The ability to conduct high-resolution fluorescence imaging in internal organs of small animal models in situ and over time can make a significant impact in biomedical research. Toward this goal, we developed a real-time confocal and multiphoton endoscopic imaging system. Using 1-mm-diameter endoscopes based on gradient index lenses, we demonstrate video-rate multicolor multimodal imaging with cellular resolution in live mice.

Spectral reflectance measurements of biological tissues have been studied for early diagnoses of several pathologies such as cancer. These measurements are often performed with a fiber optic probe in contact with the tissue surface. We report a study in which reflectance measurements are obtained in vivo from mouse thigh muscle while varying the contact pressure of the fiber optic probe. It is determined that the probe pressure is a variable that affects the local optical properties of the tissue. The reflectance spectra are analyzed with an analytical model that extracts the tissue optical properties and facilitates the understanding of underlying physiological changes induced by the probe pressure.

Laser trapping of micrometer-sized objects floating in water is investigated through the use of a tilted laser beam. With a change in the tilt direction, the orientation of the trapped object can be easily controlled when the object has an asymmetric body or nonuniform refractive index, such as nanowires, living cells, and so on. The method enables efficient orientation control under laser trapping through a simple setup. This method for tilt control may be useful for high-performance laser trapping in bioengineering and microsurgery in single living cells.

We present a novel optical sensor able to measure the distance between the tip of an endoscopic probe and the anatomical object under examination. In medical endoscopy, knowledge of the real distance from the endoscope to the anatomical wall provides the actual dimensions and areas of the anatomical objects. Currently, endoscopic examination is limited to a direct and qualitative observation of anatomical cavities. The major obstacle to quantitative imaging is the inability to calibrate the acquired images because of the magnification system. However, the possibility of monitoring the actual size of anatomical objects is a powerful tool both in research and in clinical investigation. To solve this problem in a satisfactory way we study and realize an absolute distance sensor based on fiber optic low-coherence interferometry (FOLCI). Until now the sensor has been tested on pig trachea, simulating the real humidity and temperature (37°C) conditions. It showed high sensitivity, providing correct and repeatable distance measurements on biological samples even in case of very low reflected power (down to 2 to 3 nW), with an error lower than 0.1 mm.

Noninvasive cardiovascular imaging could lead to the early detection and timely treatment of complex atherosclerotic lesions responsible for major cardiovascular events. Recent investigations have suggested that optical coherence tomography (OCT) is an ideal diagnostic tool due to the high resolution this technology achieves in discriminating the different features of atherosclerotic lesions based on structural imaging. We explore the capability of OCT for functional imaging of normal and atherosclerotic aortic tissues based on time- and depth-resolved quantification of the permeability of biomolecules through these tissues. The permeability coefficient of 20% aqueous solution of glucose was found to be (6.80±0.18)×10^-6 cm/s in normal aortas and (2.69±0.42)×10^-5 cm/s in aortas with atherosclerotic disease. The results suggest that this new OCT functional imaging method—the assessment of the permeability coefficients of various physiologically neutral biomolecules in vascular tissues—could assist in early diagnosing and detecting the different components of atherosclerotic lesions.

This PDF file contains the editorial “Small-Animal Optical Imaging” for JBO Vol. 13 Issue 01

Brain pathologies, including stroke and tumors, are associated with a variable degree of breakdown of the blood-brain barrier (BBB), which can usefully be studied in animal models. We describe a new optical technique for quantifying extravasation in the cortex of the living mouse and for imaging intraparenchymal tissue. Leakiness of the BBB was induced by microbeam x-irradiation. Two fluorescent dyes were simultaneously infused intravenously, one of high molecular weight (fluorescein-labeled dextran, 70 kDa, green fluorescence) and one of low molecular weight (sulforhodamine B, 559 Da, red fluorescence). A two-photon microscope, directed through a cranial window, obtained separate images of the two dyes in the cortex. The gains of the two channels were adjusted so that the signals coming from within the vessels were equal. Subtraction of the image of the fluorescein-dextran from that of the sulforhodamine B gave images in which the vasculature was invisible and the sulforhodamine B in the parenchyma could be imaged with high resolution. Algorithms are presented for rapidly quantifying the extravasation without the need for shape recognition and for calculating the permeability of the BBB. Sulforhodamine B labeled certain intraparenchymal cells; these cells, and other details, were best observed using the subtraction method.

Three-dimensional optical coherence tomography (3D-OCT) is used to evaluate the structure and pathology of regenerating mouse skeletal muscle autografts for the first time. The death of myofibers with associated inflammation and subsequent new muscle formation in this graft model represents key features of necrosis and inflammation in the human disease Duchenne muscular dystrophy. We perform 3D-OCT imaging of excised autografts and compare OCT images with coregistered histology. The OCT images readily distinguish the necrotic and inflammatory tissue of the graft from the intact healthy muscle fibers in the underlying host tissue. These preliminary findings suggest that, with further development, 3D-OCT could be used as a tool for the evaluation of small-animal muscle morphology and pathology, in particular, for analysis of mouse models of muscular dystrophy.

