Intracoronary Raman spectroscopy could open new avenues for the study and management of coronary artery disease due to its potential to measure the chemical and molecular composition of coronary atherosclerotic lesions. We have fabricated and tested a 1.5-mm-diameter (4.5 Fr) Raman catheter capable of collecting Raman spectra in both the fingerprint (400–1800 cm^-1) and high-wavenumber (2400–3800 cm^-1) regions. Spectra were acquired in vivo, using a human-swine xenograft model, in which diseased human coronary arteries are grafted onto a living swine heart, replicating the disease and dynamic environment of the human circulatory system, including pulsatile flow and motion. Results show that distinct spectral differences, corresponding to the morphology and chemical composition of the artery wall, can be identified by intracoronary Raman spectroscopy in vivo.

Intravital imaging using confocal microscopy facilitates high-resolution studies of cellular and molecular events in vivo. We use this, complemented by Doppler optical coherence tomography (OCT), to assess blood flow in a mouse dorsal skin-fold window chamber model to image the response of individual blood vessels to localized photodynamic therapy (PDT). Specific fluorescent cell markers were used to assess the effect on the vascular endothelial cell lining of the treated vessels. A fluorescently tagged antibody against an endothelial transmembrane glycoprotein (CD31) was used to image endothelial cell integrity in the targeted blood vessel. A cell permeability (viability) indicator, SYTOX Orange, was also used to further assess damage to endothelial cells. A fluorescently labeled anti-CD41 antibody that binds to platelets was used to confirm platelet aggregation in the treated vessel. These optical techniques enable dynamic assessment of responses to PDT in vivo, at both the vascular endothelial cell and whole vessel levels.

The ability to perform high-resolution imaging of microvasculature and its oxygenation is very important in studying early tumor development. Toward this goal, we improved upon our excited state absorption (ESA)–based imaging technique to allow us to not only image hemoglobin directly but also differentiate between oxy- and deoxyhemoglobin in tissue. We demonstrate the separation of arterioles from venules in a live nude mouse ear using our imaging technique.

A functional Fourier domain optical coherence tomography instrument offering spectral Doppler imaging of in vivo pulsatile human retinal blood flow was constructed. An improved phase-resolved algorithm was developed to correct bulk motion artifacts. Spectral Doppler imaging provides complementary temporal flow information to the spatially distributed flow information of the color Doppler image by providing direct visualization of the Doppler spectrum of the flow whose pattern can be further quantified with various velocity envelope curves and their corresponding flow indices. The coefficient of repeatability on resistance index measurement was assessed by analyzing 14 measurements on two vessels within two normal subjects.

This PDF file contains the editorial “SPECIAL SECTION GUEST EDITORIAL” for JBO Vol. 13 Issue 04

A tutorial introduction to diffuse light transport is presented. The basic analytic equations of time-resolved, steady-state and modulated light transport are introduced. The perturbation method for handling slight heterogeneities in optical properties is outlined. The treatment of boundary conditions such as an air/tissue surface is described. Finite mesh-based numerical methods are introduced to calculate the diffuse light field in complex tissues with arbitrary boundaries. Applications in tissue spectroscopy and imaging illustrate these theoretical and computational tools.

We compare and contrast the development of optical molecular imaging techniques with nuclear medicine with a didactic emphasis for initiating readers into the field of molecular imaging. The nuclear imaging techniques of gamma scintigraphy, single-photon emission computed tomography, and positron emission tomography are first briefly reviewed. The molecular optical imaging techniques of bioluminescence and fluorescence using gene reporter/probes and gene reporters are described prior to introducing the governing factors of autofluorescence and excitation light leakage. The use of dual-labeled, near-infrared excitable and radio-labeled agents are described with comparative measurements between planar fluorescence and nuclear molecular imaging. The concept of time-independent and -dependent measurements is described with emphasis on integrating time-dependent measurements made in the frequency domain for 3-D tomography. Finally, we comment on the challenges and progress for translating near-infrared (NIR) molecular imaging agents for personalized medicine.

A novel scheme for fully scalable White Monte Carlo (WMC) has been developed and is used as a forward solver in the evaluation of experimental time-resolved spectroscopy. Previously reported scaling problems are avoided by storing detection events individually, turning spatial and temporal binning into post-simulation activities. The approach is suitable for modeling of both interstitial and noninvasive settings (i.e., infinite and semi-infinite geometries). Motivated by an interest in in vivo optical properties of human prostate tissue, we utilize WMC to explore the low albedo regime of time-domain photon migration—a regime where the diffusion approximation of radiative transport theory breaks down, leading to the risk of overestimating both reduced scattering (μ^′_s) and absorption (μ_a). Experimental work supports our findings and establishes the advantages of Monte Carlo–based evaluation.

Near-infrared (NIR) region-based spectroscopy is examined for accuracy with spectral recovery using frequency domain data at a discrete number of wavelengths, as compared to that with broadband continuous wave data. Data with more wavelengths in the frequency domain always produce superior quantitative spectroscopy results with reduced noise and error in the chromophore concentrations. Performance of the algorithm in the situation of doing region-guided spectroscopy within the MRI is also considered, and the issue of false positive prior regions being identified is examined to see the effect of added wavelengths. The results indicate that broadband frequency domain data are required for maximal accuracy. A broadband frequency domain experimental system was used to validate the predictions, using a mode-locked Ti:sapphire laser for the source between 690- and 850-nm wavelengths. The 80-MHz pulsed signal is heterodyned with photomultiplier tube detection, to lower frequency for data acquisition. Tissue-phantom experiments with known hemoglobin absorption and tissue-like scatter values are used to validate the system, using measurements every 10 nm. More wavelengths clearly provide superior quantification of total hemoglobin values. The system and algorithms developed here should provide an optimal way to quantify regions with the goal of image-guided breast tissue spectroscopy within the MRI.

Diffuse reflectance (DR) spectroscopy is a simple, low-cost, and noninvasive modality with potential for distinguishing oral precancer. Recently, in an ex vivo study, the DR spectral ratio (R545/R575) of oxygenated hemoglobin bands at 545 and 575 nm was used for grading malignancy. This work presents the results of clinical trials conducted in 29 patients to detect oral precancer using this ratio. We use site-specific normal spectra from a group of 36 healthy volunteers for comparison with those of patients. Toward this, in vivo DR spectra from 14 anatomical sites of the oral cavity of healthy volunteers are recorded on a miniature fiber optic spectrometer with white light excitation. The R545/R575 ratio is lowest for healthy tissues and appears to increase with the grade of malignancy. As compared to scatter plots that use the mean DR ratio from all anatomical sites, those using site-specific data show improved sensitivity and specificity for early diagnosis and grading of oral cancer. In the case of buccal mucosa, using scatter plots of R545/R575 ratio, we obtain a sensitivity of 100% and specificity of 86% for discriminating precancer (dysplasia) from hyperplasia, and a sensitivity of 97% and specificity of 86% for discriminating hyperplasia from normal.

We present the mathematical foundation and the experimental validation of a technique that utilizes pass-through (ballistic) photons in a partial coherence interferometric transillumination setup for biomedical analyses. We demonstrate that the implementation depends closely on tissue under test, incident power, spatial and spectral characteristics of the radiation source, and detection electronics. With the aid of the complex material coherence function concept, we foresee tissue characterization and diagnostic imaging as potential applications for the technique. We propose a normalization procedure for in vitro and in vivo measurements, where nontissue-related quantities are canceled out. The validation of the proposal is achieved by obtaining the sample coherence function of a tissue phantom. The expected exponential attenuation is confirmed, and the corresponding scattering coefficients are determined. A good agreement between theory and experiment, for the initial set of samples, serves to establish that pass-through photon-based transillumination is feasible for selected biomedical applications.

