We have developed a multiphoton microscopy (MPM) system using a 12-fs Ti:sapphire laser with adjustable dispersion precompensation in order to examine the impact of pulse duration on nonlinear optical signals. The efficiencies of two-photon-excited fluorescence (TPEF) and second harmonic generation (SHG) were studied for various pulse durations, measured at the sample, ranging from ~400 fs to sub-20 fs. Both TPEF and SHG increased proportionally to the inverse of the pulse duration for the entire tested range. Because of improved signal-to-noise ratio, sub-20-fs pulses were used to enhance MPM imaging depth by approximately 160%, compared to 120-fs pulses, in human skin.

A 12-fs broadband (100-nm) source is used to combine multiphoton microscopy (MPM) and optical coherence tomography (OCT) in a single platform. An ultrafast Ti:sapphire laser simultaneously provides short pulses necessary for efficient MPM excitation and the broad bandwidth required for high-resolution OCT. Using 0.3-µm microspheres and a 63×, 0.95 numerical aperture objective, we demonstrate that MPM and OCT channels are coregistered with lateral resolution of approximately 0.5 µm and axial resolution of approximately 1.5 µm. Preliminary studies of a 3-D organotypic epithelial tissue model show that multiphoton images of fluorescence and second harmonic signals are derived from cellular and extracellular matrix structures, respectively, while OCT images are generated from scattering interfaces due to tissue variations in refractive index. The combined MPM/OCT microscope is capable of providing simultaneous functional and structural information from cells and extracellular matrix and is potentially a powerful tool for studying biological processes in thick tissues.

Bioluminescence tomography (BLT) allows in vivo localization and quantification of bioluminescent sources inside a small animal to reveal various molecular and cellular activities. We develop a reconstruction method to identify such a bioluminescent source distribution using the boundary integral method. Based on the diffusion model of the photon propagation in the biological tissue, this method incorporates a priori knowledge to define the permissible source region, and establish a direct linear relationship between measured body surface data and an unknown bioluminescent source distribution to enhance numerical stability and efficiency. The feasibility of the proposed BLT algorithm is demonstrated in heterogeneous mouse chest phantom studies.

Spectral-domain low-coherence interferometry (LCI) was used to measure the thickness of microbicidal gels applied to a cylindrical calibration test socket. Microbicides are topical formulations containing active ingredients targeted to inhibit specific pathogens that are currently under development for application to the epithelial lining of the lower female reproductive tract to combat sexually transmitted infections such as HIV. Understanding the deployment and drug delivery of these formulations is vital to maximizing their effectiveness. Previously, in vivo measurements of microbicidal formulation thickness were assessed using fluorescence measurements of fluorescein-labeled gels via an optical endoscope-based device. Here we present an LCI-based device that measures the thickness of a formulation without the use of any exogenous agents by analyzing the interference pattern generated between the reflections from the front and back surface of the sample. Results are presented that validate the effectiveness and performance of the LCI measurement in a clinically relevant system as compared to an existing fluorescence-based method. The impact of the new LCI-based design on in vivo measurements is discussed.

This PDF file contains the editorial “Special Section Guest Editorial: Cardiovascular Photonics” for JBO Vol. 11 Issue 02

While our understanding of vulnerable coronary plaque is still at an early stage, the concept that certain types of plaques predispose patients to developing an acute myocardial infarction continues to be at the forefront of cardiology research. Intracoronary optical coherence tomography (OCT) has been developed to both identify and study these lesions due to its distinct resolution advantage over other imaging modalities. We review clinical research conducted at the Massachusetts General Hospital over the past five years to develop, validate, and utilize this technology to improve our understanding of vulnerable plaque. Our results show that intracoronary OCT may be safely conducted in patients and that it provides abundant information regarding plaque microscopic morphology, which is essential to the identification and study of high-risk lesions. Even though many basic biological, clinical, and technological challenges must be addressed prior to widespread use of this technology, the unique capabilities of OCT ensure that it will have a prominent role in shaping the future of cardiology.

The rupture of vulnerable atherosclerotic plaque accounts for the majority of clinically significant acute cardiovascular events. Because stability of these culprit lesions is directly related to chemical and morphological composition, Raman spectroscopy may be a useful technique for their study. Recent developments in optical fiber probe technology have allowed for the real-time in vivo Raman spectroscopic characterization of human atherosclerotic plaque demonstrated in this work. We spectroscopically examine 74 sites during carotid endarterectomy and femoral artery bypass surgeries. Of these, 34 are surgically biopsied and examined histologically. Excellent signal-to-noise ratio spectra are obtained in only 1 s and fit with an established model, demonstrating accurate tissue characterization. We also report the first evidence that Raman spectroscopy has the potential to identify vulnerable plaque, achieving a sensitivity and specificity of 79 and 85%, respectively. These initial findings indicate that Raman spectroscopy has the potential to be a clinically relevant diagnostic tool for studying cardiovascular disease.

