of twenty CA9-22 cells is performed. (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

Rapid detection for infectious diseases is highly demanded in diagnosis and infection prevention. In this work, we introduced a plasmonic enhanced digitizing biosensor for the rapid detection of nucleic acids. The sensor successfully achieved the detection of loop-mediated isothermal amplification for the hepatitis virus in this work. The sensor comprised a nanodisc array and Bst polymerases conjugated on the rough surface of a nanodisc. The rough surface of the nanodisc provided plasmonic hot spots to enhance the fluorescence signal. The virus DNA was detected by conducting a modified loop-mediated isothermal amplification with fluorescence resonance energy transfer reporter conjugated primers on the sensor. The modified isothermal amplification improved the signal contrast and detection time compared to the original assay. By integrating the modified amplification assay and plasmonic enhancement sensor, we achieved rapid detection of the hepatitis virus. Nucleic acid with a concentration of 10−3 to 10−4 mg/mL was detected within a few minutes by our design. Our digitizing plasmonic nanoarray biosensor also showed 20–30 min earlier detection compared to conventional loop-mediated isothermal amplification sensors. [ABSTRACT FROM AUTHOR]

In vivo endoscopic optical microscopy provides a tool to assess tissue architecture and morphology with contrast and resolution similar to that provided by standard histopathology – without need for physical tissue removal. In this article, we focus on optical imaging technologies that have the potential to dramatically improve the detection, prevention, and therapy of epithelial cancers. Epithelial pre-cancers and cancers are associated with a variety of morphologic, architectural, and molecular changes, which currently can be assessed only through invasive, painful biopsy. Optical imaging is ideally suited to detecting cancer-related alterations because it can detect biochemical and morphologic alterations with sub-cellular resolution throughout the entire epithelial thickness. Optical techniques can be implemented non-invasively, in real time, and at low cost to survey the tissue surface at risk. Our manuscript focuses primarily on modalities that currently are the most developed: reflectance confocal microscopy (RCM) and optical coherence tomography (OCT). However, recent advances in fluorescence-based endoscopic microscopy also are reviewed briefly. We discuss the basic principles of these emerging technologies and their current and potential applications in early cancer detection. We also present research activities focused on development of exogenous contrast agents that can enhance the morphological features important for cancer detection and that have the potential to allow vital molecular imaging of cancer-related biomarkers. In conclusion, we discuss future improvements to the technology needed to develop robust clinical devices. [ABSTRACT FROM AUTHOR]

Significance: Label-free quantitative phase imaging is a promising technique for the automatic detection of abnormal red blood cells (RBCs) in real time. Although deep-learning techniques can accurately detect abnormal RBCs from quantitative phase images efficiently, their applications in diagnostic testing are limited by the lack of transparency. More interpretable results such as morphological and biochemical characteristics of individual RBCs are highly desirable. Aim: An end-to-end deep-learning model was developed to efficiently discriminate thalassemic RBCs (tRBCs) from healthy RBCs (hRBCs) in quantitative phase images and segment RBCs for single-cell characterization. Approach: Two-dimensional quantitative phase images of hRBCs and tRBCs were acquired using digital holographic microscopy. A mask region-based convolutional neural network (Mask R-CNN) model was trained to discriminate tRBCs and segment individual RBCs. Characterization of tRBCs was achieved utilizing SHapley Additive exPlanation analysis and canonical correlation analysis on automatically segmented RBC phase images. Results: The implemented model achieved 97.8% accuracy in detecting tRBCs. Phase-shift statistics showed the highest influence on the correct classification of tRBCs. Associations between the phase-shift features and three-dimensional morphological features were revealed. Conclusions: The implemented Mask R-CNN model accurately identified tRBCs and segmented RBCs to provide single-RBC characterization, which has the potential to aid clinical decision-making. [ABSTRACT FROM AUTHOR]