Tongue inspection (TI) is an important and unique diagnostic method in traditional Chinese medicine (TCM), because significant connections between various viscerae diseases and abnormalities in the tongue have been verified. In TCM, TI is simple and non invasive, but in clinical applications, TI is subjectively based on the experience and technique of physicians. To avoid this problem, optical coherence tomography (OCT) imaging is introduced here for TI. We study OCT imaging in rats in vivo from chronic gastritis group (model) and normal group (control) and quantitatively analyze the relative parameters, such as the thickness and the moisture degree of TI. Our results show that OCT images properly demonstrate the thickness of the tongue coating and the moisture degree of the tongue in both groups, and the thickness is increased in the model group from that in the normal group, while the moisture degree decreases. As a result, OCT technology has the potential to provide physicians with an objective diagnostic standard for visual TI in TCM clinical practice and research.

Laser-based photoacoustic tomography (PAT), a novel, nonionizing, noninvasive, laser-based technology, has been adapted to the diagnosis and imaging of inflammatory arthritis. A commonly used adjuvant induced arthritis model using carrageenan was employed to simulate acute rheumatoid arthritis in rat tail joints. Cross-sectional photoacoustic images of joints affected by acute inflammation were compared to those of the control. The diameter of the periosteum and the optical absorption of intra-articular tissue were measured on each joint image. Significant differences were found on PAT imaging between the affected joints and the control for both variables measured, including enlarged periosteum diameter and enhanced intra-articular optical absorption occurring in the joints affected with carrageenan-induced arthritis. Anatomical correlation with histological sections of imaged joints and microMRI results verified the findings of PAT. This suggests that PAT has the potential for highly sensitive diagnosis and evaluation of pathologic hallmarks of acute inflammatory arthritis.

Matrix metalloproteinases (MMPs) are a kind of secretory proteinases. Degradation of the extracellular matrix (ECM) by MMPs enhances tumor invasion and metastasis. To monitor MMPs activity and assess the MMP inhibitor effects in vivo, we constructed a plasmid that encoded a secretory fluorescent sensor named DMC (DsRed2-MSS-CFP expressed from pDisplay vector) that DsRed2 and cyan fluorescent protein (CFP) linked by MMP substrate site (MSS). MDA-MB 435s cells highly expressing endogenetic secretory MMP were transfected with the DMC plasmid so that the DMC could be cleaved by endogenetic MMP and the fluorescence ratio of DsRed2 to CFP was decreased. Treating the cells with GM6001, an MMP inhibitor, blocked the cleavage of DMC and caused an increase of the DsRed2/CFP ratio. The same result was achieved by using an in vivo tumor model that stable DMC-expressing MDA-MB 435s cells inoculated onto the chorioallantoic membrane of developing chick embryos to form primary tumors on the membrane. Thus, the fluorescent sensor DMC is able to sensitively monitor MMP activity and assess MMP inhibitors for anticancer research in vivo. This proves a novel method to efficiently screen and assess the anticancer drug MMP inhibitor in living cells and in vivo tumor models.

The metalloexopeptidase CD13/aminopeptidase N (APN) has been shown to be involved in cancer angiogenesis, invasion, and metastasis. Therefore, a CD13/APN–targeted NGR-peptide was labeled with the cyanine dye Cy 5.5 and applied to image tumor xenografts with different APN-expression levels using both planar and tomographic optical imaging methods. In vitro, the peptide-dye conjugate showed a clear binding affinity to APN-positive HT-1080 cells, while negative MCF-7 cells and predosing with the free NGR-peptide revealed little to no fluorescence. In vivo, tumor xenografts (n≥5) were clearly visualized by two-dimensional (2-D) planar fluorescence reflectance imaging (FRI) and three-dimensional (3-D) fluorescence mediated tomography (FMT) up to 24 h after injection. FMT also allowed us to quantify fluorochrome distribution in deeper tissue sections, showing an average fluorochrome concentration of 306.7±54.3 nM Cy 5.5 (HT-1080) and 116.0±18.3 nM Cy 5.5 (MCF-7) in the target tissue after 5 h. Competition with the free NGR-peptide resulted in a reduction of fluorochrome concentration in HT-1080 tumor tissue (195.3±21.9 nM; 5 h). We thus conclude that NGR-Cy 5.5 combined with novel tomographic optical imaging methods allows us to image and quantify tumor-associated CD13/APN expression noninvasively. This may be a promising strategy for a sensitive evaluation of tumor angiogenesis in vivo.

We present in vivo experiments conducted with a new fluorescence diffuse optical tomographic (fDOT) system on cancerous mice bearing mammary murine tumors. We first briefly present this new system that has been developed and its associated reconstruction method. Its main specificity is its ability to reconstruct the fluorescence yield even in heterogeneous and highly attenuating body regions such as lungs and to enable mouse inspection without immersion in optical index matching liquid (Intralipid and ink). Some phantom experiments validate the performance of this new system for heterogeneous media inspection. Its use for a mice study is then related. It consists in the follow-up of the lungs at different stages of tumor development after injection of RAFT-(cRGD)₄-Alexa700. As expected, the reconstructed fluorescence increases along with the tumor stage. These results validate the use of our system for biological studies of small animals.

