This PDF file contains the front matter associated with SPIE Proceedings Volume 6863, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

We report on the development of a Forster resonance energy transfer (FRET) enabled atomic force microscope (AFM) for the study of biomechanics and mechanobiology at the cellular level. The hybrid microscopy system combines the spatial resolution and control of the AFM with the nanoscale sensing capabilities of FRET to enable simultaneous detection of cell mechanical responses and correlation of those responses with cellular biochemistry. Here, we show FRET signal from donor-coated microspheres, that are attached to an AFM cantilever, to acceptor-labeled integrins in a fixed cell system. Additionally, we demonstrate and discuss the attachment of quantum dots to silica microspheres as the FRET donor.

Optical resonances in microspheres have recently been applied to biosensing applications. The resonances, known as Whispering Gallery Modes (WGMs) result from the total internal reflection of the light propagating inside the high index spherical surface within a lower index medium. The evanescent field of the WGM, which extends beyond the microsphere surface, is sensitive to the changes in the local refractive index in the surrounding medium. The high Q factor associated with WGMs enables a highly sensitive sensor element. Here we present a refractometric sensor with high sensitivity based on quantum dot (QD) embedded polystyrene microspheres. Ultrashort laser pulses are used to induce two photon excited luminescence from the QDs inside the microspheres. By optimizing the detection area of the microsphere, an enhanced resonance signal to background ratio can be achieved. Theoretical calculations of the resonance peak shifts are compared with the experimental data. Refractometric sensing based on WGMs is a technique that does not require analyte labeling. In this work, QDs are used as a local, broadband light source within the microspheres to enable remote excitation of the resonant modes. The sensor has great potential in many biosensing applications.

We have applied optical tweezers to measure the free-protein concentration of a microscopic sample in the nM range by measuring the optical tweezer laser power at which protein coated beads are ruptured from an antibody coated coverslip surface. We used silica beads that were covalently coated with a target protein and a glass coverslip coated with antibodies specific against the target protein, causing the coated beads to stick to the surface. The unknown unlabelled target protein concentration was added, which then competed with the bead-bound target protein for antibody binding sites on the coverslip surface. In this way the number of bead-surface bonds were modulated by the free protein concentration in solution affecting the threshold laser power necessary to rupture the bead from the surface. An optical tweezer was used to probe the number of bead-surface bonds by measuring the threshold power required to pull the bead away from the surface. We positioned an optical tweezer (1064 nm) slightly above the bead and linearly ramped the laser power until the bead ruptured from the surface. The power at which this occurred was used to determine the free protein concentration. Our measured calibration curve of threshold power versus free protein concentration was fitted to a single binding site equilibrium model which yielded an estimate for the equilibrium dissociation coefficient that is comparable to literature values.

The reliability of optical techniques for non-invasive monitoring of glucose can be significantly improved by the deployment of a subcutaneous implantable sensor that can closely track the changes in the local concentration of glucose in skin. We have developed a novel implantable sensor that can track glucose-induced changes in the optical turbidity of the implant. In this sensor, optical turbidity decreases significantly with increased glucose concentrations. We performed comparative measurements by optical coherence tomography (OCT) used to monitor backscattering or specular reflection originated from specific structures within the sensor and by collimated light transmission measurement technique to measure the changes in light attenuation as function of glucose concentration within the sensor as well as when the sensor was implanted in phantom media or in tissue samples. These measurements showed that glucose-induced changes in the transmission values derived from OCT monitoring of the sensor turbidity differed up two times from those obtained by collimated transparency measurement (CTM) technique. These results were used to determine the values for scattering coefficients of tissue and the sensor and to estimate the relative loss in sensor sensitivity as a function of implantation depth in tissue. The results suggest that the implantable sensor can be placed in turbid medium such as skin up to an optical depth of 12 mean free paths (mfp), one could expect. For a turbid medium such as skin with a scattering coefficient (µs ) of 10mm-1, this would result in geometrical depth of implantation at 1.2 mm beneath the tissue where sensor sensitivity of 50% or higher is expected. The study demonstrates that it could be feasible to engineer a novel optical sensor for glucose monitoring that can be implanted under the skin while providing a high degree of sensitivity and specificity for non-invasive glucose monitoring.