We report the application of the Laguerre deconvolution technique (LDT) to the analysis of in-vivo time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data and the diagnosis of atherosclerotic plaques. TR-LIFS measurements were obtained in vivo from normal and atherosclerotic aortas (eight rabbits, 73 areas), and subsequently analyzed using LDT. Spectral and time-resolved features were used to develop four classification algorithms: linear discriminant analysis (LDA), stepwise LDA (SLDA), principal component analysis (PCA), and artificial neural network (ANN). Accurate deconvolution of TR-LIFS in-vivo measurements from normal and atherosclerotic arteries was provided by LDT. The derived Laguerre expansion coefficients reflected changes in the arterial biochemical composition, and provided a means to discriminate lesions rich in macrophages with high sensitivity (<85%) and specificity (<95%). Classification algorithms (SLDA and PCA) using a selected number of features with maximum discriminating power provided the best performance. This study demonstrates the potential of the LDT for in-vivo tissue diagnosis, and specifically for the detection of macrophages infiltration in atherosclerotic lesions, a key marker of plaque vulnerability.

Heart disease is the primary cause of death in the western world. Many of these deaths are caused by the rupture of vulnerable plaque. Vulnerable plaques are characterized by a large lipid core covered by a thin fibrous cap. One method for detecting these plaques is reflection spectroscopy. Several studies have investigated this method using statistical methods. A more analytic and quantitative study might yield more insight into the sensitivity of this detection modality. This is the approach taken in this work. Reflectance spectra in the spectral region from 400 to 1700 nm are collected from 77 measurement points from 23 human aortas. A measure of lipid content in a plaque based on reflection spectra is presented. The measure of lipid content is compared with the thickness of the lipid core, determined from histology. Defining vulnerable plaque as having a lipid core >500 µm and fibrous cap <500 µm, vulnerable plaques are detected with a sensitivity of 88% and a specificity of 94%. Although the method can detect lipid content, it is not very sensitive to the thickness of the fibrous cap. Another detection modality is necessary to detect this feature.

Necrotic-core fibroatheromas (NCFA) with thin, mechanically weak fibrous caps overlying lipid cores comprise the majority of plaques that rupture and cause acute myocardial infarction. Laser speckle imaging (LSI) has been recently demonstrated to enable atherosclerotic plaque characterization with high accuracy. We investigate spatio-temporal analysis of LSI data, in conjunction with diffusion theory and Monte Carlo modeling of light transport, to estimate fibrous cap thickness in NCFAs. Time-varying laser speckle images of 20 NCFAs are selected for analysis. Spatio-temporal intensity fluctuations are analyzed by exponential fitting of the windowed normalized cross-correlation of sequential laser speckle patterns to obtain the speckle decorrelation time constant, τ(ρ), as a function of distance ρ from the source entry location. The distance, ρ′, at which τ(ρ) dropped to 65% of its maximum value is recorded. Diffusion theory and Monte Carlo models are utilized to estimate the maximum photon penetration depth, z_max(ρ′), for a distance equal to ρ′, measured from LSI. Measurements of z_max(ρ′) correlate well with histological measurements of fibrous cap thickness (R=0.78,p<0.0001), and paired t-tests show no significant difference between the groups (p=0.4). These results demonstrate that spatio-temporal LSI may allow the estimation of fibrous cap thickness in NCFAs, which is an important predictor of plaque stability.

Vulnerable plaques, which are responsible for most acute ischemic events, are presently invisible to x-ray angiography. Their primary morphological features include a thin or ulcerated fibrous cap, a large necrotic core, superficial foam cells, and intraplaque hemorrhage. We present evidence that multimodal spectroscopy (MMS), a novel method that combines diffuse reflectance spectroscopy (DRS), intrinsic fluorescence spectroscopy (IFS), and Raman spectroscopy (RS), can detect these markers of plaque vulnerability. To test this concept, we perform an MMS feasibility study on 17 human carotid artery specimens. Following the acquisition of spectra, each specimen is histologically evaluated. Two parameters from DRS, hemoglobin concentration and a scattering parameter, are used to detect intraplaque hemorrhage and foam cells; an IFS parameter that relates to the amount of collagen in the topmost layers of the tissue is used to detect the presence of a thin fibrous cap; and an RS parameter related to the amount of cholesterol and necrotic material is used to detect necrotic core. Taken together, these spectral parameters can generally identify the vulnerable plaques. The results indicate that MMS provides depth-sensitive and complementary morphological information about plaque composition. A prospective in vivo study will be conducted to validate these findings.