Skin consists of epidermis and dermis layers that have distinct optical properties. The quantification of skin optical properties is commonly achieved by modeling photon propagation in tissue using Monte Carlo (MC) simulations and iteratively fitting experimentally measured diffuse reflectance spectra. In order to speed up the inverse fitting process, time-consuming MC simulations have been replaced by artificial neural networks to quickly calculate reflectance spectra given tissue geometric and optical parameters. In this study the skin was modeled to consist of three layers and different scattering properties of the layers were considered. A new inverse fitting procedure was proposed to improve the extraction of chromophore-related information in the skin, including the hemoglobin concentration, oxygen saturation and melanin absorption. The performance of the new inverse fitting procedure was evaluated on 40 sets of simulated spectra. The results showed that the fitting procedure without knowing the epidermis thickness extracted chromophore information with accuracy similar to or better than fitting with known epidermis thickness, which is advantageous for practical applications due to simpler and more cost-effective instruments. In addition, the melanin volume fraction multiplied by the thickness of the melanin-containing epidermis layer was estimated more accurately than the melanin volume fraction itself. This product has the potential to provide a quantitative indicator of melanin absorption in the skin. In-vivo cuff occlusion experiments were conducted and skin optical properties extracted from the experiments were comparable to the results of previously reported in vivo studies. The results of the current study demonstrated the applicability of the proposed method to quantify the optical properties related to major chromophores in the skin, as well as scattering coefficients of the dermis. Therefore, it has the potential to be a useful tool for quantifying skin optical properties in vivo. [ABSTRACT FROM AUTHOR]

Central venous oxygen saturation (ScvO2) is an important parameter for assessing global oxygen usage and guiding clinical interventions. However, measuring ScvO2 requires invasive catheterization. As an alternative, we aim to noninvasively and continuously measure changes in oxygen saturation of the internal jugular vein (SijvO2) by a multi-channel near-infrared spectroscopy system. The relation between the measured reflectance and changes in SijvO2 is modeled by Monte Carlo simulations and used to build a prediction model using deep neural networks (DNNs). The prediction model is tested with simulated data to show robustness to individual variations in tissue optical properties. The proposed technique is promising to provide a noninvasive tool for monitoring the stability of brain oxygenation in broad patient populations.

The mass and concentration of hemoglobin per erythrocyte are important hematological parameters. Measuring these parameters from intact erythrocytes requires the value of specific refraction-index increment (RII) of oxygenated hemoglobin, which diverges in the literature. Refractive indices of hemoglobin solutions are measured directly by digital holographic microscopy on a microfluidic channel filled with hemoglobin solutions prepared by hemolysis of fresh human erythrocytes and refractive-index standards sequentially. Hemoglobin extracted from thalassemic patients shows 3-4% higher RII than that from healthy volunteers, but the difference is not significant in comparison to inter-subject variations within each group. The quantified RIIs are applied to quantify mean corpuscular hemoglobin mass of blood from 37 human subjects, and results are in accord with standard clinical test results.

Clinical studies have demonstrated that epidermal pigmentation level can affect cerebral oximetry measurements. To evaluate the robustness of these devices, we have developed a phantom-based test method that includes an epidermis-simulating layer with several melanin concentrations and a 3D-printed cerebrovascular module. Measurements were performed with neonatal, pediatric and adult sensors from two commercial oximeters, where neonatal probes had shorter source-detector separation distances. Referenced blood oxygenation levels ranged from 30 to 90%. Cerebral oximeter outputs exhibited a consistent decrease in saturation level with simulated melanin content; this effect was greatest at low saturation levels, producing a change of up to 15%. Dependence on pigmentation was strongest in a neonatal sensor, possibly due to its high reflectivity. Overall, our findings indicate that a modular channel-array phantom approach can provide a practical tool for assessing the impact of skin pigmentation on cerebral oximeter performance and that modifications to algorithms and/or instrumentation may be needed to mitigate pigmentation bias., Competing Interests: The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services. This article reflects the views of the authors and should not be construed to represent FDA’s views or policies., (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Lipid droplets have gained strong interest in recent years to comprehend how they function and coordinate with other parts of the cell. However, it remains challenging to study the regulation of lipid droplets in live preadipocytes using conventional microscopic techniques. In this paper, we study the effects of fatty acid stimulation and cell starvation on lipid droplets using optical diffraction tomography and Raman spectroscopy by measuring size, refractive index, volume, dry mass and degree of unsaturation. The increase of fatty acids causes an increase in the number and dry mass of lipid droplets. During starvation, the number of lipid droplets increases drastically, which are released to mitochondria to release energy. Studying lipid droplets under different chemical stimulations could help us understand the regulation of lipid droplets for metabolic disorders, such as obesity and diabetes.

