We developed a multimodality fluorescence and optical coherence tomography probe based on a double-clad fiber (DCF) combiner. The probe is composed of a DCF combiner, grin lens, and micromotor in the distal end. An integrated swept-source optical coherence tomography and fluorescence intensity imaging system was developed based on the combined probe for the early diagnoses of atherosclerosis. This system is capable of real-time data acquisition and processing as well as image display. For fluorescence imaging, the inflammation of atherosclerosis and necrotic core formed with the annexin V-conjugated Cy5.5 were imaged. Ex vivo imaging of New Zealand white rabbit arteries demonstrated the capability of the combined system.

Several in vitro and in vivo studies have established accelerated thrombolysis using ultrasound (US) induced microbubble (MB) cavitation. However, the mechanisms underlying MB mediated sonothrombolysis are still not completely elucidated. We performed three-dimensional (3-D) volumetric optical coherence tomography (OCT) imaging before and after the application of contrast US to thrombus. The most dramatic reduction in clot volume was observed with US + MB + recombinant tissue plasminogen activator (rt-PA). Thrombus surface erosion in this group on the side of the thrombus exposed to MB and ultrasound was evident on the OCT images. This technique may assist in clarifying the mechanisms underlying sonothrombolysis, especially regarding the importance of US transducer orientation on lytic efficacy and the effects of MB cavitation on thrombus structure.

Early patterning and polarity is of fundamental interest in preimplantation embryonic development. Label-free subcellular 3D live imaging is very helpful to its related studies. We have developed a novel system of full-field optical coherence tomography (FF-OCT) for noninvasive 3D subcellular live imaging of preimplantation mouse embryos with no need of dye labeling. 3D digitized embryos can be obtained by image processing. Label-free 3D live imaging is demonstrated for the mouse embryos at various typical preimplantation stages with a spatial resolution of 0.7 µm and imaging rate of 24 fps. Factors that relate to early patterning and polarity, such as pronuclei in zygote, shapes of zona pellucida, location of second polar body, cleavage planes, and the blastocyst axis, can be quantitatively measured. The angle between the two second cleavage planes is accurately measured to be 87 deg. It is shown that FF-OCT provides a potential breakthrough for early patterning, polarity formation, and many other preimplantation-related studies in mammalian developmental biology.

Blood pulse wave velocity (PWV) is an important physiological parameter that characterizes vascular stiffness. In this letter, we present electrocardiogram-synchronized, photoacoustic microscopy for noninvasive quantification of the PWV in the peripheral vessels of living mice. Interestingly, blood pulse wave-induced fluctuations in blood flow speed were clearly observed in arteries and arterioles, but not in veins or venules. Simultaneously recorded electrocardiograms served as references to measure the travel time of the pulse wave between two cross sections of a chosen vessel and vessel segmentation analysis enabled accurate quantification of the travel distance. PWVs were quantified in ten vessel segments from two mice. Statistical analysis shows a linear correlation between the PWV and the vessel diameter which agrees with known physiology.

Retinal and choroidal vascular imaging is an important diagnostic benefit for ocular diseases such as age-related macular degeneration. The current gold standard for vessel visualization is fluorescence angiography. We present a potential non-invasive alternative to image blood vessels based on functional Fourier domain optical coherence tomography (OCT). For OCT to compete with the field of view and resolution of angiography while maintaining motion artifacts to a minimum, ultrahigh-speed imaging has to be introduced. We employ Fourier domain mode locking swept source technology that offers high quality imaging at an A-scan rate of up to 1.68 MHz. We present retinal angiogram over ∼ 48  deg acquired in a few seconds in a single recording without the need of image stitching. OCT at 1060 nm allows for high penetration in the choroid and efficient separate characterization of the retinal and choroidal vascularization.

Abstract unavailable.