The quantitative accuracy of fluorescence and bioluminescence imaging of small animals can be improved by knowledge of the in situ optical properties of each animal. Obtaining in situ optical property maps is challenging, however, due to short propagation distances, requirements for high dynamic range, and the need for dense spatial, temporal, and spectral sampling. Using an ultrafast gated image intensifier and a pulsed laser source, we have developed a small animal diffuse optical tomography system with multiple synthetic modulation frequencies up to >1 GHz. We show that amplitude and phase measurements with useful contrast-to-noise ratios can be obtained for modulation frequencies over the range of ~250 to 1250 MHz. Experiments with tissue simulating phantoms demonstrate the feasibility of reconstructing the absorption and scattering optical properties in a small animal imaging system.

Red blood cell (RBC) aggregation in the blood stream is prevented by the zeta potential created by its negatively charged membrane. There are techniques, however, to decrease the zeta potential and allow cell agglutination, which are the basis of most of antigen-antibody tests used in immunohematology. We propose the use of optical tweezers to measure membrane viscosity, adhesion, zeta potential, and the double layer thickness of charges (DLT) formed around the cell in an electrolytic solution. For the membrane viscosity experiment, we trap a bead attached to RBCs and measure the force to slide one RBC over the other as a function of the velocity. Adhesion is quantified by displacing two RBCs apart until disagglutination. The DLT is measured using the force on the bead attached to a single RBC in response to an applied voltage. The zeta potential is obtained by measuring the terminal velocity after releasing the RBC from the trap at the last applied voltage. We believe that the methodology proposed here can provide information about agglutination, help to improve the tests usually performed in transfusion services, and be applied for zeta potential measurements in other samples.

We combine laser tweezers with custom computer tracking software and robotics to analyze the motility [swimming speed, VCL (curvilinear velocity), and swimming force in terms of escape laser power (Pesc)] and energetics [mitochondrial membrane potential (MP)] of individual sperm. Domestic dog sperm are labeled with a cationic fluorescent probe, DiOC₂(3), that reports the MP across the inner membrane of the mitochondria located in the sperm's midpiece. Individual sperm are tracked to calculate VCL. Pesc is measured by reducing the laser power after the sperm is trapped using laser tweezers until the sperm is capable of escaping the trap. The MP is measured every second over a 5-s interval during the tracking phase (sperm is swimming freely) and continuously during the trapping phase. The effect of the fluorescent probe on sperm motility is addressed. The sensitivity of the probe is measured by assessing the effects of a mitochondrial uncoupling agent (CCCP) on MP of free swimming sperm. The effects of prolonged exposed to the laser tweezers on VCL and MP are analyzed. The system's capabilities are demonstrated by measuring VCL, Pesc, and MP simultaneously for individual sperm. This combination of imaging tools is useful to quantitatively assess sperm quality and viability.

Excisional biopsies and routine histology remains the gold standard for the histomorphologic evaluation of normal and diseased skin. However, there is increasing interest in the development of noninvasive optical technologies for evaluation, diagnosis, and monitoring of skin disease in vivo. Fluorescent confocal microscopy is an innovative optical technology that has previously been used for morphologic evaluation of live human tissue. We evaluate the clinical applicability of a fluorescent confocal laser scanning microscope (FLSM) for a systematic evaluation of normal and diseased skin in vivo and in correlation with routine histology. A total of 40 patients were recruited to participate in the study. Skin sites of 10 participants with no prior history of skin disease served as controls and to evaluate topographic variations of normal skin in vivo. Thirty patients with a suspected diagnosis of nonmelanoma skin cancer were evaluated, whereby FLSM features of actinic keratoses (AK) and basal cell carcinoma (BCC) were recorded in an observational analysis. Selected BCCs were monitored for their skin response to topical therapy using Imiquimod as an immune-response modifier. A commercially available fluorescence microscope (OptiScan Ltd., Melbourne, Australia) was used to carry out all FLSM evaluations. Common FLSM features to AK and BCC included nuclear pleomorphism at the level of the granular and spinous layer and increased vascularity in the superficial dermal compartment. Even though the presence of superficial disruption and mere atypia of epidermal keratinocytes was more indicative of AK, the nesting of atypical basal cells, increased blood vessel tortuosity, and nuclear polarization were more typical for BCC. All diagnoses were confirmed by histology. FLSM allowed a monitoring of the local immune response following therapy with Imiquimod and demonstrated a continuous normalization of diseased skin on repeated evaluations over time. (Partial Abstract)

We present an evaluation of time-resolved fluorescence measurements on human skin for screening type 2 diabetes. In vivo human skin is excited with a pulse diode at 375 nm and pulse width of 700 ps. Fluorescence decays are recorded at four different emission wavelengths: 442, 460, 478, and 496 nm. Experiments are performed at various locations, including the palms, arms, legs, and cheeks of a healthy Caucasian subject to test single-subject variability. The fluorescence decays obtained are modeled using a three-exponential decay. The variations in the lifetimes and amplitudes from one location to another are minimal, except on the cheek. We compare the fluorescent decays of 38 diabetic subjects and 37 nondiabetic subjects, with different skin complexions and of ages ranging from 6 to 85 yr. The average lifetimes for nondiabetic subjects were 0.5, 2.6, and 9.2 ns with fractional amplitudes of 0.78, 0.18, and 0.03, respectively. The effects of average hemoglobin A1c (HbA1c) from the previous 4 yr and diabetes duration are evaluated. While no significant differences between the fluorescence lifetimes of nondiabetic and diabetic subjects are observed, two of the fractional amplitudes are statistically different. Additionally, none of the six fluorescence parameters correlated with diabetes duration or HbA1c. One of the lifetimes as well as two of the fractional amplitudes differ between diabetic subjects with foot ulcers and nondiabetic subjects.

