Commercial laser Doppler perfusion monitors are calibrated using the perfusion value, i.e. the first order moment of the Doppler power spectrum, from a measurement in a standardized microsphere colloidal suspension under Brownian motion. The calibration perfusion value depends on several parameters of the suspension that are difficult to keep constant with adequate accuracy, such as the concentration, temperature and the microsphere size distribution. The calibration procedure itself may therefore introduce significant errors in the measured values. An altered calibration procedure, where the zero order moment is used is described and demonstrated in this paper. Since the above mentioned parameters only affect the frequency content of the Doppler power spectrum and not the total power, the zero order moment will be independent of those parameters. It is shown that the variation in the calibration value, as given by measurements on different scattering liquids with a wide range of scattering properties and temperatures, is only a few percent using the proposed method. For the conventional calibration procedure, this variation corresponds to an error introduced by merely a 1°C variation in the reference liquid temperature. The proposed calibration method also enables absolute level comparisons between measured and simulated Doppler power spectra.

Researchers employ increasingly complex sub-micron particles for oncological applications to deliver bioactive therapeutic or imaging compounds to known and unknown in vivo tumor targets. These particles are often manufactured using a vast array of compounds and techniques resulting in a complex architecture, which can be quantified ex vivo by conventional metrology and chemical assays. In practice however, experimental homogeneity using nanoparticles can be difficult to achieve. While several imaging techniques have been previously shown to follow the accumulation of nanoparticles into tumor targets, a more rapid sensor that provides a quantifiable estimate of dose delivery and short-term systemic response could increase the clinical efficacy and greatly reduce the variability of these treatments. We have developed an optical device, the pulse photometer, that when placed on an accessible location will estimate the vascular concentration of near-infrared extinguishing nanoparticles in murine subjects. Using a technique called multi-wavelength photoplethysmography, the same technique used in pulse oximetry, our pulse photometer requires no baseline for each estimate allowing it to be taken on and off of the subject several times during experiments employing long circulating nanoparticles. We present a formal study of our prototype instrument in which circulation half-life and nanoparticle concentration of gold nanorods is determined in murine subjects with the aid of light anesthesia. In this study, we show good agreement between vascular nanorod concentrations (given in optical density) as determined by our device and with UV-VIS spectrophotometry using low volume blood samples.

Each year thousands of patients are added to the waiting list for liver transplants. The first 7-10 days after transplant have proven to be the most critical in patient recovery and it is hypothesized that monitoring organ vital signals in this period can increase patient and graft survival rates. An implantable sensor to monitor the organ perfusion and oxygenation signals following surgery is being developed by our group. The sensor operates based on measuring diffuse reflection from three light emitting diodes (735, 805 and 940 nm). In this work the optimal source detector spacing to maximize oxygenation signal level is investigated for a portal vein model. Monte Carlo simulations provided signal levels and corresponding penetration depths as a function of separation between a point optical source and detector. The modeling results indicated a rapid decay in the optical signal with increasing distance. Through further analysis, it was found that there exists an optimal range of point source to detector spacing, between roughly 1 and 2 mm, in which the blood signal from the simulated portal vein was maximized. Overall, these results are being used to guide the placement and configuration of our probe for in vivo animal studies.

Lensless imaging has recently attracted a lot of attention as a compact, easy-to-use method to image or detect biological objects like cells, but failed at detecting micron size objects like bacteria that often do not scatter enough light. In order to detect single bacterium, we have developed a method based on a thin wetting film that produces a micro-lens effect. Compared with previously reported results, a large improvement in signal to noise ratio is obtained due to the presence of a micro-lens on top of each bacterium. In these conditions, standard CMOS sensors are able to detect single bacterium, e.g. E.coli, Bacillus subtilis and Bacillus thuringiensis, with a large signal to noise ratio. This paper presents our sensor optimization to enhance the SNR; improve the detection of sub-micron objects; and increase the imaging FOV, from 4.3 mm² to 12 mm² to 24 mm², which allows the detection of bacteria contained in 0.5μl to 4μl to 10μl, respectively.