An iterative curve fitting method has been applied in both simulation [J. Biomed. Opt.17, 107003 (2012)JBOPFO1083-366810.1117/1.JBO.17.10.107003] and phantom [J. Biomed. Opt.19, 077002 (2014)JBOPFO1083-366810.1117/1.JBO.19.7.077002] studies to accurately extract optical properties and the top layer thickness of a two-layered superficial tissue model from diffuse reflectance spectroscopy (DRS) data. This paper describes a hybrid two-step parameter estimation procedure to address two main issues of the previous method, including (1) high computational intensity and (2) converging to local minima. The parameter estimation procedure contained a novel initial estimation step to obtain an initial guess, which was used by a subsequent iterative fitting step to optimize the parameter estimation. A lookup table was used in both steps to quickly obtain reflectance spectra and reduce computational intensity. On simulated DRS data, the proposed parameter estimation procedure achieved high estimation accuracy and a 95% reduction of computational time compared to previous studies. Furthermore, the proposed initial estimation step led to better convergence of the following fitting step. Strategies used in the proposed procedure could benefit both the modeling and experimental data processing of not only DRS but also related approaches such as near-infrared spectroscopy.

A robust modelling method was proposed to extract chromophore information in multi-layered skin tissue with spatially-resolved diffuse reflectance spectroscopy. Artificial neural network models trained with a pre-simulated database were first built to map geometric and optical parameters into diffuse reflectance spectra. Nine fitting parameters including chromophore concentrations and oxygen saturation were then determined by solving the inverse problem of fitting spectral measurements from three different parts of the skin. Compared to the Monte Carlo simulation accelerated by a graphics processing unit, the proposed modelling method not only reduced the computation time, but also achieved a better fitting performance., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

Complete blood count is the most common test to detect anemia, but it is unable to obtain the abnormal shape of erythrocytes, which highly correlates with the hematologic function. Tomographic diffractive microscopy (TDM) is an emerging technique capable of quantifying three-dimensional (3-D) refractive index (RI) distributions of erythrocytes without labeling. TDM was used to characterize optical and morphological properties of 172 erythrocytes from healthy volunteers and 419 erythrocytes from thalassemic patients. To efficiently extract and analyze the properties of erythrocytes, we developed an adaptive region-growing method for automatically delineating erythrocytes from 3-D RI maps. The thalassemic erythrocytes not only contained lower hemoglobin content but also showed doughnut shape and significantly lower volume, surface area, effective radius, and average thickness. A multi-indices prediction model achieved perfect accuracy of diagnosing thalassemia using four features, including the optical volume, surface-area-to-volume ratio, sphericity index, and surface area. The results demonstrate the ability of TDM to provide quantitative, hematologic measurements and to assess morphological features of erythrocytes to distinguish healthy and thalassemic erythrocytes., ((2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

Confocal imaging techniques offer an optical sectioning capability to acquire three-dimensional information from various volumetric samples by discriminating the desired in-focus signals from the out-of-focus background. However, confocal, in general, requires a point-by-point scan in both the lateral and axial directions to reconstruct three-dimensional images. In addition, axial scanning in confocal is slower than scanning in lateral directions. In this Letter, a non-axial-scanning multifocal confocal microscope incorporating multiplexed holographic gratings in illumination and dual detection for depth discrimination is presented. Further, we demonstrate the ability of the proposed confocal microscopy to image ex vivo tissue structures simultaneously at different focal depths without mechanical or electro-optic axial scanning.