Red blood cells (RBCs) in the presence of plasma proteins or other macromolecules have a tendency to form aggregates. Light-scattering technique was used to investigate the RBC aggregation process. A highly diluted suspension of RBCs was illuminated with a 632.8-nm HeNe laser. Angular-resolved measurements of light intensity scattered by an RBC suspension from a 200-μm thick optical glass cuvette during 10 min of their aggregation process were performed at 1 to 4 off-axis deg with a very high angular resolution, at hematocrits in the range of 3.5·10^-2 to 10^-1. The angular spreading of forward-scattered light at small angles during the RBC aggregation process was described in terms of a new, effective phase function model that has been used for fitting the experimental data. The aggregated RBCs' optical properties, such as effective scattering anisotropy and scattering cross section, were determined. The results were compared with prediction of Mie theory for equivolumetric spherical particles. The time dependence of the aggregates mean radius and of the mean number of cells per aggregate was also calculated. Last, the potential of the proposed technique (forward-scattering light technique) as a new quantitative investigation of cellular aggregation process was estimated.

Separation of the two optical scattering properties, the scattering coefficient (μ_s) and the anisotropy of scattering (g), has been experimentally difficult in tissues. A new method for measuring these properties in tissues uses reflectance-mode confocal scanning laser microscopy (rCSLM). Experimentally, the focus at depth z is scanned down into the tissue. The measured data is the exponential decay of the confocal reflectance signal as a function of the depth of the focal volume, R(z)=ρ exp(-μz), summarized as a local reflectivity (ρ) and an exponential decay constant (μ). The ρ and μ map uniquely into the μs and g of the tissue. The method was applied to three mouse skin tissues: one wild-type (wt/wt), one heterozygous mutant (oim/wt), and one homozygous mutant (oim/oim), where oim indicates the mutation for osteogenesis imperfecta, a bone disease that affects type I collagen structure. The mutation affects the collagen fibrils of the skin and the assembly of collagen fiber bundles. The anisotropy of scattering (g) at 488 nm wavelength decreased from 0.81 to 0.46 with the added mutant allele. There was a slight increase in the scattering coefficient (μ_s) with the mutation from 74 to 94 cm^-1. The decrease in g (toward more isotropic scattering) is likely due to the failure of the mutant fibrils to assemble into the larger collagen fiber bundles that yield forward scattering.

A fluorescence diffuse tomography (FDT) setup for monitoring tumor growth in small animals has been created. In this setup an animal is scanned in the transilluminative configuration by a single source and detector pair. To remove stray light in the detection system, we used a combination of interferometric and absorption filters. To reduce the scanning time, an experimental animal was scanned using the following algorithm: (1) large-step scanning to obtain a general view of the animal (source and detector move synchronously); (2) selection of the fluorescing region; and (3) small-step scanning of the selected region and different relative shifts between the source and detector to obtain sufficient information for 3D reconstruction. We created a reconstruction algorithm based on the Holder norm to estimate the fluorophore distribution. This algorithm converges to the solution with a minimum number of fluorescing zones. The use of tumor cell lines transfected with fluorescent proteins allowed us to conduct intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Ibr, Mel-Kor, and human embryonic kidney HEK293 Phoenix were transfected with DsRed-Express and Turbo-RFP genes. The emission of red fluorescent proteins (RFPs) in the long-wave optical range permits detection of deep-seated tumors. In vivo experiments were conducted immediately after subcutaneous injection of fluorescing cells into small animals.

In vivo molecular fluorescence tomography of brain disease mouse models has two very specific demands on the optical setup: the use of pigmented furry mice does not allow for a purely noncontact setup, and a high spatial accuracy is required on the dorsal side of the animal due to the location of the brain. We present an optimized setup and tomographic scheme that meet these criteria through a combined CW reflectance-transmittance fiber illumination approach and a charge-coupled device contactless detection scheme. To consider the anatomy of the mouse head and take short source detector separations into account, the forward problem was evaluated by a Monte Carlo simulation input with a magnetic resonance image of the animal. We present an evaluation of reconstruction performance of the setup under three different condition. (i) Using a simulated dataset, with well-defined optical properties and low noise, the reconstructed position accuracy is below 0.5 mm. (ii) Using experimental data on a cylindrical tissue-simulating phantom with well-defined optical properties, a spatial accuracy of about 1 mm was found. (iii) Finally, on an animal model with a fluorescent inclusion in the brain, the target position was reconstructed with an accuracy of 1.6 mm.

Current techniques to assess lymph node metastases in cancer patients include lymphoscintigraphy after administration of a nonspecific radiocolloid in order to locate and resect lymph nodes for pathological examination of harbored cancer cells. Clinical trials involving intradermal or subcutaneous injection of antibody-based nuclear imaging agents have demonstrated the feasibility for target-specific, molecular imaging of cancer-positive lymph nodes. The basis for employing near-infrared (NIR) optical imaging for assessing disease is evidenced by recent work showing functional lymph imaging in mice, swine, and humans. We review antibody-based immunolymphoscintigraphy with an emphasis on the use of trastuzumab (or Herceptin) to target human epidermal growth factor receptor-2 (HER2) overexpressed in some breast cancers. Specifically, we review in vitro and preclinical imaging data from our laboratory that show how the dual-labeled agent (¹¹¹In-DTPA)_n-trastuzumab-(IRDye800)_m utilizes the high photon count provided by an NIR fluorescent dye, IRDye 800CW, and the radioactive signal from a gamma emitter, Indium-111, for possible detection of HER2 metastasis in lymph nodes. We show that the accumulation and clearance of (111In-DTPA)_n-trastuzumab-(IRDye800)_m from the axillary nodes of mice occurs 48 h after intradermal injection into the dorsal aspect of the foot. The requirement for long clearance times from normal, cancer-negative nodes presents challenges for nuclear imaging agents with limited half-lives but does not hamper NIR optical imaging.

Preferential tumor localization and the aggregation state of photosensitizers (PSs) can depend on the hydrophilic/hydrophobic nature of the molecule and affect their phototoxicity. In this study, three PSs of different hydrophilicity are introduced in liposomes to understand the structure-photochemistry relationship of PSs in this cellular model system. Absorbance and fluorescence spectra of amphiphilic aluminum (III) phthalocyanine disulfonate chloride adjacent isomer (Al-2), hydrophilic aluminum (III) phthalocyanine chloride tetrasulfonic acid (Al-4), and lipophilic 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH) are compared in a liposomal confined state with free PS in bulk solution. For fluorescence measurements, a broad range of concentrations of both bulk and liposomal confined PSs are examined to track the transition from monomers to dimers or higher order aggregates. Epifluorescence microscopy, absorbance, and fluorescence measurements all confirm different localization of the PSs in liposomes, depending on their hydrophilicity. In turn, the localization affects the aggregation of molecules inside the liposome cell model. Data obtained with such cellular models could be useful in optimizing the photochemical properties of photosensitizing drugs based on their structure-dependent interactions with cellular media and subcellular organelles.