We have developed novel combinations of glucose receptors, luminescent molecules, and nanoengineered materials to enable accurate and sensitive determination of glucose using microparticles. This paper will report the results of modeling and experimental work to determine two key properties of these systems: (1) maximal stability of the response under continuous operation; (2) maximal efficiency of transdermal excitation and collection of emission.

Implementing strict glycemic control can reduce the risk of serious complications in both diabetic and critically ill patients. For this purpose, many different blood glucose monitoring techniques and insulin infusion strategies have been tested towards the realization of an artificial pancreas under closed loop control. In contrast to competing subcutaneously implanted electrochemical biosensors, microdialysis based systems for sampling body fluids from either the interstitial adipose tissue compartment or from venous blood have been developed, which allow an ex-vivo glucose monitoring by mid-infrared spectrometry. For the first option, a commercially available, subcutaneously inserted CMA 60 microdialysis catheter has been used routinely. The vascular body interface includes a double-lumen venous catheter in combination with whole blood dilution using a heparin solution. The diluted whole blood is transported to a flow-through dialysis cell, where the harvesting of analytes across the microdialysis membrane takes place at high recovery rates. The dialysate is continuously transported to the IR-sensor. Ex-vivo measurements were conducted on type-1 diabetic subjects lasting up to 28 hours. Experiments have shown excellent agreement between the sensor readout and the reference blood glucose concentration values. The simultaneous assessment of dialysis recovery rates renders a reliable quantification of whole blood concentrations of glucose and metabolites (urea, lactate etc) after taking blood dilution into account. Our results from transmission spectrometry indicate, that the developed bed-side device enables reliable long-term glucose monitoring with reagent- and calibration-free operation.

Over the last two decades, optical polarimetry method has been applied for glucose concentration monitoring in biological media such as aqueous humor and cell culture media as a non-invasive alternative method. Compared to glucose analyzer and electronic analyte sensor, the advantages of polaimetry method are non-ionizing radiation to interrogate the sample, non-interference with the sample, needless of consumable reagents, use of readily available sources, and prospect of miniaturizing the optics. Commercial polarimeter is widely used to measure the concentration of chemical compounds with optical activity. However, this device was based on off-line measurement so that it needs sample extraction process to measure the concentration of sample. This process does not reflect the real-time status of sample concentration and sample contamination can be occurred during sample extraction when applied to cell culture process. In polarimetry method, the measurement sensitivity can be controlled by varying optical path length. However, in current polarimeter, the sample cell should be exchanged to vary the optical path length. This process is a time consuming and might cause sample contamination in cell culture process. Therefore, it is necessary to develop a new polarimetry method which can measure the real-time status of sample concentration without sample extraction. In this paper, we introduce a new polarimetry probe system which might be utilized to monitor glucose concentration during cell culture process. It was designed to have variable optical path lengths to control the optical rotation angle of polarized light. We describe the feasibility of the system and the preliminary results.

We report on the successful development of a custom in vitro system that provides a physiologically relevant means of demonstrating optical methodologies for the calibration and validation of oxygen delivery and hemoglobin oxygen binding dynamics in the brain. While measured optical signals have generally been equated to heme absorbance values that are, in turn, presumed to correspond to oxygen delivery, there has been little specific study of the sigmoidal oxygen binding dynamics of hemoglobin, a tetrameric protein, within physiologically relevant parameters. Our development of this novel analytical device addresses this issue, and is a significant step towards the minimally invasive and real-time monitoring of spatially resolved cognitive processes. As such, it is of particular interest for the detection of autistic brain activity in infants and young children. Moreover, our device and approach bring with them the ability to quantify and spatially resolve oxygen delivery down to volumes relevant to individual cell oxygen uptake, without any oxygen consumption, and with a temporal resolution that is physically unachievable by any oxygen tracking modality such as fMRI etc. Such a capability opens up myriad possibilities for further investigation, such as real-time tumor biopsy and resection; the tracking and quantification of cellular proliferation, as well as metabolic measures of tissue viability, to name but a few. Our system has also been engineered to be synergistic with virtually all imaging techniques, optical and otherwise.