The progression of epithelial precancers into cancer is accompanied by changes of tissue and cellular structures in the epithelium. Correlations between the structural changes and scattering coefficients of esophageal epithelia were investigated using quantitative phase images and the scattering-phase theorem. An ex vivo study of 14 patients demonstrated that the average scattering coefficient of precancerous epithelia was 37.8% higher than that of normal epithelia from the same patient. The scattering coefficients were highly correlated with morphological features including the cell density and the nuclear-to-cytoplasmic ratio. A high interpatient variability in scattering coefficients was observed and suggests identifying precancerous lesions based on the relative change in scattering coefficients.

A calculation based on surface plasmon coupling condition and Maxwell-Garnett equation was performed for predicting the coupling angle shift and thin film thickness in scanning surface plasmon microscopy (SSPM). The refractive index sensitivity and lateral resolution of an SSPM system was also investigated. The limit of detection of angle shift was 0.01°, the limit of quantification of angle shift was 0.03°, and the sensitivity was around 0.12° shift per nm ZnO film when the film thickness was less than 22.6 nm. Two partially connected Au nano-discs with a center-to-center distance of 1.1 μm could be identified as two peaks. The system was applied to image nanostructure defects and a virus-probe functionalized nanoarray. We expect the potential application in nanobiosensors with further optimization in the future.

A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW. We then used polymerase-functionalized tips for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA. The amplicon-SYBR® Green I complex was detected and compared to real-time loop-mediated isothermal amplification. The signals of the reaction using 4 and 0.004 ng∕μl templates were detected 10 and 30 min earlier, respectively. The results showed the potential of TEF in developing a nanobiosensor for real-time DNA amplification., (© 2015 Society of Photo-Optical Instrumentation Engineers (SPIE))

We demonstrate a common-path tomographic diffractive microscopy technique for three-dimensional (3D) refractive-index (RI) imaging of unstained living cells. A diffraction grating is utilized to generate a reference beam that traverses a blank region of the sample in a common-path off-axis interferometry setup. Single-shot phase images captured at multiple illumination angles are used for 3D RI reconstruction based on optical diffraction tomography. The common-path configuration shows lower temporal phase fluctuations and better RI resolution than a Mach-Zehnder configuration. 3D subcellular RI distributions of live HeLa cells are quantified.

We are reporting on an experimental investigation of a movable diffuse reflectance spectroscopy system to extract diagnostically relevant optical properties of two-layered tissue phantoms simulating mucosae that are covered with stratified squamous epithelium. The reflectance spectra were measured at multiple sourcedetector separations using two imaging fiber bundles in contact with the phantoms, one with its optical axis perpendicular to the sample surface (perpendicular probe) and the other with its distal end beveled and optical axis tilted at 45 deg (oblique probe). Polystyrene microspheres and purified human hemoglobin were used to make tissue phantoms whose scattering and absorption properties could be well controlled and theoretically predicted. Monte Carlo simulations were used to predict the reflectance spectra for system calibration and an iterative curve fitting that simultaneously extracted the top layer reduced scattering coefficient, thickness, bottom layer reduced scattering coefficient, and hemoglobin concentration of the phantoms. The errors of the recovered parameters ranged from 7% to 20%. The oblique probe showed higher accuracy in the extracted top layer reduced scattering coefficient and thickness than the perpendicular probe. The developed system and data analysis methods provide a feasible tool to quantify the optical properties in vivo.