In imaging anisotropic samples with optical microscopy, a controlled, polarized light source can be used to gain molecular information of fibrous materials such as muscles and collagen fibers. However, the delivery of the polarized excitation light source in a system such as a laser scanning optical microscope often encounters the problem of the polarization ellipticity altering effects of the optical components. Using a half-wave plate and a quarter-wave plate, we demonstrate that the polarization ellipticity altering effect of the dichroic mirror in an epi-illuminated multiphoton laser scanning microscope can be corrected, and that this approach can be used to obtain polarized second-harmonic generation (SHG) images of rat tail tendon and mouse leg muscle. The excitation polarization dependence of the SHG intensity is fitted to determine the ratio of the second-order susceptibility tensor elements associated with type I collagen in the rat tail tendon and myofibril in the mouse leg muscle. Our methodology can be applied to polarized SHG imaging without sample rotation. This approach has great potential for imaging noncentrosymmetric biological samples, providing structural information on the molecular scale in addition to morphological information of tissues.

While quantitative analysis of dynamic biological cell motions in vivo is of great biomedical interest, acquiring 3-D (plus time) information is difficult due to the lack of imaging tools with sufficient spatial and temporal resolution. A novel 3-D high-speed microscopic imaging system is developed to enable 3-D time series data acquisition, based on a defocusing technique (DDPIV). Depth coordinate Z is resolved by the triangular image patterns generated by a mask with three apertures forming an equilateral triangle. Application of this technique to microscale imaging is validated by calibration of targets spread over the image field. 1-µm fluorescent tracer particles are injected into the blood stream of 32 h post-fertilization developing zebrafish embryos to help describe cardiac cell motions. 3-D and velocity fields of cardiovascular blood flow and trajectories of heart-wall motions are obtained.

Digital in-line holography offers some significant advantages over conventional optical holography and microscopy to image biological specimens. By combining holography with digital video microscopy, an in-line holographic video microscope is developed and is capable of recording spatial 3D holographic images of biological specimens, while preserving the time dimension. The system enables high-speed video recording of fast cell movement, such as the rapid movement of blood cells in the blood stream in vivo. This capability is demonstrated with observations of fast 3-D movement of live cells in suspension cultures in response to a gentle shake to the Petri dish. The experimental and numerical procedures are incorporated with a fast reconstruction algorithm for reconstruction of holographic video frames at various planes (z axis) from the hologram and along the time axis. The current system enables both lateral and longitudinal resolutions down to a few micrometers. Postreconstruction processing of background subtraction is utilized to eliminate noise caused by scattered light, thereby enabling visualization of, for example, blood streams of live Xenopos tadpoles. The combination of digital holography and microscopy offers unique advantages for imaging of fast moving cells and other biological particles in three dimensions in vivo with high spatial and temporal resolution.

The analysis of fluorescence lifetime imaging microscopy (FLIM) data under complex biological conditions can be challenging. Particularly, the presence of short-lived autofluorescent aggregates can confound lifetime measurements in fluorescence energy transfer (FRET) experiments, where it can become confused with the signal from exogenous fluorophores. Here we report two techniques that can be used to discriminate the contribution of autofluorescence from exogenous fluorphores in FLIM. We apply the techniques to transgenic mice that natively express yellow fluorescence protein (YFP) in a subset of cortical neurons and to histological slices of aged human brain tissue, where we study the misfolding of intracellular tau protein in the form of neurofibrillary tangles.

The cell nucleus is often considered a spherical structure. However, the visualization of proteins associated with the nuclear envelope in rat hippocampal neurons indicates that the geometry of nuclei is far more complex. The shape of cell nuclei is likely to influence the nucleo-cytoplasmic exchange of macromolecules and ions, in particular calcium, a key regulator of neuronal gene expression. We developed a tool to retrieve the 3-D view of cell nuclei from laser scanning confocal microscopy data. By applying an inertia-based filter, based on a special structure detection mechanism, the signal-to-noise ratio of the image is enhanced, the signal is smoothed, gaps in the membrane are closed, while at the same time the geometric properties, such as diameters of the membrane, are preserved. After segmentation of the image data, the microscopy data are sufficiently processed to extract surface information of the membrane by creating an isosurface with a marching tetrahedra algorithm combined with a modified Dijkstra graph-search algorithm. All methods are tested on artificial data, as well as on real data, which are recorded with a laser scanning confocal microscope. Significant advantages of the inertia-based filter can be observed when comparing it to other state of the art nonlinear diffusion filters. An additional program is written to calculate surface and volume of cell nuclei. These results represent the first step toward establishing a geometry-based model of the-dynamics of cytoplasmic and nuclear calcium.