We have previously shown that a conjugate (MA-ce6) between maleylated serum albumin and the photosensitizer chlorin(e6) (ce6) is targeted in vitro to macrophages via class A scavenger receptors. We now report on the ability of this conjugate to localize in macrophage-rich atherosclerotic plaques in vivo. Both the conjugate and the free photosensitizer ce6 are studied after injection into New Zealand White rabbits that are rendered atherosclerotic by a combination of aortic endothelial injury and cholesterol feeding into normal rabbits. Rabbits are sacrificed at 6 and 24 h after injection and intravascular fluorescence spectroscopy is carried out by fiber-based fluorimetry in intact blood-filled arteries. Surface spectrofluorimetry of numbered excised aortic segments together with injured and normal iliac arteries is carried out, and quantified ce6 content by subsequent extraction and quantitative fluorescence determination of the arterial segments and also of nontarget organs. There is good agreement between the various techniques for quantifying ce6 localization, and high contrast between arteries from atherosclerotic and normal rabbits is obtained. Fluorescence correlates with the highest burden of plaque in the aorta and the injured iliac artery. The highest accumulation in plaques is obtained using MA-ce6 at 24 h. Free ce6 gives better accumulation at 6 h compared to 24 h. The liver, spleen, lung, and gall bladder have the highest uptake in nontarget organs. Macrophage-targeted photosensitizer conjugates may have applications in both detecting and treating inflamed vulnerable plaque.

Molecular and cellular mechanisms of atherogenesis and its treatment are largely being unraveled by in vitro techniques. We describe methodology to directly image macrophage cell activity in vivo in a murine model of atherosclerosis using laser scanning fluorescence microscopy (LSFM) and a macrophage-targeted, near-infrared fluorescent (NIRF) magnetofluorescent nanoparticle (MFNP). Atherosclerotic apolipoprotein E deficient (apoE -/-) mice (n=10) are injected with MFNP or 0.9% saline, and wild-type mice (n=4) are injected with MFNP as additional controls. After 24 h, common carotid arteries are surgically exposed and prepared for LSFM. Multichannel LSFM of MFNP-enhanced carotid atheroma (5×5-µm in-plane resolution) shows a strong focal NIRF signal, with a plaque target-to-background ratio of 3.9±1.8. Minimal NIRF signal is observed in control mice. Spectrally resolved indocyanine green (ICG) fluorescence angiograms confirm the intravascular location of atheroma. On ex vivo fluorescence reflectance imaging, greater NIRF plaque signal is seen in apoE -/- MFNP mice compared to controls (p<0.01). The NIRF signal correlates well with immunostained macrophages, both by stained surface area (r=0.77) and macrophage number (r=0.86). The validated experimental methodology thus establishes a platform for investigating macrophage activity in atherosclerosis in vivo, and has implications for the detection of clinical vulnerable plaques.

Diagnosis and monitoring of critical diseases such as acute myocardial infarction (AMI) require a quantitative analysis of biological molecules. A high-throughput identification of these biological molecules can be generated by using micro-electro-mechanical systems (MEMS) structures like simple cantilever beams, which respond to the intermolecular forces resulting from binding these molecules. Biochemical markers like troponin C are considered the primary markers for myocardial injury and have generated considerable interest. A 26-residue lytic membrane protein of bee venom melittin (ME) is chosen to interact with rabbit skeletal muscle troponin C (TnC) on the surface of the cantilever beams. An optical beam deflection method is employed to identify the enzymatic reaction on the surface of the cantilever due to these proteins. Identification of these proteins is also done using fluorescence spectroscopy (FS) to compliment the optical monitored deflection method. A second set of proteins like horse raddish peroxide (HRP) and hydrogen peroxide (H₂O₂) are applied to atomic force microscopy (AFM) cantilever beams to study their behavior under the enzymatic reactions of proteins. Identification of these proteins is done using Fourier transform infrared spectroscopy (FTIR). An analytical model of the cantilever beam is developed, and its mode shapes are studied by employing orthogonal polynomials in the classic Rayleigh-Ritz method. The surface stress caused by the enzymatic reaction of the proteins that leads to pure bending on the top surface of the cantilever is evaluated. The information provided by the experimental and analytical modeling reported in this work will be useful in the development of a portable biosensor for the detection of AMI.

A prototype of a fiber-optic, multi-analyte, immunobiosensing system was developed to simultaneously quantify disease-representing biomarkers in blood plasma. This system was for simultaneous quantification of two different groups of multi-biomarkers related to cardiovascular diseases (CVD): anticoagulants (protein C, protein S, antithrombin III, and plasminogen) for deficiency diagnosis; and cardiac markers (B-type natriuretic peptide, cardiac troponin I, myoglobin, and C-reactive protein) for coronary heart disease diagnosis. As an initial effort towards the development of a disposable and easy-to-use sensing cartridge as a rapid diagnostic tool for CVD related diseases, a prototype of a flow control system was also developed to automatically perform simultaneous four-analyte quantification. Currently, the system is capable of quantifying the multiple anticoagulants in their clinically significant sensing ranges within 5 minutes, at an average signal-to-noise (S/N) ratio of 25. A simultaneous assay of the four cardiac markers can be performed within 10 min, at an average S/N ratio of 20. When this highly portable multi-analyte sensing system is completed and successfully tested for CVD patient's plasma, it can provide rapid (<10 min) and reliable diagnostic and prognostic information at a patient's bedside.