A compact and convenient system is designed and realized for high-resolution simultaneous imaging hemodynamic parameters and recording electrophysiological signals in the brain by the combination of multi-wavelength optical imaging and electrophysiological recording system. Multi-wavelength optical imaging system uses an integration of light-emitting diode (LED which has three wavelengths) and laser diode (LD) as imaging illuminants to combine the laser speckle imaging and optical intrinsic signal imaging. Electrophysiological recording system is based on the virtual instrument technology. The spatial and temporal changes in oxy-hemoglobin, deoxy-hemoglobin, total hemoglobin concentration, cerebral blood flow, and the cerebral metabolic rate of oxygen in response to the brain activities are monitored by the multi-wavelength optical imaging system. Meanwhile the electrophysiological recording system can simultaneously collect the extra-cellular electrophysiological signals. The combination system provides the capability to simultaneously investigate hemodynamic parameters and electrophysiological signals, which may lead to a better understanding of the coupling between neuronal activation and vascular responses.

Laser Speckle Contrast Analysis (LASCA) and Laser Doppler Perfusion Imaging (LDPI) are techniques widely used for determining cerebral blood flow, the skin perfusion in burns and during drug uptake, and cerebral blood flow. Both techniques are based on the dynamic speckle pattern on the detector generated by the sample under investigation. In LASCA the speckle pattern is recorded using a long exposure time (i.e. milliseconds) resulting in a blurred image, the perfusion map is obtained by calculating the contrast in the blurred image over small areas (e.g. 5x5 or 7x7 pixels). In LDPI a series of speckle patterns are recorded using a short integration time (i.e. microseconds). By determining the power spectrum of the intensity fluctuations per pixel and calculating the first moment, the perfusion map is obtained. Because both techniques are based on the same phenomenon we show it is possible to relate the outputs of LASCA and LDPI. Such a connection is important because of the growing interest in LASCA techniques. Here we perform the first steps in the comparison of both techniques, using both simulated signals and signals measured with a high speed camera which can perform LDPI as well as LASCA.

Doppler Optical Coherence Tomography (DOCT) is a useful technique for flow measurements. Its potential applications include industrial suspension viscosity measurements and blood flow measurements. In this work, a flow velocity profile of 1% Intralipid was measured in a capillary with an inner diameter of 0.8 mm and in a microfluidic channel with a cross-section of 1000 μmx100 μm. Two different DOCT measurement systems were utilized in the experiments: a commercial conventional OCT system and a laboratory-built DOCT system, intended particularly for flow velocity measurements. In the laboratory-built DOCT system, depth scanning was achieved by moving the whole measurement system with the reference mirror fixed. This modification from a conventional OCT system improves lateral resolution during the scanning process. A syringe pump was used to induce flow in the capillary. Flow velocity was measured with flow rates from 1 ml/min to 3.33 ml/min using both measurement systems. For a flow rate of 3.33 ml/min, both systems gave reasonable results. For flow rates lower than 3.33 ml/min, however, the laboratory-built DOCT system gave much better results. Its mean measurement error was as low as 0.8%, while that of the commercial OCT was 6.8%. Measured with the laboratory-built DOCT system, capillary force-induced flow velocity in the microfluidic channel was around 2 mm/s. The commercial OCT system, on the other hand, proved unsuitable for flow measurements in the microfluidic channel due to its high scanning speed.

It is demonstrated that the inherent fluorescence of a dental composite resin can be utilized to monitor the curing status, i.e. degree of conversion of the resin. The method does not require any sample preparation and is potentially very fast for real time cure monitoring. The method is verified by Raman spectroscopy analysis.

By using two-photon autofluorescence (TPAF) and second harmonics generation (SHG), we imaged hepatocellular carcinoma (HCC) biopsies from the human patients and compared them with the conventional histological biopsies. We found that multiphoton microscopy may be used to obtain label-free images of liver tissues and may be developed into an effective diagnostic tool for liver diseases.