The time-resolved fluorescence of photosensitizers (PSs) of varying hydrophobicities, di-and tetrasulfonated Al phthalocyanines (Al-2 and Al-4), and Photochlor® (HPPH), was investigated in liposomes used as cell-mimetic models. Using frequency-and time-domain apparatus, the fluorescence lifetime, τ fluo, was compared for PSs free in aqueous solution and in a liposome-associated state at varied temperatures (25 to 78°C) and oxygen concentrations (0–190 μM). The analysis of τ fluo revealed different decay behaviors for the free-solution and liposome-confined PSs, most significantly for the lipophilic HPPH. Hydrophilic PS drugs (Al-4, Al-2) were less affected by the liposomal confinement, depending on the relative hydrophilicity of the compound and the consequent localization in lipsomes. Changes in the emission decay due to confinement were detected as differences in the lifetime between the bulk solution and the liposome-localized PS in response to heating and deoxygenation. Specifically, hydrophilic Al-4 produced an identical lifetime trend as a function of temperature both in solu and in a liposome-confined state. Hydrophobic HPPH exhibited a fundamental transformation in its fluorescence decay kinetics, transitioning from a multiexponential (in free solution) to single-exponential (in liposome) decay. Deoxygenation resulted in a ubiquitous τ fluo increase for all PSs in free solution, while the opposite, a τ fluo decrease, occurred in all liposomal PSs.

In the crowded cellular milieu, biological processes require coordinated intermolecular interactions, conformational changes, and molecular transport that span a wide range of spatial and temporal scales. This complexity requires an integrated, noninvasive, multiscale experimental approach. Here, we develop a multimodal fluorescence microspectroscopy system, integrated on a single platform, to gain information about molecular interactions and their dynamics with high spatio-temporal resolution. To demonstrate the versatility of our experimental approach, we use rhodamine 123-labeled mitochondria in breast cancer cells (Hs578T), verified using differential interference contrast (DIC) and fluorescence (confocal and two-photon) microscopy, as a model system. We develop an assay to convert fluorescence intensity to actual concentrations in intact, individual living cells, which contrasts with conventional biochemical techniques that require cell lysates. In this assay, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) to quantify the fluorescence quantum yield variations found within individual cells. Functionally driven changes in cell environment, molecular conformation, and rotational diffusion are investigated using fluorescence polarization anisotropy imaging. Moreover, we quantify translational diffusion and chemical kinetics of large molecular assemblies using fluorescence correlation spectroscopy. Our integrated approach can be applied to a wide range of molecular and cellular processes, such as receptor-mediated signaling and metabolic activation.

Fluorescence lifetime imaging (FLIM) is very demanding from a technical and computational perspective, and the output is usually a compromise between acquisition/processing time and data accuracy and precision. We present a new approach to acquisition, analysis, and reconstruction of microscopic FLIM images by employing a digital micromirror device (DMD) as a spatial illuminator. In the first step, the whole field fluorescence image is collected by a color charge-coupled device (CCD) camera. Further qualitative spectral analysis and sample segmentation are performed to spatially distinguish between spectrally different regions on the sample. Next, the fluorescence of the sample is excited segment by segment, and fluorescence lifetimes are acquired with a photon counting technique. FLIM image reconstruction is performed by either raster scanning the sample or by directly accessing specific regions of interest. The unique features of the DMD illuminator allow the rapid on-line measurement of global good initial parameters (GIP), which are supplied to the first iteration of the fitting algorithm. As a consequence, a decrease of the computation time required to obtain a satisfactory quality-of-fit is achieved without compromising the accuracy and precision of the lifetime measurements.

A high content screening (HCS) system for fluorescence measurements at surfaces, in particular the plasma membrane of living cells, is described. The method is based on multiple total internal reflections (TIRs) of an incident laser beam within the glass bottom of a microtiter plate such that up to 96 individual samples could be illuminated by an evanescent electromagnetic field. Fluorescence lifetimes and time-resolved fluorescence anisotropies of these samples were assessed. While fluorescence lifetime represents a general measure for the interaction of a marker molecule with its microenvironment, the rotational diffusion time corresponds to the relaxation time of a molecule from a position with a defined orientation into a position with an arbitrary orientation. Thus, time-resolved fluorescence anisotropy reflects the viscosity of the microenvironment, i.e., membrane fluidity in the case of living cells. For all measurements in this study, either human glioblastoma cells incubated with the fluorescent membrane marker NBD-cholesterol or human breast cancer cells expressing a membrane-associating fluorescent protein were used.

Drosophila melanogaster larva myocytes are imaged with second harmonic generation (SHG) microscopy undergoing forced stretching and rhythmic contractions to determine the nature of the SHG signal. During stretching, double peaked SHG profiles of the anisotropic (A-) bands evolve into single peaks with a higher SHG intensity. The dip in the intensity profile at the center of the A-band is attributed to destructive interference from out-of-phase second harmonic radiating myosin molecules that, in the central region of myofilaments, are arranged antiparallel. An intensity increase at the center of the A-band appears during forced stretching due to a small, less than 100 nm, intermyofilament separation of the antiparallel myosin molecules leading to constructive interference of the SHG radiation. In addition, the same phenomenon occurs during periodic contractions of the myocyte, where an SHG intensity increase with the lengthening of sarcomeres is observed. The SHG intensity dependence on sarcomere length can be used for imaging myocyte contractions with low resolution microscopy, and can be applied for the development of diagnostic tools where monitoring of muscle contraction dynamics is required.

We demonstrate enhanced sensitivity in two-photon flow cytometry with an extended cavity laser excitation source. At low power, the home-built 20-MHz oscillator was able to detect a significantly larger fraction, in either phosphate buffered saline (PBS) or whole blood, of green fluorescent protein (GFP)–expressing MCA-207 cells cross-labeled with the membrane-binding lipophilic dye DiD. A geometrical model is used to explain unique features of the signals resulting from the different spatial distribution of DiD and GFP. These unique features include sub-square law scaling of unsaturated two-photon signal, a sigmoidal sensitivity curve for detection under varying powers for cell detection thresholds as low as a single photon, and uncorrelated signal strengths in two detection channels.

New imaging techniques using near-infrared (NIR) femtosecond lasers (fs-lasers) in multiphoton laser scanning microscopy (MPLSM) have great potential for in vivo applications, particularly in human skin. However, little is known about possible risks. In order to evaluate the risk, a “biological dosimeter” was used. We irradiated fresh human skin samples with both an fs-laser and a solar simulator UV source (SSU). DNA damage introduced in the epidermis was evaluated using fluorescent antibodies against cyclobutane-pyrimidin-dimers (CPDs) in combination with immunofluorescence image analysis. Four fs-irradiation regimes (at 800-nm wavelength) were evaluated differing in laser power and step width of horizontal scans. Fs-irradiation did not give CPDs at 15-mW or 30-mW irradiation power using 10 horizontal scans every 5 microns. CPDs could be seen at 60-mW laser power and 5-µm step size and at 35-mW using 1-micron step width. Quantitative comparison of SSU-induced CPDs showed that the 60-mW laser irradiation regime is comparable to UV-irradiation, giving 0.6 minimal erythemal dose (MED). The 1-micron irradiation regime was comparable to 0.45 MED. Under these experimental conditions, the risk of DNA damage due to fs-laser irradiation on skin is in the range of natural UV-exposure.