Reactive oxygen species (ROS) are short-lived, highly reactive chemical entities that play significant roles in all levels of biology. However, their measurement requires destructive preparation, thereby limiting the continuous measurement of ROS in a living tissue. We develop an optical mapping system to visualize ROS production in an isolated and perfused rat heart. By staining the heart with dihydroethidium (DHE), a 532-nm laser beam is directed to the epicardial surface, where we collect the red fluorescence (>600 nm) for semiquantitative analysis. With this system, ROS production as well as ventricular pressure and ECG in isolated perfused rat hearts are monitored throughout the reperfusion of global ischemia. Ischemia would decrease myocardial ROS production, while reperfusion would immediately result in sustained ROS overproduction. Optical mapping would provide information regarding the spatial distribution and temporal evolution of myocardial ROS production, which would enhance knowledge of the role of free radicals in cardiovascular biology.

Low-energy defibrillation is very desirable in cardiac rhythm management. We previously reported that ventricular fibrillation (VF) can be synchronized with a novel synchronized pacing technique (SyncP) using low-energy pacing pulses. This study sought to create a line of block during VF using SyncP. SyncP was performed in six isolated rabbit hearts during VF using optical recording to control the delivery of pacing pulses in real time. Four pacing electrodes with interelectrode distances of 5 mm were configured in a line along and across the myocardial fiber direction. The electrodes were controlled independently (independent mode) or fired together (simultaneous mode). Significant wavefront synchronization was observed along the electrode line as indicated by a decrease in variance. With the independent SyncP protocol, the decrease in the variance was 19.3 and 13.7% (P<0.001) for the along-, and across-fiber configurations, respectively. With the simultaneous SyncP protocol, the variance was reduced by 24.2 and 10.7% (P<0.001) in the along- and across-fiber configurations. The effect of synchronization dropped off with distance from the line of pacing. We conclude that SyncP can effectively create a line of functional block that isolates regions of VF propagation. Further optimization of this technique may prove useful for low-energy ventricular defibrillation.

Optical coherence tomography (OCT) is an emerging high-resolution real-time biomedical imaging technology that has potential as a novel investigational tool in developmental biology and functional genomics. In this study, murine embryos and embryonic hearts are visualized with an OCT system capable of 2-µm axial and 15-µm lateral resolution and with real-time acquisition rates. We present, to our knowledge, the first sets of high-resolution 2- and 3-D OCT images that reveal the internal structures of the mammalian (murine) embryo (E10.5) and embryonic (E14.5 and E17.5) cardiovascular system. Strong correlations are observed between OCT images and corresponding hematoxylin- and eosin-stained histological sections. Real-time in vivo embryonic (E10.5) heart activity is captured by spectral-domain optical coherence tomography, processed, and displayed at a continuous rate of five frames per second. With the ability to obtain not only high-resolution anatomical data but also functional information during cardiovascular development, the OCT technology has the potential to visualize and quantify changes in murine development and in congenital and induced heart disease, as well as enable a wide range of basic in vitro and in vivo research studies in functional genomics.

We fabricate scaffolds with a fine linear grating (periodicity of 1 to 8 µm and height of 1 µm) on a glass substrate for controlling cardiomyocyte orientation. The fabrication is done by the solidification of a liquid photopolymerizable material using two laser beam interference. As the photopolymerizable material, we use acrylated trimethylene-carbonate-based oligomers initiated with trimethylolpropane (T/TMP), followed by acrylation at terminal ends. Rat cardiomyocytes cultured on the fabricated scaffolds exhibit cell elongation, orientation, and contraction along the scaffold grating. Fluorescence observation of bundles of actin filaments of the cultured cells show that the cytoskeleton of the cells is also generated and oriented parallel to the grating. With a change in grating periodicity from 8 to 1 µm, the percentage of the cells that show orientation along the grating increase from approximately 40 to 70%. The cell orientation along the grating is observed 18 h after seeding the cells on the scaffold. This result implies that the attachment between a cell and a well-defined microarchitectural substrate at an early stage of culture is a significant determinant of cell morphology.