Photodynamic therapy (PDT) is an effective and minimally invasive treatment modality with relatively less side effects, which is approved by FDA for the treatment of esophageal cancer. Maximum therapeutic outcome of the PDT protocol for each individual patient requires optimization of the components of PDT operating at their highest efficacy. Tumor necrosis, the method of malignant tissue destruction by PDT, is carried out by the toxic singlet oxygen molecules that are being formed from the molecular oxygen in the tumor. The availability of molecular oxygen, hence being the rate limiting step for PDT plays a key role in the treatment protocol. Currently the PDT of esophageal carcinoma is rather a blind process since there is no method to monitor the tumor oxygen level during the treatment. In this paper we present an optical technique to monitor molecular oxygen level in the PDT milieu. The technique described herein is a reflection oximetry technique designed with small semiconductor lasers and a silicon photodiode. The light used for monitoring system comes from two semiconductor diode lasers of 650 nm and 940 nm wavelengths. The two lasers and the photodiode are mounted onto a small package which is to be imprinted onto a balloon catheter containing the PDT light delivery system. Lasers and the photodiode are powered and controlled by a control box that is connected via a cable. Light sources and the respective photodiode output are controlled by the LabVIEW virtual instrumentation. The sequential on and off light source and the respective reflective signal are processed with MATLAB. The latter code integrates with LabVIEW to make an automatic calculation of the corresponding light absorption by each chromophore and to calculate the change in oxygen level as well as the amount of blood and oxygen present in the treatment area. The designed system is capable of monitoring the change in oxygen level and the blood flow in any part of the human body where the package is possible to place.

Quantitative non-invasive assessment of the wound healing process in chronic wounds may assist in selection and monitoring of expensive treatments. The Diffuse Photon Density Wave (DPDW) methodology at near infrared wavelengths can be used to non-invasively measure the optical absorption and reduced scattering coefficients of tissue at depths of several millimeters. Changes in the optical properties of tissue at near-infrared wavelengths (685nm-950nm) are caused by changes in blood volume, oxygenation, and tissue hydration. A four-wavelength DPDW system with a single source position and four detectors was used to monitor the optical properties of wounds in healthy and streptozotocin-induced diabetic rats. Optical data obtained after inflicting full-thickness wounds on the dorsal region of diabetic and control rats indicate that DPDW technology can be used to monitor wound healing and differentiate the rate of impaired vs. normal wound healing. The concentrations of oxyhemoglobin, deoxyhemoglobin and water were calculated from the optical absorption coefficients. Changes in hemoglobin concentration may indicate increased vascularization throughout the wound healing process, while changes in water content may reflect inflammation following tissue injury. These physiological changes are supported by qualitative immunohistochemical analysis of wound biopsies.

The concentration of glucose in interstitial fluid determined by using the surface plasmon resonance (SPR) biosensor with chemical bonding D-Galactose/D-Glucose Binding Protein (GGBP) is proposed in this paper. D-Galactose/D-Glucose Binding Protein (GGBP), a kind of protein which has the ability to absorb the glucose specifically, is immobilized on the gold film of the SPR sensor to improve the sensitivity of glucose detecting. The GGBPs mutated at different points have different association abilities with glucose, which bring different measurement range and precision. So the selection of proteins is a critical problem of the determination of glucose by using SPR biosensor. Using different mutated GGBPs, the samples with different concentrations of glucose are measured in the experiment, and the prediction error and precision are discussed. Furthermore, the light intensity of sensor is instable, so the baseline of SPR responses is tracked and adjusted accordingly using the methods - fixing points and fixing areas' ratio. The experiment results show that GGBPs mutated at different points have its corresponding working curves and different measurement precision. In conclusion, the study is significant for the application of SPR biosensor to the minimally invasive diabetes testing and other detection of human body components.

Photoexcitation and luminescence spectra of dried urine sample under laser excitation were studied. Luminescence spectra of urine are determined by luminescence of urea which is the main component of urine. The presence of pathological salts in urine leads to the long-wave shifting of maxima of luminescence and to the decreasing of luminescence intensity.