An automated procedure for detecting breast cancer using near-infrared (NIR) tomographic images is presented. This classification procedure automatically extracts attributes from three imaging parameters obtained by an NIR imaging system. These parameters include tissue absorption and reduced scattering coefficients, as well as a tissue refractive index obtained by a phase-contrast-based reconstruction approach. A support vector machine (SVM) classifier is utilized to distinguish the malignant from the benign lesions using the automatically extracted attributes. The classification results of in vivo tomographic images from 35 breast masses using absorption, scattering, and refractive index attributes demonstrate high sensitivity, specificity, and overall accuracy of 81.8%, 91.7%, and 88.6% respectively, while the classification sensitivity, specificity, and overall accuracy are 63.6%, 83.3%, and 77.1%, respectively, when only the absorption and scattering attributes are used. Furthermore, the automated classification procedure provides significantly improved specificity and overall accuracy for breast cancer detection compared to those by an experienced technician through visual examination.

Shallow lesions less than 1.5-cm deep are frequently seen in breast patients when they are scanned in reflection geometry. Two boundary conditions are compared for imaging shallow lesions, and a new probe design is introduced. A partial reflection boundary condition is suitable for imaging shallow lesions less than 1.0-cm deep; whereas an absorption boundary condition is desirable for imaging lesions more than 1.5-cm deep. Our new probe design incorporates either a partial reflection boundary or an absorption boundary based on a priori knowledge of lesion depth provided by coregistered real-time ultrasound images. An angled source is introduced to further improve the illumination of the region between 1.0- to 1.5-cm depths. Simulation, phantom, and freshly excised mouse tumor experiments demonstrate that targets located at different depths can be uniformly reconstructed. A clinical example is given to demonstrate the utility of this new approach for optimally probing lesions located at different depths.

The purpose of this study is to investigate the reduced scattering coefficient of C6 glioma by the near-infrared (NIR) technique. Light scattering properties of C6 glioma in brain tissue is measured by NIR spectroscopy within the wavelength range from 700 to 850 nm. C6 gliomas were implanted in rats' right brains. The scattering properties of the left and right target corresponding to the position of normal and tumor tissue were measured by a bifurcated needle probe on postoperative days 3, 10, and 17. The results show that there was no significant difference in reduced scattering coefficient between left and right brain tissue at postoperative day 3, but significant decreases were found between left and right brains at postoperative days 10 and 17. This study proved our initial hypothesis that the NIR technique may have a potential for clinical application in brain µglioma diagnosis.

The diffuse fundus reflectance and the spectral transmittance of the swine sensory retina was measured in vivo using intravitreal illumination. Pars plana vitrectomy and intravitreal manipulations were performed on a female American Yorkshire domestic swine. Light from a scanning monochromator was coupled into a fiber optic intraocular illuminator inserted into the vitreous. A 1.93-mm² region of the illuminated fundus was imaged from an oblique illumination angle. Multispectral retinal images were acquired for four experimental conditions: the eye (1) prior to vitrectomy, (2) after vitrectomy, (3) after insertion of a Spectralon disk super-retinally, and (4) after subretinal insertion of the disk. The absorption of melanin and hemoglobin in the red wavelengths was used to convert relative spectral reflectance to absolute reflectance. The flux scattered from the super-retinal Spectralon was used to correct for scattering in the globe. The transmittance of the sensory retina was measured in vivo using the scatter corrected subretinal Spectralon disk reflectance. The hemoglobin and melanin components of the spectrum due to scattered light were removed from the retinal transmission spectrum. The in vivo spectral transmittance of the sensory retina in this swine was essentially flat across the visible spectrum, with an average transmittance >90%.

Changes of optical properties of wound tissue in hairless rats were quantified by diffuse photon density wave methodology at near-infrared frequencies. The diffusion equation for semi-infinite media was used to calculate the absorption and scattering coefficients based on measurements of phase and amplitude with a frequency domain device. There was an increase in the absorption and scattering coefficients and a decrease in blood saturation of the wounds compared with the nonwounded sites. The changes correlated with the healing stage of the wound. The data obtained were supported by immunohistochemical analysis of wound tissue. These results verified now by two independent animal studies could suggest a noninvasive method to detect the progress of wound healing.

We describe a multimodality imaging approach that combines x-ray tomosynthesis with near-infrared diffuse optical tomography (DOT) for high-resolution imaging of osteoarthritis in the finger joints. In this approach, we take advantage of high resolution x-ray imaging particularly of the bones and incorporate the fine structural maps obtained from x ray as a priori information into DOT reconstructions. To realize this multi-modality approach, we constructed a hybrid imaging platform that integrated a C-arm–based x-ray tomosynthetic system with a multichannel optic-fiber–based DOT system. We also implemented improved hybrid regularization-based reconstruction algorithms that have shown to be especially effective for high-resolution modality-guided DOT. Initial evaluation of our x-ray–guided DOT reconstruction approach in the finger joints shows that spatial resolution of DOT can be enhanced dramatically when the three-dimensional geometry of bones is known a priori. In particular, the improved quantitative capability of imaging absorption and scattering coefficients of the joint tissues allows for more accurate diagnosis of osteoarthritis over x-ray radiography or DOT alone.

Real-time investigation of cerebral blood flow (CBF), and oxy- and deoxyhemoglobin concentration (HbO, HbR) dynamics has been difficult until recently due to limited spatial and temporal resolution of techniques like laser Doppler flowmetry and magnetic resonance imaging (MRI). The combination of laser speckle flowmetry (LSF) and multispectral reflectance imaging (MSRI) yields high-resolution spatiotemporal maps of hemodynamic and metabolic changes in response to functional cortical activation. During acute focal cerebral ischemia, changes in HbO and HbR are much larger than in functional activation, resulting in the failure of the Beer-Lambert approximation to yield accurate results. We describe the use of simultaneous LSF and MSRI, using a nonlinear Monte Carlo fitting technique, to record rapid changes in CBF, HbO, HbR, and cerebral metabolic rate of oxygen (CMRO₂) during acute focal cerebral ischemia induced by distal middle cerebral artery occlusion (dMCAO) and reperfusion. This technique captures CBF and CMRO₂ changes during hemodynamic and metabolic events with high temporal and spatial resolution through the intact skull and demonstrates the utility of simultaneous LSF and MSRI in mouse models of cerebrovascular disease.

The discovery of new fluorescent proteins (FPs) that emit in the far-red part of the spectrum, where light absorption from tissue is significantly lower than in the visible, offers the possibility for noninvasive biological interrogation at the entire organ or small animal level in vivo. The performance of FPs in deep-tissue imaging depends not only on their optical characteristics, but also on the wavelength-dependent tissue absorption and the depth of the fluorescence activity. To determine the optimal choice of FP and illumination wavelength, we compared the performance of five of the most promising FPs: tdTomato, mCherry, mRaspberry, mPlum, and Katushka. We experimentally measured the signal strength through mice and employed theoretical predictions to obtain an understanding of the performance of different illumination scenarios, especially as they pertain to tomographic imaging. It was found that the appropriate combination of red-shifted proteins and illumination wavelengths can improve detection sensitivity in small animals by at least two orders of magnitude compared with green FP. It is also shown that the steep attenuation change of the hemoglobin spectrum around the 600-nm range may significantly affect the detection sensitivity and, therefore, necessitates the careful selection of illumination wavelengths for optimal imaging performance.

Photoacoustic tomography (PAT) is applied to image the brain cortex of a monkey through the intact scalp and skull ex vivo. The reconstructed PAT image shows the major blood vessels on the monkey brain cortex. For comparison, the brain cortex is imaged without the scalp, and then imaged again without the scalp and skull. Ultrasound attenuation through the skull is also measured at various incidence angles. This study demonstrates that PAT of the brain cortex is capable of surviving the ultrasound signal attenuation and distortion caused by a relatively thick skull.