Er:glass lasers have been in operation with both long pulses (hundreds of microseconds) and Q-switched pulses (50 to 100 ns) for more than 35 yr. The ocular hazards of this laser were reported early, and it was determined that damage to the eye from the 1.54-µm wavelength occurred mainly in the cornea where light from this wavelength is highly absorbed. Research on skin hazards has been reported only in the past few years because of limited pulse energies from these lasers. Currently, however, with pulse energies in the hundreds of joules, these lasers may be hazardous to the skin in addition to being eye hazards. We report our minimum visible lesion (MVL) threshold measurements for two different pulse durations and three different spot sizes for the 1.54-µm wavelength using porcine skin as an in vivo model. We also compare our measurements to results from our model, based on the heat transfer equation and the rate process equation. Our MVL-ED₅₀ thresholds for the long pulse (600 µs) at 24 h postexposure were measured to be 20, 8.1, and 7.4 J cm^–2 for spot diameters of 0.7, 1.0, and 5 mm, respectively. Q-switched laser pulses of 31 ns had lower ED50 (estimated dose for a 50% probability of laser-induced damage) thresholds of 6.1 J cm^–2 for a 5-mm-diam, top-hat spatial profile laser pulse.

We use the optical path difference (OPD) technique to quantify the organization of collagen fibers during skin repair of full-thickness burns following low-intensity polarized laser therapy with two different polarization incidence vectors. Three burns are cryogenerated on the back of rats. Lesion L_|| is irradiated using the electric field vector of the polarized laser radiation aligned in parallel with the rat's occipital-caudal direction. Lesion L_⊥ is irradiated using the electric field vector of the polarized laser radiation aligned perpendicularly to the aforementioned orientation. Lesion C is untreated. A healthy area labeled H is also evaluated. The tissue samples are collected and processed for polarized light microscopy. The overall finding is that the OPD for collagen fibers depends on the electric field vector of the incident polarized laser radiation. No significant differences in OPDs are observed between L_|| and H in the center, sides, and edges of the lesion. Lesions irradiated using the electric field vector of the polarized laser radiation aligned in parallel with the rat's occipital-caudal direction show higher birefringence, indicating that collagen bundles in these lesions are more organized.

Laser-assisted cartilage reshaping (LACR) is a relatively novel technique designed to noninvasively and permanently restructure cartilaginous tissue. It is believed that heat-induced stress relaxation, in which a temperature-mediated disruption of H₂O binding is associated with conformational alterations in the proteoglycan and collagen-rich matrix, constitutes the underlying mechanism of LACR. Several reports have suggested that laser-mediated cartilage mineralization may contribute to the permanent shape change of laser-reshaped cartilage. In an effort to validate these results in the context of Er:glass LACR, we performed a preliminary Raman microspectrometric study to characterize the crystal deposits in laser-irradiated chondrocytes and extracellular matrix. For the first time, we identified intracellular calcium sulfate deposits and extracellular calcium phosphate (apatite) crystals in laser-reshaped rabbit auricular cartilage. Calcium carbonate deposits are localized in both irradiated and nonirradiated samples, suggesting that this mineral plays no role in conformational retention. In our discussion, we elaborate on the possible molecular and cellular mechanisms responsible for intra- and extracellular crystallization, and propose a novel hypothesis on the formation of apatite, inasmuch as the biological function of this mineral (providing structure and rigidity in bones and dental enamel) may be extrapolated to the permanent shape change of laser-irradiated cartilage.

Current research has revealed the importance of a class of cell surface proteins called integrins in various vital physiological functions such as blood clotting, regulation of blood pressure, tissue blood flow, and vascular remodeling. The key to integrin functionality is its ability to mediate force transmission by interacting with the extracellular matrix and cytoskeleton. In addition, they play a role in signal transduction via their connection with the proteins in focal adhesion (FA) points. To understand the complex mechanism of cell-cell and cell-extracellular matrix (ECM) adhesion that is responsible for these diverse biochemical interactions, it is necessary to identify the integrins on cells and monitor their interaction with various ligands. To this end, for the first time, we employ surface-enhanced Raman spectroscopy (SERS) to detect integrins. The results show the capability using SERS to detect the integrins to the nanomolar concentration regime and to distinguish between two different kinds of integrins, αVβ3 and α5β1, that are present in vascular smooth muscle cells (VSMCs). It is anticipated that the SERS approach will potentially help elucidate the mechanism of integrin-ligand interactions in a variety of phenomena of physiological importance.

It is generally proposed that bonding of resins to dentin results from infiltration of the adhesive monomers into the superficially demineralized dentin. However, it is still not clear how well the mineral phase of dentin is removed and how far each monomer penetrates into the thin zone of "wet" demineralized dentin. The quality and molecular structure of adhesive/dentin interfaces formed under "wet" bonding conditions are studied using 2-D Raman microspectroscopic mapping/imaging techniques. Micro-Raman imaging analysis of the adhesive/dentin interface provides a reliable and powerful means of identifying the degree and depth of dentin demineralization, adhesive monomer distribution, and flaws or defects in the pattern of adhesive penetration. The image of mineral reveals a partially demineralized layer on the top of dentin substrate. Adhesive monomers readily penetrate into dentin tubules and spread into intertubular region through open tubules. The extent of adhesive monomer penetration is higher in the intertubular regions close to tubules as compared to the middle regions between the tubules. The diffusion of resin monomers differs substantially. In a comparison with a hydrophilic monomer, the hydrophobic monomer resists diffusion into the demineralized intertubular dentin area.