We investigate the relationship of incubation time and forward-scattering signature for bacterial colonies grown on solid nutrient surfaces. The aim of this research is to understand the colony growth characteristics and the corresponding evolution of the scattering patterns for a variety of pathogenic bacteria relevant to food safety. In particular, we characterized time-varying macroscopic and microscopic morphological properties of the growing colonies and modeled their optical properties in terms of two-dimensional (2-D) amplitude and phase modulation distributions. These distributions, in turn, serve as input to scalar diffraction theory, which is, in turn, used to predict forward-scattering signatures. For the present work, three different species of Listeria were considered: Listeria innocua, Listeria ivanovii, and Listeria monocytogenes. The baseline experiments involved the growth of cultures on brain heart infusion (BHI) agar and the capture of scatter images every 6 h over a total incubation period of 42 h. The micro- and macroscopic morphologies of the colonies were studied by phase contrast microscopy. Growth curves, represented by colony diameter as a function of time, were compared with the measured time-evolution of the scattering signatures.

We describe a combined orientation-independent differential interference contrast OI-DIC and polarization microscope and its biological applications. Several conventional DIC images were recorded with the specimen oriented in different directions followed by digital alignment and processing of the images. Then the obtained images are used for computation of the phase gradient magnitude and azimuth distribution and, further, the phase image. The OI-DIC images were obtained using optics having numerical aperture (NA) 1.4, thus achieving a level of resolution not previously achieved with phase contrast or interference microscope. The combined system yields two complementary phase images of thin optical sections of the specimen: distribution of refractive index and distribution of birefringence due to anisotropy of the cell structure. For instance, in a live dividing cell, the OI-DIC image clearly shows the detailed shape of the chromosomes, while the polarization image quantitatively depicts the distribution of birefringent microtubules in the spindle, both without any need for staining or other modifications of the cell. We present pseudo-color combined images of a crane fly spermatocyte at diakinesis and metaphase of meiosis I. Those images provide clear evidence that the proposed technique can reveal fine architecture and molecular organization in live cells without perturbation associated with staining or fluorescent labeling.

We have been developing a Mach-Zehnder type of optical frequency domain reflectometry optical coherence tomography (OFDR-OCT) that uses discretely swept superstructure-grating distributed Bragg- reflector (SSG-DBR) lasers developed for telecommunication fields and has a 12-mm-depth range. We report images obtained with L-band (1560 to 1600 nm) and C-band (1529 to 1568 nm) SSG-DBR sources at 0.5 µs/step, which is 20 times faster than the scanning speed used to obtain the images we reported previously. Despite the faster scanning, we obtain good OCT images of both hard and soft dental tissues in vitro and in vivo.

Phase retardation of in vivo human retinal nerve fiber layer (RNFL) is quantitatively measured by two methods—polarization-sensitive spectral-domain optical coherence tomography (PS-SD-OCT) and scanning laser polarimetry (SLP). An en face cumulative phase retardation map is calculated from the three-dimensional (3-D) phase retardation volume of healthy and glaucomatous eyes measured by PS-SD-OCT. It is shown that the phase retardation curves around the optic nerve head measured by PS-SD-OCT and SLP have similar values except near the retinal blood vessels. PS-SD-OCT can measure the cumulative phase retardation of RNFL as well as SLP, which will allow the evaluation of RNFL for glaucomatous eyes.

Fine needle aspiration biopsy (FNAB) is a rapid and cost-effective method for obtaining a first-line diagnosis of a palpable mass of the breast. However, because it can be difficult to manually discriminate between adipose tissue and the fibroglandular tissue more likely to harbor disease, this technique is plagued by a high number of nondiagnostic tissue draws. We have developed a portable, low coherence interferometry (LCI) instrument for FNAB guidance to combat this problem. The device contains an optical fiber probe inserted within the bore of the fine gauge needle and is capable of obtaining tissue structural information with a spatial resolution of 10 μm over a depth of approximately 1.0 mm. For such a device to be effective clinically, algorithms that use the LCI data must be developed for classifying different tissue types. We present an automated algorithm for differentiating adipose tissue from fibroglandular human breast tissue based on three parameters computed from the LCI signal (slope, standard deviation, spatial frequency content). A total of 260 breast tissue samples from 58 patients were collected from excised surgical specimens. A training set (N=72) was used to extract parameters for each tissue type and the parameters were fit to a multivariate normal density. The model was applied to a validation set (N=86) using likelihood ratios to classify groups. The overall accuracy of the model was 91.9% (84.0 to 96.7) with 98.1% (89.7 to 99.9) sensitivity and 82.4% (65.5 to 93.2) specificity where the numbers in parentheses represent the 95% confidence intervals. These results suggest that LCI can be used to determine tissue type and guide FNAB of the breast.

An optical microvascular skin model, valid at 780 nm, was developed. The model consisted of six layers with individual optical properties and variable thicknesses and blood concentrations at three different blood flow velocities. Monte Carlo simulations were used to evaluate the impact of various model parameters on the traditional laser Doppler flowmetry (LDF) measures. A set of reference Doppler power spectra was generated by simulating 7000 configurations, varying the thickness and blood concentrations. Simulated spectra, at two different source detector separations, were compared with in vivo recorded spectra, using a nonlinear search algorithm for minimizing the deviation between simulated and measured spectra. The model was validated by inspecting the thickness and blood concentrations that generated the best fit. These four parameters followed a priori expectations for the measurement situations, and the simulated spectra agreed well with the measured spectra for both detector separations. Average estimated dermal blood concentration was 0.08% at rest and 0.63% during heat provocation (44 °C) on the volar side of the forearm and 1.2% at rest on the finger pulp. The model is crucial for developing a technique for velocity-resolved absolute LDF measurements with known sampling volume and can also be useful for other bio-optical modalities.