One current challenge of studying human tympanic membranes (TM) with optical coherence tomography (OCT) is the implementation of optics that avoid direct contact with the inflamed tissue. At the moment, no commercial device is available. We report an optics design for contactless forward imaging endoscopic optical coherence tomography (EOCT) with a large working distance (WD) and a broad field of view (FOV) by restricting the overall diameter of the probe to be small (3.5 mm), ensuring a sufficient numerical aperture. Our system uses a gradient-index (GRIN) relay lens and a GRIN objective lens, and executes a fan-shaped optical scanning pattern. The WD and FOV can be adjusted by manually changing the distance between the triplet and the GRIN relay lens. The measured lateral resolution is ∼28  μm at a WD of 10 mm with a FOV of 10 mm. Additionally, a camera and an illumination beam path were implemented within the probe for image guidance during investigations of the TM. We demonstrated the performance of the EOCT design by 3-D imaging of a human TM ex vivo and in vivo with a k-linear spectral domain OCT system.

Three-dimensional (3-D) visualization of the fine structures within the lung parenchyma could advance our understanding of alveolar physiology and pathophysiology. Current knowledge has been primarily based on histology, but it is a destructive two-dimensional (2-D) technique that is limited by tissue processing artifacts. Micro-CT provides high-resolution three-dimensional (3-D) imaging within a limited sample size, but is not applicable to intact lungs from larger animals or humans. Optical reflectance techniques offer the promise to visualize alveolar regions of the large animal or human lung with sub-cellular resolution in three dimensions. Here, we present the capabilities of three optical reflectance techniques, namely optical frequency domain imaging, spectrally encoded confocal microscopy, and full field optical coherence microscopy, to visualize both gross architecture as well as cellular detail in fixed, phosphate buffered saline-immersed rat lung tissue. Images from all techniques were correlated to each other and then to corresponding histology. Spatial and temporal resolution, imaging depth, and suitability for in vivo probe development were compared to highlight the merits and limitations of each technology for studying respiratory physiology at the alveolar level.

Tissue water content and molecular microenvironment can provide important intrinsic contrast for cancer imaging. In this work, we examine the relationship between water optical spectroscopic features related to binding state and magnetic resonance imaging (MRI)-measured water diffusion dynamics. Broadband diffuse optical spectroscopic imaging (DOSI) and MR images were obtained from eight patients with locally-advanced infiltrating ductal carcinomas (tumor size = 5.5±3.2  cm). A DOSI-derived bound water index (BWI) was compared to the apparent diffusion coefficient (ADC) of diffusion weighted (DW) MRI. BWI and ADC were positively correlated (R = 0.90, p-value = 0.003) and BWI and ADC both decreased as the bulk water content increased (R = −0.81 and −0.89, respectively). BWI correlated inversely with tumor size (R = −0.85, p-value = 0.008). Our results suggest underlying sensitivity differences between BWI and ADC to water in different tissue compartments (e.g., extracellular vs cellular). These data highlight the potential complementary role of DOSI and DW-MRI in providing detailed information on the molecular disposition of water in breast tumors. Because DOSI is a portable technology that can be used at the bedside, BWI may provide a low-cost measure of tissue water properties related to breast cancer biology.

We present a simple method applicable to common-path Fourier domain optical coherence tomography (OCT) in which the tissue surface is used as the reference arm. We propose using aluminium hydroxide powder as a potential tissue surface diffuser to allow wider application of this method. This technique allows one to avoid placing a reference arm reflective element, such as glass plate, on tissue, and intrinsically avoids both coherent and complex conjugate mirror artifacts associated with glass plates. Aluminium hydroxide can be sprayed onto tissue using spray nozzles commonly found in endoscopes. The sensitivity of the tissue reference arm common-path OCT image is 94 dB for a 50-µs charge-coupled device integration time, and 97.5 dB for a 200-µs CCD integration time.

We report the tomographic imaging of a photodynamic therapy (PDT) photosensitizer, 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH) in vivo with time-domain fluorescence diffuse optical tomography (TD-FDOT). Simultaneous reconstruction of fluorescence yield and lifetime of HPPH was performed before and after PDT. The methodology was validated in phantom experiments, and depth-resolved in vivo imaging was achieved through simultaneous three-dimensional (3-D) mappings of fluorescence yield and lifetime contrasts. The tomographic images of a human head-and-neck xenograft in a mouse confirmed the preferential uptake and retention of HPPH by the tumor 24-h post-injection. HPPH-mediated PDT induced significant changes in fluorescence yield and lifetime. This pilot study demonstrates that TD-FDOT may be a good imaging modality for assessing photosensitizer distributions in deep tissue during PDT monitoring.