Early identification of high-risk disease could greatly reduce both mortality and morbidity due to oral cancer. We describe a simple handheld device that facilitates the direct visualization of oral-cavity fluorescence for the detection of high-risk precancerous and early cancerous lesions. Blue excitation light (400 to 460 nm) is employed to excite green-red fluorescence from fluorophores in the oral tissues. Tissue fluorescence is viewed directly along an optical axis collinear with the axis of excitation to reduce inter- and intraoperator variability. This robust, field-of-view device enables the direct visualization of fluorescence in the context of surrounding normal tissue. Results from a pilot study of 44 patients are presented. Using histology as the gold standard, the device achieves a sensitivity of 98% and specificity of 100% when discriminating normal mucosa from severe dysplasia/carcinoma in situ (CIS) or invasive carcinoma. We envisage this device as a suitable adjunct for oral cancer screening, biopsy guidance, and margin delineation.

A laser fluorescence (LF) device is a portable tool, but it does not measure minor mineral changes. Our in vitro study aim is to propose the association of an LF with two fluorescent dyes and to evaluate the performance in detecting and quantifying early demineralization. Artificial caries lesions are created in 40 primary canine teeth using a demineralizing solution (pH=4.8) for 12, 24, 48, and 96 h. LF measurements are performed with DIAGNOdent after demineralization in these samples and in 20 sound primary teeth. Measurements with LF with 0.2-mM tetrakis(N-methylpyridyl)porphyrin (LF TMPyP) and with 4-mM protoporphyrin IX (LF PPIX) are made. The amount of calcium loss is determined by atomic emission spectrometry. A correlation between LF and LF with dyes and mineral loss and receiver operating characteristics analysis are performed, as well as comparisons of sensitivity, specificity, and accuracy values. Significant correlation is obtained with LF TMPyP and mineral loss of lesions demineralized for 24, 48, and 96 h. Better performance is achieved with LF TMPyP for all parameters than with LF alone. LF PPIX does not present good results. In conclusion, LF TMPyP provides good performance in detecting and quantifying very early enamel caries lesions.

We present a mathematical model to calculate the relative concentration of light scatterers, light absorbers, and fluorophores in the epithelium and stroma. This mathematical description is iteratively fit to the fluorescence spectra measured in vivo, yielding relative concentrations of each molecule. The mathematical model is applied to a total of 493 fluorescence measurements of normal and dysplastic cervical tissue acquired in vivo from 292 patients. The estimated parameters are compared with histopathologic diagnosis to evaluate their diagnostic potential. The mathematical model is validated using fluorescence spectra simulated with known sets of optical parameters. Subsequent application of the mathematical model to in vivo fluorescence measurements from cervical tissue yields fits that accurately describe measured data. The optical parameters estimated from 493 fluorescence measurements show an increase in epithelial flavin adenine dinucleotide (FAD) fluorescence, a decrease in epithelial keratin fluorescence, an increase in epithelial light scattering, a decrease in stromal collagen fluorescence, and an increase in stromal hemoglobin light absorption in dysplastic tissue compared to normal tissue. These changes likely reflect an increase in the metabolic activity and loss of differentiation of epithelial dysplastic cells, and stromal angiogenesis associated with dysplasia. The model presented here provides a tool to analyze clinical fluorescence spectra yielding quantitative information about molecular changes related to dysplastic transformation.

We aim to establish a confocal spectroscopic technique able to study the features of fluorescence spectra arising from native Cr³⁺ impurity in polycrystalline alumina (Al₂O₃as a biomaterial and to use their emission lines as microscopic probes for the characterization of residual stress fields stored in artificial hip prostheses during their implantation in vivo. As an application of the technique, we report for the first time concerning the evolution of microscopic (residual) stress fields stored on the surface and in the subsurface of N=7 retrieved Al₂O₃ hip joints after exposure in the human body from a few months to 19 yr. The micrometric diameter of the laser beam waist impinging on the joint surface (typically about 1 µm in lateral resolution) enables us to estimate the patterns and magnitude of residual stress with high spatial resolution, at least comparable with the grain size of the material. In addition, a selected confocal configuration for the optical probe enables minimization of the probe size along the in-depth direction. According to a statistical collection of data on the microscopic level for retrieved femoral heads in toto, a residual stress field arising from loading history in vivo during the lifetime of the Al₂O₃ femoral head can be revealed. Finally, an interpretation is given of microscopic wear mechanisms in Al₂O₃ artificial hip joints consistent with the observed evolution of surface residual stress fields on elapsed time in vivo.