The direct simultaneous acquisition of coherent imaging and strain information is of particular importance in the biomechanical characterization of biological tissue. This type of simultaneous information acquisition can be accomplished using a coupled photorefractive holography and shearography system for imaging and strain measurements, respectively. Optical scattering in a conventional speckle shearing interferometer rapidly reduces the contrast of the shearing fringes, thereby limiting the use of such interferometers with opaque surfaces. By coupling photorefractive holography with speckle shearing interferometry, properties of the photorefractive effect (spatial high-pass filtering and temporal low-pass filtering) combine to restore the shearing fringe contrast and enable strain imaging in diffusing media. This effect is demonstrated using synthetic scattering phantoms built from suspensions of silica spheres in water.

Polarization-sensitive optical coherence tomography (PS-OCT) and near-infrared (NIR) imaging are promising new technologies under development for monitoring early carious lesions. Fluorosis is a growing problem in the United States, and the more prevalent mild fluorosis can be visually mistaken for early enamel demineralization. Unfortunately, there is little quantitative information available regarding the differences in optical properties of sound enamel, enamel developmental defects, and caries. Thirty extracted human teeth with various degrees of suspected fluorosis were imaged using PS-OCT and NIR. An InGaAs camera and a NIR diode laser were used to measure the optical attenuation through transverse tooth sections (~200 μm). A digital microradiography system was used to quantify the enamel defect severity by measurement of the relative mineral loss for comparison with optical scattering measurements. Developmental defects were clearly visible in the polarization-resolved OCT images, demonstrating that PS-OCT can be used to nondestructively measure the depth and possible severity of the defects. Enamel defects on whole teeth that could be imaged with high contrast with visible light were transparent in the NIR. This study suggests that PS-OCT and NIR methods may potentially be used as tools to assess the severity and extent of enamel defects.

We demonstrate the ex vivo imaging of an oral cancerous sample with a swept-source optical coherence tomography (SS-OCT) system. With the axial resolution of 8 µm in free space and system sensitivity of 108 dB, we can well distinguish the normal and abnormal tissue portions in a sample. In particular, we analyze the lateral variation of A-scan profiles to show two parameters of SS-OCT signal for delineating an oral cancer lesion. One of the parameters is the decay constant in the exponential fitting of the SS-OCT signal intensity along depth. This decay constant decreases as the A-scan point moves laterally across the margin of a lesion. The other parameter is the standard deviation of the SS-OCT signal intensity fluctuation in an A-scan. This parameter increases significantly when the A-scan point is moved across the transition region between the normal and abnormal portions. Such parameters are useful for determining the margins of oral cancer.

Optical coherence microscopy (OCM) is an interferometric technique that combines principles of confocal microscopy and optical coherence tomography (OCT) to obtain high-resolution en face images. Axial and lateral resolutions of several microns can be achieved using OCM depending on the numerical aperture (NA) of the objective and sample properties. We address the computational complexity that is inherent in spectral-domain OCM systems that limits its real-time capability as a microscope. An architecture that will improve the efficiency of the computation involved is presented. Currently, spectral-domain OCM images are obtained by individually taking the Fourier transform of each axial scan in cross-sectional frames and computationally slicing them to generate en face images. The real-time architecture presented here relies on the fact that only one Fourier domain point of a given axial scan needs to be computed rather than computing all the Fourier domain points, which can frequently require a significant amount of time to compute. This new realization has been shown to reduce the processing time to obtain the en face OCM images by a factor of 30.

Experimental proof-of-concept is presented for a quasi-holographic solution to polarization-sensitive optical coherence tomography (PS OCT). Due to decoupling between the reference and sample beams by polarization, the solution seems acceptable to acquisition and communication of optical data in the nonlaboratory environment. The nonlab environment implies uncontrollable disturbances, e.g., temperature changes and mechanical effects happening under shop testing in industry or routine examinations in common clinics and hospitals. For mapping the collagen-related depolarization ratio of light backscattered from the human dermis, a phenomenological model is evolved from the theory of light depolarization in crystalline polymers. The model yielded a simplified intensity-based estimation algorithm. The design concept and the model rely on a submillimeter tumor thickness as a proofed prognostic factor and an important criterion for complementary functional diagnostics of skin cancers in their early phase. Choice of the model is inspired by similarity of structural and optical properties between liquid-crystal collagen fibers in the dermis and birefringent crystalline lamellae in some polymer materials. The model gives a plausible interpretation of a peculiarity of cumulative birefringence in the abnormal skin dermis. Following a top-down approach to design, the authors attempt to contribute to bridging the gap between practitioners' concerns and academic studies.

A new approach of acquiring quasi-simultaneous optical coherence tomography (OCT) and confocal images is presented. The two images are generated using different principles, OCT and confocal microscopy. When the system is used to image the retina, the two images have depth resolutions, at present, of <20 μm and ~1 mm, respectively. The acquisition and display of en face OCT and confocal images are quasi-simultaneous, without the need of a beamsplitter. By using a chopper to periodically obstruct the reference beam in the OCT interferometer, synchronized with the XY-transversal scanner, much higher acquisition speed is obtained than in a previous report where we flipped an opaque screen in the reference arm of the interferometer. Successful operation of the novel configuration was achieved by: (1) stable synchronization of the chopper's movement with the horizontal line scanner and (2) fast self-adjusting of the gain value of avalanche photodiodes, depending on the optical power. Images from coin, leaves, and retina in vivo have been collected to demonstrate the functionality of the system.

We describe the design and operation of a multispectral confocal microendoscope. This fiber-based fluorescence imaging system consists of a slit-scan confocal microscope coupled to an imaging catheter that is designed to be minimally invasive and allow for cellular level imaging in vivo. The system can operate in two imaging modes. The grayscale mode of operation provides high resolution real-time in vivo images showing the intensity of fluorescent signal from the specimen. The multispectral mode of operation uses a prism as a dispersive element to collect a full multispectral image of the fluorescence emission. The instrument can switch back and forth nearly instantaneously between the two imaging modes (less than half a second). In the current configuration, the multispectral confocal microendoscope achieves 3-μm lateral resolution and 30-μm axial resolution. The system records light from 500 to 750 nm, and the minimum resolvable wavelength difference varies from 2.9 to 8.3 nm over this spectral range. Grayscale and multispectral imaging results from ex-vivo human tissues and small animal tissues are presented.

Failure of endodontic treatment is commonly associated with the presence of Enterococcus faecalis. Studies have highlighted that E. faecalis can form a calcified biofilm in tough environmental conditions, such as within root canals. The aims of this study were to investigate the effects of chemicals used in root-canal disinfection on the adherence of E. faecalis to collagen, as well as to estimate the force of adhesion between E.faecalis and collagen after such treatment. The number of adhering bacteria after chemical treatment was determined using confocal laser scanning microscopy–based adherence assay. It was found that the calcium hydroxide–treated group had a statistically significant (p=0.05) increase in the population of bacteria adhering. The adhesion force between bacteria and collagen of the treatment group with the highest number of bacteria adhering was determined by using optical tweezers (1064 nm) and Equipartitition theorem-based stiffness measurements. The presence of calcium hydroxide was found to significantly increase the bacterium-collagen adhesion force. These experiments highlighted the potential advantage of using optical tweezers to study bacteria-substrate interactions. The findings from the present study suggests that the presence of calcium hydroxide increased the adhesion force and adherence of E. faecalis to type-I collagen.