Our lab group is currently developing a fluorescent competitive binding assay between the Alexa fluor 647 labeled lectin, Concanavalin A, and highly structured glycosylated dendrimers to be sensitive to varying levels of glucose. Previously, this chemistry has elicited a high sensitivity to additions of physiological concentrations of glucose. However, the exact mechanism behind the sensing has not yet been well understood. This work presents a conceptual model of the response in which competitive binding results in different distributions of aggregates size to varying amounts of glucose. Preliminary experiments were performed by using Numerical Tracking Analysis (NTA) which correlates the movement of particles, positioned by light scattering, to the equivalent Brownian motion associated with particles of a certain spherical diameter. Using this method, the sensing chemistry was exposed to two different glucose concentrations and histograms of the size distribution for glucose concentrations were obtained. Herein the aggregation profile, mean aggregate size, and the number of aggregates (aggregates per mL) for two glucose concentrations are displayed, showing a correlation between the aggregation and glucose concentration.

Physiological glucose monitoring is important aspect in the treatment of individuals afflicted with diabetes mellitus. Although invasive techniques for glucose monitoring are widely available, it would be very beneficial to make such measurements in a noninvasive manner. In this study, a New Zealand White (NZW) rabbit animal model was utilized to evaluate a developed iris-based imaging technique for the in vivo measurement of physiological glucose concentration. The animals were anesthetized with isoflurane and an insulin/dextrose protocol was used to control blood glucose concentration. To further help restrict eye movement, a developed ocular fixation device was used. During the experimental time frame, near infrared illuminated iris images were acquired along with corresponding discrete blood glucose measurements taken with a handheld glucometer. Calibration was performed using an image based Partial Least Squares (PLS) technique. Independent validation was also performed to assess model performance along with Clarke Error Grid Analysis (CEGA). Initial validation results were promising and show that a high percentage of the predicted glucose concentrations are within 20% of the reference values.

Clinical guidelines dictate that frequent blood glucose monitoring in diabetic patients is critical towards proper management of the disease. Although, several different types of glucose monitors are now commercially available, most of these devices are invasive, thereby adversely affecting patient compliance. To this end, optical polarimetric glucose sensing through the eye has been proposed as a potential noninvasive means to aid in the control of diabetes. Arguably, the most critical and limiting factor towards successful application of such a technique is the time varying corneal birefringence due to eye motion artifact. We present a spatially variant uniaxial eye model to serve as a tool towards better understanding of the cornea's birefringence properties. The simulations show that index-unmatched coupling of light is spatially limited to a smaller range when compared to the index-matched situation. Polarimetric measurements on rabbits' eyes indicate relative agreement between the modeled and experimental values of corneal birefringence. In addition, the observed rotation in the plane of polarized light for multiple wavelengths demonstrates the potential for using a dual-wavelength polarimetric approach to overcome the noise due to timevarying corneal birefringence. These results will ultimately aid us in the development of an appropriate eye coupling mechanism for in vivo polarimetric glucose measurements.

There is a need to effectively and accurately monitor physiological glucose levels in individuals afflicted with diabetes mellitus. One promising noninvasive technique involves the use of optical polarimetry, in which the eye is commonly used as the sensing location. Since glucose is a chiral molecule, it has the ability to rotate plane polarized light by an amount that is proportional to glucose concentration. It has also been shown that glucose levels in the aqueous humor of the eye correlate well to those of blood. Therefore, we will report on an in vivo study that is conducted using a New Zealand White (NZW) rabbit model in conjunction with a custom developed Faradaybased optical polarimeter with sub-millidegree resolution. All animals used in this investigation were anesthetized with isoflurane and an insulin/dextrose protocol was used to control blood glucose concentration. A polarized laser light (632.8nm HeNe) signal was coupled through the anterior chamber of the eye using a custom designed ocular apparatus. System calibration was performed through measurement of the detected optical polarimetric signal and corresponding discrete blood glucose measurements taken with a handheld glucometer. Reference blood glucose samples were also measured using a YSI 2300+ glucose analyzer. The study results show that physiological glucose can be predicted with error levels on the order of 15%.