We develop a superficial diffusing probe with a 3 mm source-detector separation that can be used in combination with diffuse optical spectroscopic (DOS) methods to noninvasively determine full-spectrum optical properties of superficial in vivo skin in the wavelength range from 650 to 1000 nm. This new probe uses a highly scattering layer to diffuse photons emitted from a collimated light source and relies on a two-layer diffusion model to determine tissue absorption coefficient μ_a and reduced scattering coefficient μ_s′. By employing the probe to measure two-layer phantoms that mimic the optical properties of skin, we demonstrate that the probe has an interrogation depth of 1 to 2 mm. We carry out SSFDPM (steady state frequency-domain photon migration) measurements using this new probe on the volar forearm and palm of 15 subjects, including five subjects of African descent, five Asians, and five Caucasians. The optical properties of in vivo skin determined using the superficial diffusing probe show considerable similarity to published optical properties of carefully prepared ex vivo epidermis+dermis.

We present a new method for studying melanin in vivo based on diffuse reflectance spectroscopy of human skin. We find that the optical absorption spectrum of in vivo melanin exhibits an exponential dependence on wavelength, consistent with, but with a higher decay slope than, in vitro results. We offer theoretical justification for this exponential dependence on the basis of a recently proposed model for the structure of eumelanin protomolecules. Moreover, we report on a new method for analysis of diffuse reflectance spectra, which identifies intrinsic differences in absorption spectra between malignant melanoma and dysplastic nevi in vivo. These preliminary results are confirmed both by analysis of our own clinical data as well as by analysis of data from three independent, previously published studies. In particular, we find evidence that the histologic transition from dysplastic nevi to melanoma in situ and then to malignant melanoma is reflected in the melanin absorption spectra. Our results are very promising for the development of techniques for the noninvasive detection of melanoma and, more generally, for the study and characterization of pigmented skin lesions. It is also a promising approach for a better understanding of the biological role, structure, and function of melanin.

The Fourier transform (FT)-Raman spectroscopy technique is used to assess the biochemical alterations that occur in the degenerative process of the rotator cuff supraspinatus tendon. The main alterations observed occur in the glycine, proline, hydroxyproline, cysteine, cistine, phenylalanine, tyrosine, collagen I and III, nucleic acid, lipids, glycosaminoglycans, and metalloproteinases bands. An increasing intensity for these bands is found in degenerated tendons, a finding well correlated with hyaline state and cellular activity. Statistical analysis (principal components analysis and clustering) shows a clear separation of the spectra into nonhyalinized and hyalinized clusters, which enables the construction of a binary diagnosis model based on logistic regression. Best diagnosis provided a sensitivity of 66.0% and a specificity of 74.7% with 79.6% concordant pairs. The discriminating power of the diagnostic test is assessed by computing the area under the receiving-operator characteristic curve (AUC), which indicates good accuracy (AUC=0.81). In principle, these results indicate that Raman spectroscopy can be used as an auxiliary aid to improve shoulder tendon surgery quality by guiding anchoring onto more healthy (nonhyaline) pieces of tendons. This should contribute to a decrease in the current high rerupture rate (13 to 68%) for this procedure.

The safety of tooth bleaching, which is based upon hydrogen peroxide (HP) as the active agent, has been questioned. Our aim was to investigate the effects of 30% HP on human tooth enamel. The specimens were divided randomly into three groups and treated with distilled water, HCl, and HP, respectively. Raman scattering and laser-induced fluorescence of enamel were determined before and after treatment. Microhardness testing and scanning electron microscopy were also used. The results of Raman scattering showed that the Raman relative intensity of enamel changed significantly after HP and HCl treatment. These findings were consistent with the results of microhardness testing and morphological observations. In addition, a small band at 876 cm^-1 due to O–O stretching of HP became pronounced during HP treatment, which provided direct evidence that HP has the ability to penetrate enamel. Meanwhile, the results of laser-induced fluorescence revealed that HP caused the greatest fluorescence reduction. This suggested that the organic matter in enamel might be greatly affected by HP, which was also supported by the results of microhardness. It can be concluded, therefore, that the 30% HP may have adverse effects on the mineral and the organic matter of human tooth enamel.

This study presents the application of multivariate analyses to analyze micro-Raman spectral imaging data in reference to the adhesive/dentin interface as well as comparison with univariate analysis. The univariate statistical methods, such as mapping of specific functional group peak intensities, do not always detect functional group positions and quantities due to peak overlapping. A comprehensive chemical analysis of the adhesive/dentin interface, along with the multivariate statistical methods, principal component analysis, and fuzzy c-means clustering, is studied. Compared to univariate analysis, multivariate methods present the entire hyperspectral information from the specimen in a concise and uncorrelated way. Apart from the ease with which information can be extracted and presented, multivariate methods also highlight minute and often important variations in the spectra that are difficult to observe using univariate methods. The results show for the first time the clear chemical and structural classifications in the adhesive/dentin interface at successively greater resolutions.