Determining the health of muscle cells by in vivo imaging could impact the diagnosis and monitoring of a large number of congenital and acquired muscular or cardiac disorders. However, currently used technologies are hampered by insufficient resolution, lack of specificity, or invasiveness. We have combined intrinsic optical second-harmonic generation from sarcomeric myosin with a novel mathematical treatment of striation pattern analysis, to obtain measures of muscle contractile integrity that correlate strongly with the neuromuscular health of mice suffering from genetic, acquired, and age-related decline in skeletal muscle function. Analysis of biopsies from a pilot group of human volunteers suggests a similar power in quantifying sarcopenic changes in muscle integrity. These results provide the first strong evidence that quantitative image analysis of sarcomere pattern can be correlated with physiological function, and they invite the application of SHG imaging in clinical practice, either in biopsy samples or via microendoscopy.

Live tissue nonlinear microscopy based on multiphoton autofluorescence and second harmonic emission originating from endogenous fluorophores and noncentrosymmetric-structured proteins is rapidly gaining interest in biomedical applications. The advantage of this technique includes high imaging penetration depth and minimal phototoxic effects on tissues. Because fluorescent dyes are not used, discrimination between different components within the tissue is challenging. We have developed a nonlinear spectral imaging microscope based on a home-built multiphoton microscope, a prism spectrograph, and a high-sensitivity CCD camera for detection. The sensitivity of the microscope was optimized for autofluorescence and second harmonic imaging over a broad wavelength range. Importantly, the spectrograph lacks an entrance aperture; this improves the detection efficiency at deeper lying layers in the specimen. Application to the imaging of ex vivo and in vivo mouse skin tissues showed clear differences in spectral emission between skin tissue layers as well as biochemically different tissue components. Acceptable spectral images could be recorded up to an imaging depth of ~100 μm.

Second harmonic generation (SHG) and two-photon fluorescence (TPF) microscopy is used to image the intercellular and pericellular matrix in normal and degenerate equine articular cartilage. The polarization sensitivity of SHG can be used directly to determine fiber orientation in the superficial 10 to 20 μm of tissue, and images of the ratio of intensities taken with two orthogonal polarization states reveal small scale variations in the collagen fiber organization that have not previously been reported. The signal from greater depths is influenced by the birefringence and biattenuance of the overlying tissue. An assessment of these effects is developed, based on the analysis of changes in TPF polarization with depth, and the approach is validated in tendon where composition is independent of depth. The analysis places an upper bound on the biattenuance of tendon of 2.65×10^-4. Normal cartilage reveals a consistent pattern of variation in fibril orientation with depth. In lesions, the pattern is severely disrupted and there are changes in the pericellular matrix, even at the periphery where the tissue appears microscopically normal. Quantification of polarization sensitivity changes with depth in cartilage will require detailed numerical models, but in the meantime, multiphoton microscopy provides sensitive indications of matrix changes in cartilage degeneration.

Theileria annulata is an intracellular parasite that infects and transforms bovine leukocytes, inducing continuous proliferation of its host cell both in vivo and in vitro. Theileria-infected cells can easily be propagated in the laboratory and serve as a good model for laser ablation studies. Using single pulses from an amplified ultrashort pulse laser system, we developed a technique to introduce submicrometer holes in the plasma membrane of the intracellular schizont stage of Theileria annulata. This was achieved without compromising either the viability of the organisms or that of the host cell that harbors the parasite in its cytoplasm. Multiphoton microscopy was used to generate image stacks of the parasite before and after ablation. The high axial resolution allowed precise selection of the region of the membrane that was ablated. It also allowed observation of the size of the holes generated (in fixed, stained cells) and determination of the structural changes in the parasite resulting from the laser pulses (in living cells in vitro). This technique opens a new possibility for the transfection of Theileria or delivery of molecules to the schizont that may prove useful in the study of this special host-parasite relationship.

We used two-photon laser scanning microscopy (TPLSM) to demonstrate for the first time its potential in studying relational details at the cellular level of atherogenesis in intact, viable mouse carotid arteries. Isolated and mounted arteries of ApoE-/-mice, aged 15 or 21 weeks (7 and 13 weeks on western diet), were imaged after labeling with specific fluorescent markers for cell nuclei, inflammatory cells, collagen, and lipids. Data were compared with C57BL6/J mice fed a chow diet. Control vessels had intact endothelium without adhering blood cells or significant intimal collagen labeling. In ApoE-/-mice already at 15 weeks, inflammatory cells adhered to the endothelium and increased labeling of collagen was observed in tunica intima at both lesion-prone and non-lesion-prone sites, indicating endothelium activation. In plaques, internalized inflammatory cell density increased with age and plaque progression in tunicae adventitia and intima, but not media. In the whole plaque, aging or plaque progression did not alter the direct relationship between inflammatory cells and collagen. However, within the fibrous caps specifically, direct contact between inflammatory cells and collagen increased with age. This study demonstrates the potential of TPLSM in determining detailed information regarding the complex relationship between inflammatory cells and collagen during atherogenesis.

The effects of off-axis optical aberration in multiphoton microscopy and the resulting lateral and axial image inhomogeneity are investigated. The lateral inhomogeneity of the scanning field is demonstrated by second harmonic generation (SHG) imaging of fasciae and two-photon fluorescence (TPF) microscopy of thin fluorescent samples. Furthermore, refractive index mismatch-caused intensity attenuation of the TPF signal at central and peripheral regions of the scanning frame is measured using homogeneous 10-μM sulforhodamine B samples with refractive indexes of 1.33 and around 1.465. In addition to characterizing image field convexity, we also found that image resolution degrades away from the optical axis. These effects need to be accounted for in both qualitative and quantitative multphoton imaging applications.

We present a multifunctional darkfield microscopy using an axicon. It combines the functions of a darkfield microscope, fluorescence microscope, and microspectrophotometer in one platform. The advantage of the system over conventional darkfield microscopy includes the high transmittance of the illuminating flux, the high contrast of the image, and the convenient toggle between darkfield and brightfield microscopy. Examples of dark, bright, and fluorescent micrographs as well as concerned spectra of microsized specimens implemented in this apparatus are demonstrated.

We report on the development of a compact commercial instrument for measuring carotenoids in skin tissue. The instrument uses two light-emitting diodes (LEDs) for dual-wavelength excitation and four photomultiplier tubes for multichannel detection. Bandpass filters are used to select the excitation detection wavelengths. The f/1.3 optical system has high optical throughput and single photon sensitivity, both of which are crucial in LED-based Raman measurements. We employ a signal processing technique that compensates for detector drift and error. The sensitivity and reproducibility of the LED Raman instrument compares favorably to laser-based Raman spectrometers. This compact, portable instrument is used for noninvasive measurement of carotenoid molecules in human skin with a repeatability better than 10%.

We demonstrate dynamic imaging of molecular distribution in unstained living cells using Raman scattering. By combining slit-scanning detection and optimizing the excitation wavelength, we imaged the dynamic molecular distributions of cytochrome c, protein beta sheets, and lipids in unstained HeLa cells with a temporal resolution of 3 minutes. We found that 532-nm excitation can be used to generate strong Raman scattering signals and to suppress autofluorescence that typically obscures Raman signals. With this technique, we reveal time-resolved distributions of cytochrome c and other biomolecules in living cells in the process of cytokinesis without the need for fluorescent labels or markers.