Successful development of real-time non-invasive glucose monitoring would represent a major advancement not only in the treatment and management of patients with diabetes mellitus and carbohydrate metabolism disorders, but also for understanding in those biochemical, metabolic and (patho-)physiological processes of glucose at the molecular level in vivo. Here, ATR-FTIR spectroscopy technique has been challenged not only for in vivo measurement of interstitial glucose levels, but also for their non-invasive molecular qualitative and quantitative comparative characterization in the skin tissue. The results, based on calculated mean values of determined 5 glucose-specific peaks in the glucose-related 1000-1160 cm-1 region, showed intra- and inter-subject differences in interstitial glucose activity levels with their changes at different times and doses of OGTT, while raising questions about the relationships between interstitial and blood glucose levels. In conclusion, the introduction of ATR-FTIR spectroscopy technique has opened up an access to the interstitial fluid space in the skin tissue for interstitial glucose characterization and monitoring in vivo. Though interstitial versus blood glucose monitoring has different characteristics, it can be argued that accurate and precise measurements of interstitial glucose levels may be more important clinically.

Optical spectroscopy has the inherent advantage of directly measuring glucose levels by means of the unique nature of a spectral fingerprint for the target analyte, glucose, which is determined in the form of a calibration spectrum (a product of multivariate analysis method). The literature is replete with claims of successful in-vivo glucose measurements in terms of Clark error grid analysis. However, all of them fail to demonstrate an in vivo glucose-specific calibration spectrum. In this study we use Raman spectroscopy to capture a high quality spectral fingerprint of the glucose molecule noninvasively and demonstrate calculated correlation coefficients between a pure glucose spectrum and calibration spectra at over 0.8.

As optical technology progresses through the translational research pipeline, phantoms are becoming more important as verification tools to demonstrate proper performance of the devices before clinical studies. Because of the wide range of optical methodologies, there can be no single phantom that is useful for all modes of imaging, but protocols for phantom use should be organized to stand as standards. This paper discusses the features that phantoms must have to be considered as standards for optical measurements.

The transparent elastomer Polydimethylsiloxane (PDMS) Sylgard 184 is increasingly used in optical applications, as in the manufacture of microlens, waveguides (optical fibers) and to elaborated phantoms (simulator of biological tissue); The wide range of applications is due to its excellent physic-chemical properties, its low cost, easy operation and null toxicity. This paper describes the manufacturing process and physic-chemical characterization of Phantoms prepared with PDMS as grid and doped with some elements present as Gliceryl, ink, glucose 10% and melanin provided by sigma aldrich. We made phantoms with different concentrations and elements; we measured their profiles, and thicknesses. Finally, we obtained their Raman Spectra. We present the experimental results obtained of the physic-chemical parameters of the phantoms and the conclusions.

In this paper, we present recent development of phantoms of coronary arteries with representative mechanical properties. The phantoms were made of poly(vinyl alcohol) cryogel (PVA-C). Multilayer phantoms were fabricated by an overmoulding process. The optical properties are adjusted in each layer by the different number of freeze-thaw cycles in combination with additives. The mechanical properties of the multilayer phantoms are characterized, and various means for improving the strain hardening are investigated.

Optical tissue phantoms are very important tools for the development of biomedical imaging applications. Optical phantoms are often used as ground truth against which instruments results can be compared. It is therefore important that the optical properties of reference phantoms be measured in a manner that is traceable to the international system of units. SI traceability insures long term consistency of results and will therefore improve the effectiveness of diffuse optics research effort more effective by reducing unwanted variability in the data produced and shared by the community. The ultimate benefit of rigorous SI traceability is the reduction of variability in the data produced by novel diagnostic devices, which will in turn increase the statistical power of clinical trials aiming at validating their clinical usefulness. SI traceability, and therefore uncertainty analysis, is also relevant to traceability aspects mandated by FDA regulations. SI traceability is achieved through a thorough analysis of the measurement principle and its potential error sources. The uncertainty analysis should be ultimately validated by inter-laboratory comparison until a consensus is attained on the best practices for measuring the optical properties of tissue phantoms.