A novel technique for axial resolution improvement in Fourier domain optical coherence tomography (FDOCT) is presented. The technique is based on the deconvolution of modulated optical coherence tomography signals. In FDOCT, the real part of the Fourier transform of the interferogram is modulated by a frequency which depends on the position of the interferogram in k space. A slight numerical k shift results in a different modulation frequency. By adding two shifted signals, beating can appear in the A-scan. When the amount of shifting is appropriately selected, deconvolution of the resulting depth profile, using suitable modulated kernels, yields a narrower resolution width. A resolution improvement by a factor of ∼ 7 can be achieved without the need for a broader bandwidth light source.

The purpose of this study is to investigate the potential of intensity modulated fiber optic displacement sensor scanning system for the imaging of dental cavity. Here, we discuss our preliminary results in the imaging of cavities on various teeth surfaces, as well as measurement of the diameter of the cavities which are represented by drilled holes on the teeth surfaces. Based on the analysis of displacement measurement, the sensitivities and linear range for the molar, canine, hybrid composite resin, and acrylic surfaces are obtained at 0.09667  mV/mm and 0.45 mm; 0.775  mV/mm and 0.4 mm; 0.5109  mV/mm and 0.5 mm; and 0.25  mV/mm and 0.5 mm, respectively, with a good linearity of more than 99%. The results also show a clear distinction between the cavity and surrounding tooth region. The stability, simplicity of design, and low cost of fabrication make it suitable for restorative dentistry.

The atrial pacemaker complex is responsible for the initiation and early propagation of cardiac impulses. Optical coherence tomography (OCT), a nondestructive imaging modality with spatial resolutions of ∼ 1 to 15 μm, can be used to identify unique fiber orientation patterns in this region of the heart. Functionally characterized canine sinoatrial nodes (SAN) (n = 7) were imaged using OCT up to ∼ 1  mm below the endocardial tissue surface. OCT images were directly compared to their corresponding histological sections. Fiber orientation patterns unique to the crista terminalis (CT), SAN, and surrounding atrial myocardium were identified with dominant average fiber angles of 89±12  deg, 110±16  deg, and 95±35  deg, respectively. Both the CT and surrounding atrial myocardium displayed predominantly unidirectionally based fiber orientation patterns within each specimen, whereas the SAN displayed an increased amount of fiber disarray manifested quantitatively as a significantly greater standard deviation in fiber angle distribution within specimens [33±7  deg versus 23±5  deg, atrium (p = 0.02); 18±3  deg, CT (p = 0.0003)]. We also identified unique, local patterns of fiber orientation specific to the functionally characterized block zone. We demonstrate the ability of OCT in detecting components of the atrial pacemaker complex which are intimately involved in both normal and abnormal cardiac conduction.

Although several strategies exist for a minimal-invasive treatment of patients with lung failure, the mortality rate of acute respiratory distress syndrome still reaches 30% at minimum. This striking number indicates the necessity of understanding lung dynamics on an alveolar level. To investigate the dynamical behavior on a microscale, we used three-dimensional geometrical and functional imaging to observe tissue parameters including alveolar size and length of embedded elastic fibers during ventilation. We established a combined optical coherence tomography (OCT) and confocal fluorescence microscopy system that is able to monitor the distension of alveolar tissue and elastin fibers simultaneously within three dimensions. The OCT system can laterally resolve a 4.9 μm line pair feature and has an approximately 11 μm full-width-half-maximum axial resolution in air. confocal fluorescence microscopy visualizes molecular properties of the tissue with a resolution of 0.75 μm (laterally), and 5.9 μm (axially) via fluorescence detection of the dye sulforhodamine B specifically binding to elastin. For system evaluation, we used a mouse model in situ to perform lung distension by application of different constant pressure values within the physiological regime. Our method enables the investigation of alveolar dynamics by helping to reveal basic processes emerging during artificial ventilation and breathing.

The authors describe the development of diffuse optical imaging (DOI) technologies, specifically the use of spatial and temporal modulation to control near infrared light propagation in thick tissues. We present theory and methods of DOI focusing on model-based techniques for quantitative, in vivo measurements of endogenous tissue absorption and scattering properties. We specifically emphasize the common conceptual framework of the scalar photon density wave for both temporal and spatial frequency-domain approaches. After presenting the history, theoretical foundation, and instrumentation related to these methods, we provide a brief review of clinical and preclinical applications from our research as well as our outlook on the future of DOI technology.