We report on laser-induced breakdown spectroscopy (LIBS) of whole blood and other organic fluids. LIBS spectra, in the region 200-970 nm, are measured by recording the radiation emitted by the samples following their ablation in a helium environment. We show that these spectra, although very complex, reveal the presence of elements such as nitrogen, hydrogen, oxygen and carbon and that of important metallic elements such as iron, magnesium, calcium, potassium, and sodium. We compare the measured LIBS spectra of whole blood to that of pure carbon and pure iron and find that in the 200-300 nm region. Nearly 90% of the peaks can be assigned to only these two elements. We also report on similar studies of methanol, ethanol, isopropanol and water solutions of protein molecules of interest to cancer research. We show that using simple numerical algorithms, it is possible to distinguish between complex organic compounds that have nearly the same chemical composition.

Clinical diagnosis of infections, generally are realized by serological methods, which identifies the antibodies presents in serum or tissue fluids of the patient. Antibodies are proteins present in our bodies that aid in the elimination of pathogens or antigens. Identification of antibodies isotypes is important because can help to predict when and whether patients will recover from infections and are commonly diagnosed by means of indirect methods such as serological test. In the other hand, the majority of these methods requires specific kits for the analysis, special sample preparation, chemical reagents, expensive equipment and require long time for getting results. In this work we show the feasibility to discriminate antibody isotypes in biological fluids like human colostrum by means of Raman spectroscopy and chemometrics. Spectra were obtained using an excitation wavelength of 514 nm over dried samples of human colostrum labeled previously as positives to specific IgG and IgM antibodies against Toxoplasma Gondii by means of ELISA test. Partial least square-discriminant analysis (PLS-DA) was used to discriminate among antibody isotypes by use second derivative of Raman spectra of colostrum samples.

The use of near-infrared spectroscopy for the monitoring of blood glucose concentration is limited by many ambiguous factors, which leads to the prediction precision is not satisfied. Due to the weak interested signal and the difficulty to quantify the physiological noise directly, the absorbance induced by glucose concentration and temperature was analyzed based on Beer-Lambert Law and displacement between glucose and water. Then the transmittance of glucose aqueous solution in different temperatures was measured by spectrometer to investigate the influence of glucose concentration and temperature. As it's difficult to distinguish the influence of temperature from the diffuse reflectance, the Monte Carlo simulation was used to compute the light intensity induced by the change in glucose concentration and physiological temperature. Finally, the influence of actual physiological temperature on the prediction model of glucose concentration was estimated based on the oral glucose tolerance tests of two diabetics. The result showed that, near the normal physiological temperature, the intensity of diffuse reflectance caused by -0.1 °C change in temperature was equivalent to that caused by 2.7 mmol/L change in glucose concentration. Moreover, the proportion of prediction error induced by temperature to the total error was more than 50%.

In the noninvasive blood glucose sensing using near-infrared spectroscopy, the physiological noise was one of the biggest challenges. In order to efficiently reduce the influence of the physiological background variations on the diffuse reflectance spectra, the floating-reference method was used by differentially processing two signals from reference point and measuring point. In this paper, the wavelength-dependent characteristic of the floating-reference point was discussed by simulation and primary experiment. First, the wavelength-dependent characteristic of intralipid- 5% solution was investigated in the wavelength range of 1300-1600nm. And source-detector distance for reference point in the wavelength of 1300nm was conducted by different concentration of scatter media including 2%, 5% and 10% intralipid solution. Then the single-layer and three layers skin model were built to investigate the wavelength characteristic of reference point. The water displacement coefficients and relative large change in glucose concentration were considered in the simulation. Finally, the primary experiment of intralipid model was conducted to validate the wavelength dependence of reference point. The result showed that, the floating reference will not exist in the strong absorption region (near 1450nm) and the region where the change of absorption coefficient is positive (high than 1525nm) due to the corporate influence of scattering and absorption coefficient. And the wavelength-dependent characteristic is consistent for intralipid solution and the skin model.

The continuous blood glucose monitoring system using interstitial fluid (ISF) extracted by ultrasound and vacuum is proposed in this paper. The skin impedance measurement is introduced into the system to monitor the skin permeability variation. Low-frequency ultrasound is applied on skin surface to enhance the skin permeability by disrupting the lipid bilayers of the stratum corneum (SC), and then ISF is extracted out of skin continuously by vacuum. The extracted ISF is diluted and the concentration of glucose in it is detected by a biosensor and used to predict the blood glucose concentration. The skin permeability is variable during the extraction, and its variation affects the prediction accuracy. The skin impedance is an excellent indicator of skin permeability in that the lipid bilayers of the SC, which offer electrical resistance to the skin, retard transdermal transport of molecules. So the skin impedance measured during the extraction is transformed to skin conductivity to estimate correlation coefficient between skin conductivity and permeability. Skin conductivity correlates well with skin permeability. The method and experiment system mentioned above may be significative for improving the prediction accuracy of continuous blood glucose monitoring system.