We report the design, integration, and validation of a fluorescence microscopy system for imaging of electroperturbation—the effects of nanosecond, megavolt-per-meter pulsed electric fields on biological cells and tissues. Such effects have potential applications in cancer therapy, gene regulation, and biophysical research by noninvasively disrupting intracellular compartments and inducing apoptosis in malignant cells. As the primary observing platform, an epifluorescence microscope integrating a nanosecond high-voltage pulser and a micrometer electrode chamber enable in situ imaging of the intracellular processes triggered by high electric fields. Using specific fluorescence molecular probes, the dynamic biological responses of Jurkat T lymphocytes to nanosecond electric pulses (nanoelectropulses) are studied with this system, including calcium bursts, the polarized translocation of phosphatidylserine (PS), and nuclear enlargement and chromatin/DNA structural changes.

Caspase-2 is important for the mitochondrial apoptotic pathway, however, the mechanism by which caspase-2 executes apoptosis remains obscure. We carry out the first measurements of the dynamics of caspase-2 activation in a single living cell by a FRET (fluorescence resonance energy transfer) probe. Two FRET probes are constructed that each encoded a CRS (caspase-2 or caspase-3 recognition site) fused with a cyan fluorescent protein (CFP) and a red fluorescent protein (DsRed) (CFP-CRS-DsRed). Using these probes, we found that during cisplatin-induced apoptosis, caspase-2 activation occurred more slowly than did activation of caspase-3; additionally, caspase-2 activation was initiated much earlier than that of caspase-3.

Quantitative distance measurements are difficult to obtain in spite of the strong distance dependency of the energy transfer efficiency. One problem for the interpretation of the Förster resonant energy transfer (FRET) efficiency is the so-called zero-efficiency peak caused by FRET pairs with missing or nonfluorescent acceptors. Other problems occurring are direct excitation of the acceptor, spectral crosstalk, and the determination of the quantum efficiency of the dyes as well as the detector sensitivity. Our approach to overcome these limitations is based on the pulsed-interleaved excitation (PIE) of both the acceptor and the donor molecule. PIE is used to excite the acceptor dye independently of the FRET process and to prove its existence via fluorescence. This technique enables us to differentiate a FRET molecule, even with a very low FRET efficiency, from a molecule with an absent or non-fluorescent acceptor. Crosstalk, direct acceptor excitation, and molecular brightness of acceptor and donor molecules are determined by analyzing the data with fluorescence correlation spectroscopy (FCS). FRET efficiencies of the same data set are also determined by analyzing the lifetimes of the donor fluorophores. The advantages of the PIE-FRET approach are demonstrated on a polyproline assay labeled with Alexa-555 and Alexa-647 as donor and acceptor, respectively.

We establish, for the first time, a simulation model for dealing with the second-harmonic signals under a microscope through a tissue-like turbid medium, based on the Monte Carlo method. With this model, the angle-resolved distribution and the signal level η of second-harmonic light through a slab of the turbid medium are demonstrated and the effects of the thickness (d) of the turbid medium, the numerical aperture (NA) of the objective as well as the size (ρ) of the scatterers forming the turbid medium are explored. Simulation results reveal that the use of a small objective NA results in a narrow angle distribution but strong second-harmonic signals. A turbid medium consisting of large scattering particles has a strong influence on the angle distribution and the signal level η, which results in a low penetration limit for second-harmonic signals made up of ballistic photons. It is approximately 30 µm in our situation.

Spectral domain phase microscopy (SDPM) is a function extension of spectral domain optical coherence tomography. SDPM achieves exquisite levels of phase stability by employing common-path interferometry. We discuss the theory and limitations of Doppler flow imaging using SDPM, demonstrate monitoring the thermal contraction of a glass sample with nanometer per second velocity sensitivity, and apply this technique to measurement of cytoplasmic streaming in an Amoeba proteus pseudopod. We observe reversal of cytoplasmic flow induced by extracellular CaCl₂, and report results that suggest parabolic flow of cytoplasm in the A. proteus pseudopod.

Simultaneous transcranial imaging of two functional parameters, the total concentration of hemoglobin and the hemoglobin oxygen saturation, in the rat brain in vivo is realized noninvasively using laser-based photoacoustic tomography (PAT). As in optical diffusion spectroscopy, PAT can assess the optical absorption of endogenous chromophores, e.g., oxygenated and deoxygenated hemoglobins, at multiple optical wavelengths. However, PAT can provide high spatial resolution because its resolution is diffraction-limited by photoacoustic signals rather than by optical diffusion. Laser pulses at two wavelengths are used sequentially to acquire photoacoustic images of the vasculature in the cerebral cortex of a rat brain through the intact skin and skull. The distributions of blood volume and blood oxygenation in the cerebral cortical venous vessels, altered by systemic physiological modulations including hyperoxia, normoxia, and hypoxia, are visualized successfully with satisfactory spatial resolution. This technique, with its prominent sensitivity to endogenous contrast, can potentially contribute to the understanding of the interrelationship between neural, hemodynamic, and metabolic activities in the brain.