We describe the auto-oxidation of 3, 4-dihydroxy-L-phenylalanine (L-DOPA) in the synthesis of eumelanin to spontaneously produce fibrils upon drying. The self-assembled fibrils are of characteristic diameter ~ 1 to 2 μm, composed of filaments, and are unidirectional, apart from branches that are formed at typically an angle of 20 to 22 deg. The fibrils are characterized using fluorescence spectroscopy, fluorescence decay times, scanning electron microscopy, atomic force microscopy, and fluorescence lifetime imaging microscopy. The fibrils mimic natural melanin in consisting of core eumelanin with efficient nonradiative properties, but they also display pockets of electronically isolated species with higher radiative rates on the nanosecond timescale. Eumelanin fibrils formed occasionally in solution are tentatively attributed to a scaffold of bacteria or fungus. Fabricating and characterizing novel synthetic eumelanin structures such as fibrils are of interest in helping to reveal a functional structure for eumelanin, in understanding its photophysics, in learning more about L-DOPA as it is used in the treatment of Parkinson's disease, and in producing novel materials which might embody some of the diverse properties of eumelanin.

A Monte Carlo model has been developed to simulate light transport and absorption in neural tissue during infrared neural stimulation (INS). A range of fiber core sizes and numerical apertures are compared illustrating the advantages of using simulations when designing a light delivery system. A range of wavelengths, commonly used for INS, are also compared for stimulation of nerves in the cochlea, in terms of both the energy absorbed and the change in temperature due to a laser pulse. Modeling suggests that a fiber with core diameter of 200 μm and NA = 0.22 is optimal for optical stimulation in the geometry used and that temperature rises in the spiral ganglion neurons are as low as 0.1°C. The results show a need for more careful experimentation to allow different proposed mechanisms of INS to be distinguished.

The open question regarding the compensation of the ocular aberrations between the cornea and the lens is currently being investigated. We report additional insights considering the role of the lens gradient-index (GRIN) profile in third-order ocular aberrations, since this profile changes through life. Thus, we have calculated the contribution of that profile to the ocular aberrations with aging by applying the Seidel third-order theory of tilted and decentered elements, and by using a schematic-eye model. The results show the GRIN profile is needed to account for the decoupling of the aberrations between the cornea and the lens because the geometrical changes of the ocular surfaces with aging are not enough. Therefore, the current developments of aging human-eye models, as well as the experimental studies, cannot neglect the changes of the lens GRIN structure through life when modelling mechanisms of the compensation of ocular aberrations.

We used a liquid-crystal spatial light modulator to project 473 nm light patterns surrounding a region of adherent cells and achieved an arbitrarily micropatterned cell culture. For a group of ~ 60 cells, the cell boundaries fit the pattern of light within 15% deviation of the side length. We also demonstrated a wound-healing experiment with a definite starting temporal point by using this technique. While observing mitochondrial structures in the illuminated cells, we found that the 473 nm light damaged the integrity of mitochondria and thus prohibited cell proliferation in the illuminated region.

We propose a method of calculating the corneal asphericity (Q) and analyze the characteristics of the anterior corneal shape using the tangential radius. Fifty-eight right eyes of 58 subjects were evaluated using the Orbscan II corneal topographer. The Q-values of the flat principal semi-meridians calculated by the sagittal radius were compared to those by the tangential radius. Variation in the Q-value with semi-meridian in the nasal and temporal cornea calculated by the tangential radius was analyzed. There were significant differences in Q-values (P<0.001) between the two methods. The mean Q-values of the flat principal semi-meridians calculated by tangential radius with −0.33±0.10 in the nasal and −0.22±0.12 in the temporal showed more negative than the corresponding Q-values calculated by the sagittal radius. The Q-values calculated by tangential radius became less negative gradually from horizontal semi-meridians to oblique semi-meridians in both nasal and temporal cornea. Variation in Q-value with semi-meridian was more obvious in the nasal cornea. The method of calculating corneal Q using the tangential radius could provide more reasonable and complete Q-value than that by the sagittal radius. The model of a whole anterior corneal surface could be reconstructed on the basis of the above method.