Laser speckle imaging (LSI) technique is considered as a promising method of accessing cerebral blood flow (CBF) of animals for its high spatiotemporal resolution and simplicity. It is important in LSI that optimum imaging parameters and limited noises should be confirmed to promote the imaging precision. We investigated in this paper different factors which may affect the imaging results with a moving white plate model, and then proposed a method of enlarging the linear response range of velocity. Through experiment, we proposed in our LSI system the optimum imaging parameters, including the numerical aperture and magnification of microscopy, the integration time, the gain mode of CCD camera. The average intensity was found optimum at about 800 counts out of 4096 grey level, which permits the highest contrast in our experiment. To eliminate the influence of uneven illumination, a direct current weight of 27 counts was subtracted during data processing. The result indicated that the relationship between measured velocity and the real one remained linear with R² equaling to 0.99 throughout the scale of 80 mm/s.

Cortical spreading depression (CSD) is an important pathological model of migraine and is related to other neural disorders, such as cerebral ischemia and epilepsy. It has been reported that brain stimulation is a quite effective way to treat neural diseases. However, direct stimulation could cause harm to brain. If peripheral nerve stimulation could have the same treatment, it would be essential to investigate the mechanisms of peripheral nerve and the study of sciatic nerve stimulation would have profound clinical meaning. In this paper, we used optical intrinsic signal imaging (OISI) and extracellular electrophysiologic recording techniques to study the effects of sciatic nerve stimulation on the propagation of CSD. We found that: (1) continuous sciatic nerve stimulation on rats caused a decrease in light intensity on the whole cortex, which meant an increase in cerebral blood volume(CBV); (2) the spreading velocity of CSD declined from 3.63± 0.272 mm/min to 3.06±0.260 mm/min during sciatic nerve stimulation, compared with that without sciatic nerve stimulation. In summary, data suggests that sciatic nerve stimulation elicits a response of cortex and causes a slowdown in the propagation of CSD.

We have analyzed infrared spectra of microtome sections of frozen cervical tissues from the malignant (cancer) and normal histopathological groups. Intensity ratios of the different bands throughout the fingerprint region in the mid-IR are compared for the above groups. The bands are evaluated to show that the peak absorbances for the different groups exhibit statistically significant differences. Among the significant changes observed is the increase in the peak intensity of the 1400 cm^-1 band of the normal tissue group. Also, the contribution of the 1240 cm^-1 band which is ^*due to the phosphatediester group of nucleic acids was greater for the malignant tissue group compared to normal tissues. Partial-Least Squares (PLS) factors are used to further analyze the tissues to access the more subtle differences between the cancer and normal groups and to help determine the spectral bands that are most useful for cancer diagnosis. Also, we utilized Linear Discriminant Analysis (LDA) as well as the supervised learning method of Support Vector Machines (SVM) to study our tissue samples and develop diagnostic algorithms. PLS was used to decompose the spectra to reduce the spectral variables. The PLS components were then used as variables in SVM procedures to construct algorithms that produce specificity and sensitivity values greater than 90%.

Portable LIBS, (Laser Induced Breakdown Spectroscopy) systems are capable of real-time material analysis without sample preparation. LIBS systems focus a high peak power laser pulse onto a targeted material to produce a laser spark or plasma. Elemental line spectra is created, collected and analyzed by a fiber spectrophotometer. The line spectra emission data is quickly displayed on a laptop computer display. "Eye-safe" Class I lasers provide for practical in-situ LIBS applications such as detection of malignant skin tissues without the need for eye-protection goggles. This is due to the fact that Megawatt peak power Q-switched lasers operating at 1.54um in the narrow spectral window between 1.5um and 1.6um are approximately 8000 times more "eye-safe" than other laser devices operating in the visible and near infrared.