The scattering properties and refractive indices (RI) of tissue are important parameters in tissue optics. These parameters can be determined from quantitative phase images of thin slices of tissue blocks. However, the changes in RI and structure of cells due to fixation and paraffin embedding might result in inaccuracies in the estimation of the scattering properties of tissue. In this study, three-dimensional RI distributions of cells were measured using digital holographic microtomography to obtain total scattering cross sections (TSCS) of the cells based on the first-order Born approximation. We investigated the slight loss of dry mass and drastic shrinkage of cells due to paraformaldehyde fixation and paraffin embedding removal processes. We propose a method to compensate for the correlated changes in volume and RI of cells. The results demonstrate that the TSCS of live cells can be estimated using restored cells. The percentage deviation of the TSCS between restored cells and live cells was only −8%. Spatially resolved RI and scattering coefficients of unprocessed oral epithelium ranged from 1.35 to 1.39 and from 100 to 450 cm−1, respectively, estimated from paraffinembedded oral epithelial tissue after restoration of RI and volume.

We have successfully performed localized loop-mediated isothermal reactions of hepatitis B virus (HBV) and hepatitis C virus (HCV) on the apex (50~100 nm) of metallic tips coated with Bst polymerases. The SYBR green molecules binding to the new formed HBV DNA inside the optical near fields were excited by two-photon fluorescence microscopy, and directly imaged in far field. Another reporter primer is used for HCV replication detection. Preliminary results are presented in this manuscript.

Diffuse reflectance spectroscopy has been applied to detect tissue absorption and scattering properties associated with dysplasia, which is a potential precursor of epithelial cancers. The ability of DRS techniques to detect dysplasia could be improved by enhancing the detection of optical properties of the thin epithelial layer where dysplasia occurs. We propose a beveled fiber bundle probe consisting of a source fiber and multiple detection fibers parallel to each other and oriented obliquely to the tissue surface and investigate the sensitivity of reflectance measured with the probe to optical properties of a two-layered normal oral mucosa model. A scalable Monte Carlo method is employed to speed up analyzing spatially resolved reflectance spectra. Results reveal that the oblique probe is more sensitive to epithelial scattering and less sensitive to both stromal absorption and scattering than conventional perpendicular fiber configuration. The clinical relevance of the enhanced sensitivity to epithelial scattering by the proposed probe is demonstrated by quantifying optical properties of the two-layered tissue model from simulated data. The average error of extracted epithelial scattering coefficient is 1.5% and 32% using the oblique and perpendicular probe, respectively. The errors in other optical properties are all below 10% using the oblique probe.

We applied hyperspectral imaging to measure spatially-resolved diffuse reflectance spectra in the visible range and an iterative inversion method based on forward Monte Carlo modeling to quantify optical properties of two-layered tissue models. We validated the inversion method using spectra experimentally measured from liquid tissue mimicking phantoms with known optical properties. Results of fitting simulated data showed that simultaneously considering the spatial and spectral information in the inversion process improves the accuracies of estimating the optical properties and the top layer thickness in comparison to methods fitting reflectance spectra measured with a single source-detector separation or fitting spatially-resolved reflectance at a single wavelength. Further development of the method could improve noninvasive assessment of physiological status and pathological conditions of stratified squamous epithelium and superficial stroma.

To facilitate the application of plasmonic nanoparticles (PNPs) in high-throughput detection, we develop a hyperspectral imaging system (HSIS) combining dark-filed microscopy and imaging Fourier transform spectrometry to measure scattering spectra from immobilized PNPs. The current setup has acquisition time of 5 seconds and spectral resolution of 21.4 nm at 532.1 nm. We demonstrate the applicability of the HSIS in conjunction with spectral data analysis to quantify multiple types of PNPs and detect small changes in localized surface plasmon resonance wavelengths of PNPs due to changes in the environmental refractive index.

Tip-enhanced fluorescence of localized DNA replication by loop-mediated isothermal amplification (LAMP) is a potential way to observe real-time biological reaction confined in nanometer scale. We successfully coated Bst polymerase on the apex (~100 nm) of an atomic force microscope (AFM) tip and performed localized LAMP reaction of hepatitis B virus (HBV). By using this tip-based reaction, the replicated HBV DNA can be directly imaged to be 400~500 nm spots by using two-photon excitation fluorescence microscopy.