A reference standard for tissue-simulating phantoms, i.e., a phantom with well known and stable optical properties, reproducible, and easy to be found, would be very useful for many applications based on measurements of diffused light. Although many tissue-equivalent phantoms have been proposed, to our knowledge none of them has been characterized sufficiently well to be suggested as a reference standard. Based on the results of measurements of optical properties we carried out at visible and NIR wavelengths, the use of Intralipid 20% diluted in water as diffusive medium, and of India ink as absorber, is here suggested as a first step towards a diffusive reference standard for tissue-simulating phantoms. As for Intralipid 20%, measurements carried out on samples from nine different batches with expiry dates spreading over ten years showed surprisingly small batch-to-batch variations. For the reduced scattering coefficient the maximum deviation from the value averaged over the nine batches was of about 2%, and the results for the absorption coefficient were very close to those for pure water. As for India ink measurements on samples from different batches and from five different brands showed large inter-brand and inter-batch variations for both the absorption and the extinction coefficient. On the contrary, small variations have been observed for the ratio between the absorption and the extinction coefficient. Intralipid 20% and Indian ink can be therefore easily mixed to obtain liquid phantoms with well known optical properties. This phantom can be a first step towards a reference standard for optical tissue phantoms.

Tissue mimicking phantoms are important for performance evaluation of imaging systems: in photoacoustics they need to accomplish both soft tissue optical and acoustic properties. PVA gels by freezing-thawing (F-T) have been used to simulate tissue properties. The microstructure, dependent on rate and extent of temperature cycling, is responsible for optical and acoustic properties. In F-T of larger phantoms, temperature differences in the mass are expected, resulting in a likely inhomogeneous distribution of properties in the phantom. We investigated speed of sound, acoustic attenuation, reduced optical scattering coefficient and microstructure: their variations are correlated with thermal differences between phantom surface and bulk.

We present a optical phantom approach for the characterization of OCT axial resolution and contrast via multilayered "bar charts." We explored two methods to fabricate these phantoms: the first is based on monolayers of light-scattering microspheres with an intervening layer of transparent silicone, and the second involves alternating layers of scatteringenhanced silicone and transparent silicone. Varying the diameter of the microspheres and the thickness of the silicone layers permits different spatial frequencies to be realized in the axial dimension of the phantoms. Because the phantom's dimensions are accurately known independent of the OCT system, no information about the system's spatial calibration is required. We quantified the degree to which the bars in each phantom could be resolved with OCT, which provides insights into the axial contrast transfer function. Initial testing of these phantoms on time-resolved and Fourier-domain OCT platforms indicated that our approach can provide accurate, system-independent estimates of resolution.

The aim of this project was to develop a skin phantom that resembles the epidermis including the lipid matrix of the stratum corneum and the dermis. The main intent was to achieve optical properties similar to skin tissue. Therefore, two compartments of the skin, dermis and epidermis, were examined regarding their optical properties. Based on these results, the skin phantom was designed using relevant skin components. The scattering coefficient was measured by using Reflectance-based Confocal Microscopy (RCM) and the fluorescence spectrum was detected via confocal laser-scanning microscopy (CLSM). Prospective, the skin phantom can be used to incorporate various fluorescing chemicals, such as fluorescent dyes and fluorescent-labeled drugs to perform calibration measurements in wide-field and laser-scanning microscopes to provide a basis for the quantification of skin penetration studies.

Due to the great number of new clinical applications of Low-Level-Laser-Therapy (LLLT), the development of precise, stable and low cost solid phantoms of skin, fat, muscle and bone becomes extremely important. The aim is to find the best combination of matrix, absorber and scatterers, which simulate skin, fat, muscle and bone tissues to build LLLT phantoms. Eight cylindrical phantoms simulating various human fingers were constructed and tested. Matrixes of polyester resins and paraffin were used with various concentrations of dyes and scatterers (Al₂O₃ nanoparticles) to adjust the optical parameters. A CCD camera was used to obtain transmission and scattering images of the phantoms, and of swine tissues and volunteer's fingers illuminated by lasers (diode 635 and 820 nm, and HeNe, 633 nm). The light fluence transmitted through the sample form Gaussian shaped profiles. Light scattered at 90 degrees shows an intensity profile with a steep growth followed by an exponential attenuation. The comparison of these two kinds of profiles for phantoms and swine tissue was used to evaluate the concentrations that better simulate different kinds of tissues. The outcomes of this study point to a reliable tool to aid clinicians with LLLT dosimetry.