To develop a fast and easy clinical method for glucose measurements on whole blood samples, changes in glucose spectra are investigated varying temperature, glucose concentration, and solvent using attenuated total reflection Fourier transform infrared (ATR- FTIR) measurements. The results show a stability of the spectra at different temperatures and wavelength shifts of the absorption bands when water is replaced by blood. Because the ATR measurements are influenced by sedimentation of the red blood cells, a two-wavelength CO₂ laser is used to determine the glucose concentration in whole blood samples. For this purpose, the first laser wavelength λ1 is tuned to the maximum of the glucose absorption band in blood at 1080 cm^-1, and the second laser wavelength λ2 is tuned to 950 cm^-1 for background measurements. The transmitted laser power through the optical cell containing the whole blood sample at λ1 and λ2 is used to determine the ratio. This signal correlates well with the glucose concentration in the whole blood samples. The CO₂ laser measurement is too fast to be influenced by the red blood cell sedimentation, and will be a suitable method for glucose determination in whole blood.

We present a study using plasmonic nanoparticles (NPs) to image epidermal growth factor receptor (EGFR) in live cells. Through detailed analysis of the NP scattering spectra, we determine the intracellular refractive index (RI) within attoliter volumes inside of the living cells. Molecular imaging is demonstrated using anti-EGFR labeled gold nanospheres delivered to cancer cells that overexpress EGFR, with targeted binding confirmed by appropriate control experiments. RI determination is achieved by measurement of the bound NPs' scattering spectra, acquired using a precision dark-field microspectroscopy system and through careful characterization of the NP properties throughout the immuno-labeling procedure. To demonstrate the effect of receptor-mediated uptake, the data are compared to similar spectral measurements using antibody-free NPs, taken up by the cells through nonspecific mechanisms. In these experiments, NP aggregation introduces interparticle effects in the scattering spectra, suggesting that EGFR-mediated internalization of NPs may provide an advantage for maintaining NP isolation upon uptake. The results of this study show the potential utility of dark-field microspectroscopy and labeled NPs for live cell imaging. By demonstrating RI sensitivity over nanometer length scales, this study also presents a potential new avenue for assessing the structure and dynamics of live cells.

Recently it has become possible to study single protein molecules in a cell. However, such experiments are plagued by rapid photobleaching. We recently showed that the interaction of fluorophores with localized surface plasmon polaritons (LSPs) induced in the metallic nanoparticles led to a substantial reduction of photobleaching. We now investigate whether the photobleaching could be further reduced when the excited fluorophore interacts with the LSP excited in the metallic nanoparticles resident on mirrored surface. As an example we use myofibrils, subcellular structures within skeletal muscle. We compare nanoparticle-enhanced fluorescence of myofibrils in the presence and in the absence of a mirrored surface. The proximity of the mirrored surface led to enhancement of fluorescence and to a decrease in fluorescent lifetime, much greater than that observed in the presence of nanoparticles alone. We think that the effect is caused by the near-field interactions between fluorophores and LSP, and between fluorophores and propagating surface plasmons (PSPs) produced in the metallic surface by the nanoparticles. Photobleaching is decreased because fluorescence enhancement enables illumination with a weaker laser beam and because the decrease in fluorescence lifetime minimizes the probability of oxygen attack during the time a molecule is in the exited state.

Fluorescence quantification in tissues using conventional techniques can be difficult due to the absorption and scattering of light in tissues. Our previous studies have shown that a single-mode optical fiber (SMF)–based, two-photon optical fiber fluorescence (TPOFF) probe could be effective as a minimally invasive, real-time technique for quantifying fluorescence in solid tumors. We report improved results with this technique using a solid, double-clad optical fiber (DCF). The DCF can maintain a high excitation rate by propagating ultrashort laser pulses down an inner single-mode core, while demonstrating improved collection efficiency by using a high–numerical aperture multimode outer core confined with a second clad. We have compared the TPOFF detection efficiency of the DCF versus the SMF with standard solutions of the generation 5 poly(amidoamine) dendrimer (G5) nanoparticles G5-6TAMRA (G5-6T) and G5-6TAMRA-folic acid (G5-6T-FA). The DCF probe showed three- to five-fold increases in the detection efficiency of these conjugates, in comparison to the SMF. We also demonstrate the applicability of the DCF to quantify the targeted uptake of G5-6T-FA in mouse tumors expressing the FA receptor. These results indicate that the TPOFF technique using the DCF probe is an appropriate tool to quantify low nanomolar concentrations of targeted fluorescent probes from deep tissue.