During cardiopulmonary bypass (CPB) because of weak arterial pulsation, near-IR spectroscopy (NIRS) is almost the only available method to monitor cerebral oxygenation noninvasively. Our group develops a NIRS oximeter to monitor regional cerebral oxygenation especially its oxygen saturation (rScO₂). To achieve optimal coupling between the sensor and human brain, the distances between the light source and the detectors on it are properly chosen. The oximeter is calibrated by blood gas analysis, and the results indicate that its algorithm is little influenced by either background absorption or overlying tissue. We used it to measure the rScO₂ of 15 patients during CPB. It is shown that rScO₂ is negatively correlated with body temperature and positively with perfusion rate. There are two critical stages during CPB when rScO₂ might be relatively low: one is the low-perfusion-rate stage, the other is the early rewarming stage. During cooling, the changes of total hemoglobin concentration (C_tHb) compared with its original value is also monitored. It is shown that C_tHb decreases to a small extent, which may mainly reflect cerebral vasoconstriction induced by cooling. All these results indicate that NIRS can be used to monitor cerebral oxygenation to protect cerebral tissue during CPB.

We propose automated identification of microorganisms using three-dimensional (3-D) complex morphology. This 3-D complex morphology pattern includes the complex amplitude (magnitude and phase) of computationally reconstructed holographic images at arbitrary depths. Microscope-based single-exposure on-line (SEOL) digital holography records and reconstructs holographic images of the biological microorganisms. The 3-D automatic recognition is processed by segmentation, feature extraction by Gabor-based wavelets, automatic feature vector selection by graph matching, training rules, and a decision process. Graph matching combined with Gabor feature vectors measures the similarity of complex geometrical shapes between a reference microorganism and unknown biological samples. Automatic selection of the training data is proposed to achieve a fully automatic recognition system. Preliminary experimental results are presented for 3-D image recognition of Sphacelaria alga and Tribonema aequale alga.

We present a two-axis goniometer for measuring the phase function of scattering media with an angular resolution of about 0.2 deg having 12 decades of dynamic range and covering almost the full solid angle. The setup is evaluated with polystyrene spheres and with perpendicularly and obliquely illuminated thin glass cylinders. The scattering pattern and its intensity distribution are in excellent agreement with analytical theory. A multiple scattering configuration composed of two parallel cylinders is also examined. Finally, the phase function of dentin slabs is measured and its dependence on the dental microstructure is discussed.

Cardiac optical mapping currently provides 2-D maps of transmembrane voltage-sensitive fluorescence localized near the tissue surface. Methods for interrogation at different depths are required for studies of arrhythmias and the effects of defibrillation shocks in 3-D cardiac tissue. We model the effects of coloading with a dye that absorbs excitation or fluorescence light on the radius and depth of the interrogated region with specific illumination and collection techniques. Results indicate radii and depths of interrogation are larger for transillumination versus epi-illumination, an effect that is more pronounced for broad-field excitation versus laser scanner. Coloading with a fluorescence absorber lessens interrogated depth for epi-illumination and increases it for transillumination, which is confirmed with measurements using transillumination of heart tissue slices. Coloading with an absorber of excitation light consistently decreases the interrogated depths. Transillumination and coloading also decrease the intensities of collected fluorescence. Thus, localization can be modified with wavelength-specific absorbers at the expense of a reduction in fluorescence intensity.

A classifier capable of ranking structural alterations of the cytoskeleton is developed. Images of cytoskeletal microtubules obtained from the epifluorescence microscopy of primary culture rat hepatocytes are analyzed. Morphological descriptors are extracted by contour and mass fractal analysis, direct methods, and spectrum enhancement. All methods are designed and tuned to make the extracted morphological descriptors insensitive to absolute fluorescence intensities. Spectrum enhancement is a nonlinear filter that involves spatial differentiation of the gray-scale image followed by conversion of power spectral density to the logarithmic scale and averaging over arcs in the reciprocal domain. Enhanced spectra exhibit local maxima that correspond to the structured microtubule bundles of a normal cytoskeleton. Descriptor fusion for classification is achieved by means of multivariate analysis. The classifier is trained by image sets representing normal ("negative control") microtubules and those altered by exposure to a fungicide at the highest dose of the experiment design. Some sensitivity and validation tests, including discriminant functions analysis, are applied to the classifier. The latter is applied to recognize images of microtubules not used in the training stage and comes from treatments at lower concentrations and shorter times. As a result, structural alterations are ranked and structural recovery after treatment is quantified. The method has potential use in quantitative, morphology-based tests on the cytoskeleton treated either by anticancer drugs or by cytotoxic agents.