Hyperspectral image projection applied to optical medical imaging can provide a means to evaluate imager performance. This allows repeated viewing of unique surgical scenes without the need for costly experiments on patients. Additionally, the generated scene can be well characterized and used repeatedly as a standard for many different imagers at different times and locations. This paper describes the use of a hyperspectral image of a pig kidney. The scene of the kidney is projected with the full spectral content allowing the oxygenation status of the tissue to be observed and evaluated spatially.

Non-invasive blood glucose monitoring using NIR light has been suffered from the variety of optical background that is mainly caused by the change of human body, such as the change of temperature, water concentration, and so on. In order to eliminate these internal influence and external interference a so called floating-reference method has been proposed to provide an internal reference. From the analysis of the diffuse reflectance spectrum, a position has been found where diffuse reflection of light is not sensitive to the glucose concentrations. Our previous work has proved the existence of reference position using diffusion equation. However, since glucose monitoring generally use the NIR light in region of 1000-2000nm, diffusion equation is not valid because of the high absorption coefficient and small source-detector separations. In this paper, steady-state high-order approximate model is used to further investigate the existence of the floating reference position in semi-infinite medium. Based on the analysis of different optical parameters on the impact of spatially resolved reflectance of light, we find that the existence of the floating-reference position is the result of the interaction of optical parameters. Comparing to the results of Monte Carlo simulation, the applicable region of diffusion approximation and higher-order approximation for the calculation of floating-reference position is discussed at the wavelength of 1000nm-1800nm, using the intralipid solution of different concentrations. The results indicate that when the reduced albedo is greater than 0.93, diffusion approximation results are more close to simulation results, otherwise the high order approximation is more applicable.

Non-invasive blood glucose sensing by near-infrared spectroscopy is easily interrupted by the strong background variations compared to the weak glucose signals. In this work, according to the distribution of diffuse reflectance intensity at different source-detector separations, a method based on a reference point and a measuring point, where the diffuse reflectance intensity is insensitive and most sensitive to the variation of glucose concentration, respectively, is applied. And the data processing method based on the information of two points is investigated to improve the precision of glucose sensing. Based on the Monte Carlo simulation in 5% intralipid solution model, the corresponding optical probe is designed which includes two detecting points: a reference point located at 1.3-1.7mm and a measuring point located at 1.7-2.1mm. Using the probe, the in vitro experiment with different glucose concentrations in the intralipid solution is conducted at 1100-1600nm. As a result, compared to the PLS model built by the signal of the measuring point, the root mean square error of prediction (RMSEP) and root mean square error of cross calibration (RMSEC) of the corrected model built by reference point and measuring point reduces by 45.10%, and 32.15% respectively.

Since the beginning of the 21st century, the issue of food safety is becoming a global concern. It is very important to develop a rapid, cost-effective, and widely available method for food adulteration detection. In this paper, near-infrared spectroscopy techniques and pattern recognition were applied to study the qualitative discriminant analysis method. The samples were prepared and adulterated with one of the three adulterants, urea, glucose and melamine with different concentrations. First, the spectral characteristics of milk and adulterant samples were analyzed. Then, pattern recognition methods were used for qualitative discriminant analysis of milk adulteration. Soft independent modeling of class analogy and partial least squares discriminant analysis (PLSDA) were used to construct discriminant models, respectively. Furthermore, the optimization method of the model was studied. The best spectral pretreatment methods and the optimal band were determined. In the optimal conditions, PLSDA models were constructed respectively for each type of adulterated sample sets (urea, melamine and glucose) and all the three types of adulterated sample sets. Results showed that, the discrimination accuracy of model achieved 93.2% in the classification of different adulterated and unadulterated milk samples. Thus, it can be concluded that near-infrared spectroscopy and PLSDA can be used to identify whether the milk has been adulterated or not and the type of adulterant used.