Reflectance spectra measured from epithelial tissue have been used to extract size distribution and refractive index of cell nuclei for noninvasive detection of precancerous changes. Despite many in vitro and in vivo experimental results, the underlying mechanism of sizing nuclei based on modeling nuclei as homogeneous spheres and fitting the measured data with Mie theory has not been fully explored. We describe the implementation of a three-dimensional finite-difference time-domain (FDTD) simulation tool using a Gaussian pulse as the light source to investigate the wavelength-dependent characteristics of backscattered light from a nuclear model consisting of a nucleolus and clumps of chromatin embedded in homogeneous nucleoplasm. The results show that small-sized heterogeneities within the nuclei generate about five times higher backscattering than homogeneous spheres. More interestingly, backscattering spectra from heterogeneous spherical nuclei show periodic oscillations similar to those from homogeneous spheres, leading to high accuracy of estimating the nuclear diameter by comparison with Mie theory. In addition to the application in light scattering spectroscopy, the reported FDTD method could be adapted to study the relations between measured spectral data and nuclear structures in other optical imaging and spectroscopic techniques for in vivo diagnosis.

This paper presents a micro-/nanofluidic biochip for enhanced sensitivity of immunoassay. By using a protein preconcentrator based on nanofluidic filters, we greatly improve the binding kinetics of immunoassay. A shallow nanofluidic channel connecting two microchannels for ion-selective filter is etched on the glass surface and sealed by PDMS/Glass irreversible bonding treated with O2 plasma. Our device achieved a 10(5) fold increase in protein concentration with 12 minutes of preconcentration. For applying the preconcentrator to immunoassay, we fabricated a novel device with a thin layer of Au sputtered near the nanochannel to provide a substrate for antibody immobilization and a sensing area at the site of highly concentrated proteins. After increasing the concentration of antibody and target antigen by multiple sample preconcentration steps, the binding kinetics of immunoassay can be significantly improved.

Composite organic-inorganic nanoparticles (COINs) are novel optical labels for detection of biomolecules. We have previously developed methods to encapsulate COINs and to functionalize them with antibodies. Here we report the first steps toward application of COINs to the detection of proteins in human tissues. Two analytes, PSA and CK18, are detected simultaneously using two different COINs in a direct binding assay, and two different COINs are shown to simultaneously label PSA in tissue samples.

We present a fiber optic confocal reflectance microscope (FCRM) which can be used to image epithelial tissue with sub-cellular resolution in vivo. Confocal images of normal and abnormal appearing cervical tissue were obtained in vivo from eighteen patients undergoing colposcopic examination of the cervix; biopsy specimens were taken from imaged sites. The measured lateral and axial resolutions of the system were 1.6 microm and 3 microm, respectively. Morphologic features, including nuclear size and nuclear-to-cytoplasmic ratio, were extracted from confocal images obtained at various depths beneath the epithelial surface. Image features extracted from confocal images compared well with features extracted from confocal images obtained in vitro and from previous histopathologic studies. This study shows that fiber optic confocal reflectance microscopy can be used to visualize the morphology of cervical epithelium in vivo.

We have built a fiber-optic confocal reflectance microscope capable of imaging human tissues in near real time. Miniaturization of the objective lens and the mechanical components for positioning and axially scanning the objective enables the device to be used in inner organs of the human body. The lateral resolution is 2 micrometers and axial resolution is 10 micrometers. Confocal images of fixed tissue biopsies and the human lip in vivo have been obtained at 15 frames/s without any fluorescent stains. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope.

An endoscopic confocal microscope requires a high-performance, miniaturized microscope objective. We present the design of a miniature water-immersion microscope objective that is approximately 10 times smaller in length than a typical commercial objective. The miniature objective is 7 mm in outer diameter and 21 mm in length (from object to image). It is used in a fiber confocal reflectance microscope. The miniature microscope objective has a numerical aperture of 1.0, a field of view of 250 microm, and a working distance of 450 microm. It delivers diffraction-limited performance at lambda = 1064 nm. Micrometer-level resolution has been experimentally demonstrated.