Variation in the level of the carotenoid antioxidant substances beta-carotene and lycopene in the human skin of ten healthy volunteers was measured with resonance Raman spectroscopy in an in vivo experiment over the course of 12 months. Information on the lifestyle of the volunteers concerning dietary supplementation and stress factors was obtained daily by the completion of questionnaires. The results showed individual variations in the levels of carotenoid antioxidant substances in the skin of the volunteers, which strongly correlated to specific lifestyles, such as the intake of dietary supplementations rich in carotenoids, and the influence of stress factors. A carotenoid-rich nutrition, based on large amounts of fruit and vegetables, increased the measured carotenoid levels of skin, while stress factors such as fatigue, illness, smoking, and alcohol consumption gave rise to a decrease in carotenoid levels of the skin. These decreases occured relatively quickly over the course of one day, while the subsequent increases lasted for up to 3 days. During the summer and autumn months, an increase in the level of carotenoids in the skin was measured for all volunteers. The average "seasonal increase" of the carotenoid content in the skin was determined to be 1.26-fold.

Ultraviolet A (UVA) radiation has been known to generate reactive oxygen species, such as singlet oxygen, in skin, leading to the oxidation of lipids and proteins. This oxidation influences cellular metabolism and can trigger cellular signaling cascades, since cellular membranes and the stratum corneum contain a substantial amount of fatty acids and lipids. Using highly sensitive IR-photomultiplier technology, we investigated the generation of singlet oxygen by fatty acids and lipids. In combination with their oxidized products, the fatty acids or lipids produced singlet oxygen under UVA radiation at 355 nm that is directly shown by luminescence detection. Linolenic or arachidonic acid showed the strongest luminescence signals, followed by linoleic acid and docohexaenoic acid. The amount of singlet oxygen induced by lipids such as phosphatidylcholine was significantly higher compared to the corresponding fatty acids within phospholipids. This result indicates a synergistic process of oxygen radicals and singlet oxygen during irradiation. UVA radiation initiates singlet oxygen generation, which subsequently oxidizes other fatty acids that in turn produce additional singlet oxygen. This leads to an enhancement of UVA-induced damage of fatty acids and lipids, which must enhance the oxidative damages in cells.

The wavelength-dependent penetration depth of ultraviolet radiation in human skin is a fundamental parameter for the estimation of the possible photobiological impact of ultraviolet (UV) radiation. We have determined the absorption spectra of human skin in vivo in the wavelength range from 290 to 341 nm in 3-nm steps using laser optoacoustics and calculated the respective penetration depths. Data were analyzed with respect to different skin regions and skin phototype of the 20 subjects in the study (phototype I: n=3; II: n=7; III: n=5; IV: n=5), revealing large variability between individuals. The penetration depth of UV radiation in human skin is highly dependent on wavelength and skin area, but no significant dependence on skin phototype could be found.

Monitoring of the tissue response to photodynamic therapy (PDT) can provide important information to help optimize treatment variables such as drug and light dose, and possibly predict treatment outcome. A urinary bladder cancer cell line (AY-27) was used to induce orthotopic transitional cell carcinomas (TCC) in female Fischer rats, and hexyl 5-aminolevulinate (HAL, 8 mM, 1 h)-induced PDT was performed on day 14 after instillation of the cancer cells (20 J/cm² fluence at 635 nm). In vivo optical reflectance and fluorescence spectra were recorded from bladders before and after laser treatment with a fiberoptic probe. Calculated fluorescence bleaching and oxygen saturation in the bladder wall were examined and correlated to histology results. Reflectance spectra were analyzed using a three-layer optical photon transport model. Animals with TCC treated with PDT showed a clear treatment response; decreased tissue oxygenation and protoporphyrin IX (PpIX) fluorescence photobleaching were observed. Histology demonstrated that 3 of 6 animals with treatment had no sign of the tumor 7 days after PDT treatment. The other 3 animals had significantly reduced the tumor size. The most treatment-responsive animals had the highest PpIX fluorescence prior to light irradiation. Thus, optical spectroscopy can provide useful information for PDT. The model has proved to be very suitable for bladder cancer studies.

Numerous studies have shown that the use of proteinic solders during laser-assisted vascular anastomosis (LAVA) and repair (LAVR) can significantly increase welding strength, but these studies combined solder-mediated LAVA/R with the use of stay sutures, thereby defeating its purpose. In an in vitro study, we examined the leaking point pressures (LPPs) and histological damage profile of porcine carotid arteries following albumin solder-mediated CO₂ LAVR without the use of sutures. Longitudinal arteriotomies (9.1±0.8 mm in length) were sheathed with 25% liquid bovine serum albumin solder, and LAVR was performed using a micromanipulator-controlled CO₂ laser operating at 170-mW power and 1.25-mm spot size in continuous wave mode. The welding regime consisted of a transversal zigzag pass followed by one or two longitudinal zigzag passes, producing an irradiance of 13.9 W/cm² and energies of 10.5 J and 11.3 J per mm weld, respectively. LPPs were measured by the fluid infusion technique, and histological analysis was performed with light, fluorescence, and polarization microscopy. The LPP of the two-pass welds was 351±158 mmHg versus 538±155 mmHg for the three-pass welds. Thermal damage was confined primarily to the adventitial layers, with limited heat diffusion into the media below the solder around the coaptation interface.

Temperature distribution is a crucial factor in determining the outcome of laser phototherapy in cancer treatment. Magnetic resonance imaging (MRI) is an ideal method for 3-D noninvasive temperature measurement. A 7.1-T MRI was used to determine laser-induced high thermal gradient temperature distribution of target tissue with high spatial resolution. Using a proton density phase shift method, thermal mapping is validated for in vivo thermal measurement with light-absorbing enhancement dye. Tissue-simulating phantom gels, biological tissues, and tumor-bearing animals were used in the experiments. An 805-nm laser was used to irradiate the samples, with laser power in the range of 1 to 3 W. A clear temperature distribution matrix within the target and surrounding tissue was obtained with a specially developed processing algorithm. The temperature mapping showed that the selective laser photothermal effect could result in temperature elevation in a range of 10 to 45°C. The temperature resolution of the measurement was about 0.37°C with 0.4-mm spatial resolution. The results of this study provide in vivo thermal information and future reference for optimizing laser dosage and dye concentration in cancer treatment.

Currently available biomechanical models of the human eyeball focus mainly on the geometries and material properties of its components while little attention has been given to its optics—the eye's primary function. We postulate that in the evolution process, the mechanical structure of the eyeball has been influenced by its optical functions. We develop a numerical finite element analysis-based model in which the eyeball geometry and its material properties are linked to the optical functions of the eye. This is achieved by controlling in the model all essential optical functions while still choosing material properties from a range of clinically available data. In particular, it is assumed that in a certain range of intraocular pressures, the eye is able to maintain focus. This so-called property of optical self-adjustments provides a more constrained set of numerical solutions in which the number of free model parameters significantly decreases, leading to models that are more robust. Further, we investigate two specific cases of a model that satisfies optical self-adjustment: (1) a full model in which the cornea is flexibly attached to sclera at the limbus, and (2) a fixed cornea model in which the cornea is not allowed to move at the limbus. We conclude that for a biomechanical model of the eyeball to mimic the optical function of a real eye, it is crucial that the cornea is allowed to move at the limbal junction, that the materials used for the cornea and sclera are strongly nonlinear, and that their moduli of elasticity remain in a very close relationship.