Raman spectroscopy is a promising technology for noninvasive blood glucose monitoring because of its good selectivity for the glucose molecule. The low sensitivity of the Raman signal however, makes it difficult to quantify the concentration of glucose directly from the Raman spectra. To solve this, statistical methods such as PCA (principle component analysis) and PLS (partial least square) are traditionally used. These statistical methods general work very well and give highly accurate results, provided there is no interference. In the in-vivo case however, there are many interferences such as the inhomogeneity of tissue, physiological changes, and denaturation of the tissue by the light source. This study investigates the affect of in-vivo interferences on Raman glucose measurements. In this study, a high throughput dispersive Raman system was constructed with an 830nm multimode laser, a multiple conductor optical fiber bundle, and a back-illuminated CCD spectrometer. A simply phantom was devised, which was comprised of a plastic cuvette fitted with a human fingernail window and glucose doped human serum used as the sample. To test the inhomogeneity of tissue samples, different sites of the phantom were exposed to the laser. In the case of denaturation, tests were conducted under two laser power densities: low (3.7mW/mm²) and high density (110mW/mm²). To simulate the physiological change, gelatin phantoms of varied concentration were investigated. The results of the study indicate that the dominant interferers for Raman in-vivo glucose measurements are the inhomogeneity of the tissue and the denaturation by the laser power density. The next phase for this study will be the design of a high SNR Raman system which affords a low power density laser sample illumination as well as larger volumetric illumination to mitigate the effects of tissue inhomogeneity.

In this work we carried out a comparison and localization of skin Raman spectra. Measurements were made in regions where Raman scatter is caused by the excitation source; we used the spectra overlap in a comparative way. Ten volunteers with different skin colors participated in the experiment; body parts sampled were palm and dorsum of the hand. The excitation wavelength used for this work was 785nm. A spectrometer with 6cm^-1 resolution and spectral region range 0 to 2000 cm^-1 was utilized. We used Matlab® to overlap and compare the differences between Raman spectra form different samples. We found spectral variations that were caused by differences on the surface of the skin, such as scars and moles. This work helps to identify potential undesirable behavior on the epidermis.

The introduction of polarimetry in optical imaging of biological tissues provides a powerful method to enhance contrast and specificity in the characterization of anisotropic biological tissues. Moreover, the fact that Mueller calculus can deal with partially polarized light and depolarizing media enables to analyze the strong effect of scattering in light propagation through biological tissues. The inherent heterogeneity of biological tissues causes that multiple effects are overlapped in a single measurement. Regarding polarimetry, anisotropic structures can simultaneously exhibit birefringence, diattenuation and depolarization. Lu-Chipman polar decomposition has been widely used in order to isolate the effects and quantify each of them. However, it entails a limitation: as long as the original Mueller matrix is decomposed into the product of three components, the result of the decomposition varies with the order. In this work, we propose a polarimetric analysis based on differential Mueller matrices. This analysis is not affected by the order in which the effects take place within the medium. We apply it to the study of optical activity in chiral and turbid biological media, in particular to a solution of glucose mixed with an aqueous suspension of polystyrene microspheres. The results obtained by Lu-Chipman polar decomposition and by differential Mueller matrices analysis are compared. It will be shown that the results obtained by the polarimetric analysis proposed in this work are in good agreement with those obtained by polar decomposition, with the advantage that differential Mueller matrices provide additional information to further develop polarimetric analysis in a robust way.

Interaction of red blood cells (RBC) with optical tweezers has been found to differ under varied physiological and pathological conditions as compared to its normal conditions. Earlier, we reported difference in rotation of trapped RBC in hypertonic conditions for detection of malaria infection. Disk-like RBC when trapped in optical tweezers get oriented in the vertical plane to maximize interaction with trapping beam. However, classical bright field, phase contrast or epifluorescence microscopy cannot confirm its orientation, thus leading to ambiguous conclusions such as folding of RBC during trapping by some researchers. Now, with use of digital holographic microscopy (DHM), we achieved high axial sensitivity that confirmed orientation of trapped red blood cell. Further, DHM enabled quantitative phase imaging of RBC under hypertonic condition. Dynamic changes of rotating RBC under optical tweezers at different trapping laser power were evaluated by the use of DHM. The deviation from linear dependence of rotation speed of RBC on laser power, was attributed towards deformation of RBC shape due to higher laser power (or speed).