Indocyanine green (ICG) is a Federal Drug Administration-approved near-infrared imaging agent susceptible to chemical degradation, nonspecific binding to blood proteins, and rapid clearance from the body. In this study, we describe the encapsulation of ICG within polymeric micelles formed from poly(styrene-alt-maleic anhydride)-block-poly(styrene) (PSMA-b-PSTY) diblock copolymers to stabilize ICG for applications in near-infrared diagnostic imaging. In aqueous solution, the diblock copolymers self-assemble to form highly stable micelles approximately 55 nm in diameter with a critical micelle concentration (CMC) of ~1 mg/L. Hydrophobic ICG salts readily partition into the PSTY core of these micelles with high efficiency, and produce no change in micelle morphology or CMC. Once loaded in the micelle core, ICG is protected from aqueous and thermal degradation, with no significant decrease in fluorescence emission over 14 days at room temperature and retaining 63% of its original emission at 37°C. Free ICG does not release rapidly from the micelle core, with only 11% release over 24 h. The ICG-loaded micelles do not exhibit significant cell toxicity. This system has the potential to greatly improve near-infrared imaging in breast cancer detection by increasing the stability of ICG for formulation/administration, and by providing a means to target ICG to tumor tissue.

Abnormal microvasculature contributes to the pathophysiologic microenvironment of tumors. Understanding microvascular tumor oxygen transport is necessary to comprehend the factors that influence tumor biology, physiology, and therapy. Previously, we described an in vivo spectral imaging microscopy system for measurements of microvessel hemoglobin saturation (HbSat). We measure temporal fluctuations and spatial gradients in tumor microvessel oxygenation and identify instances of anastomoses between vessels with significantly different oxygenations. Slow periodic fluctuations in HbSat <0.2 cycles per minute were observed. These measurements are consistent with microelectrode measurements of fluctuating tumor oxygenation. Gradients in HbSat along individual tumor microvessels were measured that were larger in magnitude than normal tissue microvessels. Images were captured of anastomoses of tumor microvessels with diameters ≤100 μm and significantly different HbSat values (>20%). Shunting of inspired oxygen, presumably due to arteriovenous anastomoses, from tumor feeding arterioles to adjacent venules was imaged. This effect was confined to a region around the tumor and was not observed in nearby normal microvessels. Imaging measurements of tumor microvessel oxygen transport may offer insight to current questions regarding oxygen-related tumor biology and treatment responses, and spectral imaging may be a useful research tool in this regard.

We report on the development of a novel optical oxygen sensor for breath monitoring applications using the technique of phase fluorometry. The principal design criteria are that the system be compact, lightweight, and employ a disposable sensing element (while performing competitively with current commercial analyzers). The oxygen-sensitive, luminescent ruthenium complex Ru[dpp]₃²⁺ is encapsulated in a sol-gel matrix and deposited onto a custom-designed, polymer sensor chip that provides significantly improved luminescence capture efficiency. The performance of the sensor module is characterized using a commercially available lung simulator. A resolution of 0.03% O₂ is achieved, which compares well with commercial breath monitoring systems and, when combined with its immunity to humidity and ability to respond effectively across a broad range of breathing rates, makes this device an extremely promising candidate for the development of a practical, low-cost biodiagnostic tool.

The deposition of thin films of poly(D,L-lactide) (PDLLA) by using the matrix-assisted pulsed laser evaporation (MAPLE) technique is investigated. PDLLA is a highly biocompatible and biodegradable polymer, with wide applicability in the biomedical field. The laser wavelength used in the MAPLE process is optimized to obtain a good-quality deposition. The structure of the polymer film is analyzed by Fourier transform infrared spectroscopy (FTIR). It is found that the chemical structure of PDLLA undergoes little or no damage during deposition with near-infrared laser radiation (1064 nm). It is thus confirmed that at this wavelength, the MAPLE technique can be applied for fragile biopolymer molecules, which are easily damaged by other laser radiations (UV radiation). This method allows future development of tailored polymer coatings for biomedical applications.

Digital colposcopy is a promising technology for the detection of cervical intraepithelial neoplasia. Automated analysis of colposcopic images could provide an inexpensive alternative to existing screening tools. Our goal is to develop a diagnostic tool that can automatically identify neoplastic tissue from digital images. A multispectral digital colposcope (MDC) is used to acquire reflectance images of the cervix with white light before and after acetic-acid application in 29 patients. A diagnostic image analysis tool is developed to identify neoplasia in the digital images. The digital image analysis is performed in two steps. First, similar optical patterns are clustered together. Second, classification algorithms are used to determine the probability that these regions contain neoplastic tissue. The classification results of each patient's images are assessed relative to the gold standard of histopathology. Acetic acid induces changes in the intensity of reflected light as well as the ratio of green to red reflected light. These changes are used to differentiate high-grade squamous intraepithelial (HGSIL) and cancerous lesions from normal or low-grade squamous intraepithelial (LGSIL) tissue. We report diagnostic performance with a sensitivity of 79% and a specificity of 88%. We show that diagnostically useful digital images of the cervix can be obtained using a simple and inexpensive device, and that automated image analysis algorithms show a potential to identify histologically neoplastic tissue areas.

We explore the hypothesis that shape factors can be used as tools to probe disease progression in addition to being used for simple classification. We define and apply a new shape factor to digital images of tumor cross sections of progressive rat hepatoma. Using a number of standard pathological measures, each image is also associated with a "disease time," a continuous variable between 0 and 1 with 0 being disease initiation and 1 being a near-fatal condition. The images are converted to data files that represent the 2-D shapes of the tumors. Their shape factors are then determined and plotted versus disease time. The results show that the shape factor can indicate and localize the tumor structural phase transition that occurs between uniform growth and infiltration.