A multizone model for postsurgical corneal topography is presented and applied to a comparative analysis of the outcome of standard and customized myopic LASIK. The different zones are segmented automatically by a clustering algorithm. The algorithm uses a set of three local descriptors, which correspond to normalized physical magnitudes computed for each point of the corneal topography map: Gauss curvature, root-mean-square (RMS) fit error to an ellipsoid surface model, and distance to the center of the topographic map. Both presurgical and post-LASIK corneal topographies of 31 eyes were analyzed using monozone and multizone models. The patients were classified into three groups according to the different LASIK treatments applied: Allegretto, Zyoptix, and PlanoScan. For post-LASIK corneas, the multizone model provided a lower fit error, an average of 1.2±0.4 μm versus 2.4±0.7 μm (monozone). The comparative analysis of the three different LASIK treatments showed no improvement of custom over standard treatments. The outcomes of Zyoptix and PlanoScan were basically equivalent and consistent with previous findings: The higher-order aberration (HOA) increased by a factor of two. The increase in HOA was higher, by a factor of three, after the Allegretto treatment. The mutizone model shows a higher-fidelity representation and permits a deeper understanding of the postsurgical cornea.

Linear birefringence and optical activity are two common optical polarization effects present in biological tissue, and determination of these properties has useful biomedical applications. However, measurement and unique interpretation of these parameters in tissue is hindered by strong multiple scattering effects and by the fact that these and other polarization effects are often present simultaneously. We have investigated the efficacy of a Mueller matrix decomposition methodology to extract the individual intrinsic polarimetry characteristics (linear retardance δ and optical rotation ψ, in particular) from a multiply scattering medium exhibiting simultaneous linear birefringence and optical activity. In the experimental studies, a photoelastic modulation polarimeter was used to record Mueller matrices from polyacrylamide phantoms having strain-induced birefringence, sucrose-induced optical activity, and polystyrene microspheres–induced scattering. Decomposition of the Mueller matrices recorded in the forward detection geometry from these phantoms with controlled polarization properties yielded reasonable estimates for δ and ψ parameters. The confounding effects of scattering, the propagation path of multiple scattered photons, and detection geometry on the estimated values for δ and ψ were further investigated using polarization-sensitive Monte Carlo simulations. The results show that in the forward detection geometry, the effects of scattering induced linear retardance and diattenuation are weak, and the decomposition of the Mueller matrix can retrieve the intrinsic values for δ and ψ with reasonable accuracy. The ability of this approach to extract the individual intrinsic polarimetry characteristics should prove valuable in diagnostic photomedicine, for example, in quantifying the small optical rotations due to the presence of glucose in tissue and for monitoring changes in tissue birefringence as a signature of tissue abnormality.

The use of a combined spectral intensity and polarization signals optically scattered by tissue to determine analyte concentration in optically clear and turbid biological media was explored in a simulation study. Blood plasma was chosen as the biological model and glucose as the analyte of interest. The absorption spectrum and optical rotatory dispersion were modeled using experimental data and the Drude's equation, respectively, between 500 and 2000 nm. A polarization-sensitive Monte Carlo light-propagation model was used to simulate scattering media. Unfold partial least squares and multiblock partial least squares were used as regression methods to combine the spectral intensity and polarization signals, and to predict glucose concentrations in both clear and scattering models. The results show that the combined approaches produce better predictive results in both clear and scattering media than conventional partial least squares analysis, which uses intensity or polarization spectra independently. This improvement was somewhat diminished with the addition of scattering to the model, since the polarization signals were reduced due to multiple scattering. These findings demonstrate promise for the combined approach in clear or moderately scattering biological media; however, the method's applicability to highly scattering tissues is yet to be determined. The methodology also requires experimental validation.

Laser diodes (LD) are commonly used for optical neural recordings in chronically recorded animals and humans, primarily due to their brightness and small size. However, noise introduced by LDs may counteract the benefits of brightness when compared to low-noise light-emitting diodes (LEDs). To understand noise sources in optical recordings, we systematically compared instrument and physiological noise profiles in two recording paradigms. A better understanding of noise sources can help improve optical recordings and make them more practical with fewer averages. We stimulated lobster nerves and a rat cortex, then compared the root mean square (RMS) noise and signal-to-noise ratios (SNRs) of data obtained with LED, superluminescent diode (SLD), and LD illumination for different numbers of averages. The LED data exhibited significantly higher SNRs in fewer averages than LD data in all recordings. In the absence of tissue, LED noise increased linearly with intensity, while LD noise increased sharply in the transition to lasing and settled to noise levels significantly higher than the LED's, suggesting that speckle noise contributed to the LD's higher noise and lower SNRs. Our data recommend low coherence and portable light sources for in vivo chronic neural recording applications.

Four-dimensional (4D) (3D+time) measurement systems make it possible today to measure objects while moving and deforming. One of the fields where 4D systems prove themselves useful is medicine—particularly orthopedics and neural sciences—where measurement results may be used to estimate dynamic parameters of a patient's movement. Relatively new in 4D, optical full-field shape measurement systems capture more data than standard marker-based systems and open new ways for clinical diagnosis. However, before this is possible, the appropriate 4D data processing and analysis methods need to be developed. We present a new data analysis path for 4D data input as well as new shape parameters describing local features of a surface. The developed shape parameters are easier and quicker to calculate than standard surface parameters, such as curvatures, but they give results that are very similar to the latter. The presented 4D data analysis path allows characteristic areas on the body, so-called anatomical landmarks, to be located and traces them in time along the measurement sequence. We also present the general concepts and describe selected steps of the developed 4D data analysis path. The algorithms were implemented and tested on real and computer-generated data representing the surface of lower limbs. Finally, we give sample processing and analysis results.

We developed a miniature fiber optic pressure sensor system and utilized it for in vitro intradiscal pressure measurements for rodents. One of the unique features of this work is the design and fabrication of a sensor element with a multilayer polymer-metal diaphragm. This diaphragm consists of a base polyimide layer (150 nm thick), a metal reflective layer (1 μm thick), and another polyimide layer for protection and isolation (150 nm thick). The sensor element is biocompatible and can be fabricated by simple, batch-fabrication methods in a non-cleanroom environment with good device-to-device uniformity. The fabricated sensor element has an outer diameter of only 366 μm, which is small enough to be inserted into the rodent discs without disrupting the structure or altering the intradiscal pressures. In the calibration and in vitro rodent intradiscal pressure measurements, the sensor element exhibits a linear response to the applied pressure over the range of 0–70 kPa, with a sensitivity of 0.0206 μm/kPa and a resolution of 0.17 kPa. To our best knowledge, this work is the first successful demonstration of rodent intradiscal pressure measurements.

We report an optical molecular imaging technique that exploits local administration of fluorophore-conjugated antibodies and confocal fluorescence microscopy to achieve high-contrast imaging of host cell populations in normal and tumor tissue in living mice. The method achieves micron-scale spatial resolution to depths greater than 100 μm. We illustrate the capabilities of this approach by imaging two dendritic cell populations in the skin and normal and tumor vasculature in vivo.

This PDF file contains the errata for “JBO Vol. 13 Issue 04 Paper 2979676” for JBO Vol. 13 Issue 04

This PDF file contains the editorial “Fundamentals of Photonics, Second Edition” for JBO Vol. 13 Issue 04

This PDF file contains the editorial “Biomedical Optics, Principles and Imaging” for JBO Vol. 13 Issue 04