When an erbium-laser pulse is directed into water through a small-diameter fiber tip (FT), the absorption of the laser energy superheats the water and its boiling induces a vapor bubble. We present the influence of different FT geometries and pulse parameters on the vapor-bubble dynamics. In our investigation, we use a free-running erbium: yttrium aluminum garnet (Er:YAG) (λ = 2.94 μm) laser that was designed for laser dentistry. Its pulse is directed into the water through FTs with a flat and conical geometry. Our results show that in the case of the conical FT, a spherical bubble is induced, while a channel-like bubble develops for the flat FT. The ratio between the mechanical energy of the liquid medium and the pulse energy, which we call the optodynamic energy-conversion efficiency, is examined using shadow photography. The results indicate that this efficiency is significantly larger when a conical FT is used and it increases with increasing pulse energy and decreasing pulse duration. The spherical bubbles are compared with the Rayleigh model in order to present the influence of the pulse duration on the dynamics of the bubble's expansion.

Optical tomography is a medical imaging technique based on light propagation in the near infrared (NIR) part of the spectrum. We present a new way of predicting the short-pulsed NIR light propagation using a time-dependent two-dimensional-global radiative transfer equation in an absorbing and strongly anisotropically scattering medium. A cell-vertex finite-volume method is proposed for the discretization of the spatial domain. The closure relation based on the exponential scheme and linear interpolations was applied for the first time in the context of time-dependent radiative heat transfer problems. Details are given about the application of the original method on unstructured triangular meshes. The angular space (4ΠSr) is uniformly subdivided into discrete directions and a finite-differences discretization of the time domain is used. Numerical simulations for media with physical properties analogous to healthy and metastatic human liver subjected to a collimated short-pulsed NIR light are presented and discussed. As expected, discrepancies between the two kinds of tissues were found. In particular, the level of light flux was found to be weaker (inside the medium and at boundaries) in the healthy medium than in the metastatic one.

We examine the morphological and chemical changes in the pulp chamber dentin after using endodontic agents by scanning electron microscopy (SEM), Fourier transform Raman spectroscopy (FT-Raman), and micro energy-dispersive x-ray fluorescence spectrometry (μEDXRF). Thirty teeth were sectioned exposing the pulp chamber and divided by six groups (n = 5): NT-no treatment; CHX-2% chlorhexidine; CHXE-2% chlorhexidine+17% EDTA; E-17% EDTA; SH5-5.25% NaOCl; SH5E-5.25% NaOCl+17% EDTA. The inorganic and organic content was analyzed by FT-Raman. μEDXRF examined calcium (Ca) and phosphorus (P) content as well as Ca/P ratio. Impressions of specimens were evaluated by SEM. Data were submitted to Kruskal-Wallis and Dunn tests (p<0.05). Differences were observed among groups for the 960 cm^-1 peak. Ca and P content differences were significant (SH5>NT = SH5E>CHX>E>CHXE). CHXE and E presented the highest Ca/P ratio values compared to the other groups (p<0.05). The SEM images in the EDTA-treated groups had the highest number of open tubules. Erosion in the tubules was observed in CHX and SH5E groups. Endodontic agents change the inorganic and organic content of pulp chamber dentin. NaOCl used alone, or in association with EDTA, was the most effective agent considering chemical and morphological approaches.

A biometry-based human eye model was developed by using the empirical anatomic and optical data of ocular parameters. The gradient refractive index of the crystalline lens was modeled by concentric conicoid isoindical surfaces and was adaptive to accommodation and age. The chromatic dispersion of ocular media was described by Cauchy equations. The intraocular scattering model was composed of volumetric Mie scattering in the cornea and the crystalline lens, and a diffusive-surface model at the retina fundus. The retina was regarded as a Lambertian surface and was assigned its corresponding reflectance at each wavelength. The optical performance of the eye model was evaluated in CodeV and ASAP and presented by the modulation transfer functions at single and multiple wavelengths. The chromatic optical powers obtained from this model resembled that of the average physiological eyes. The scattering property was assessed by means of glare veiling luminance and compared with the CIE general disability glare equation. By replacing the transparent lens with a cataractous lens, the disability glare curve of cataracts was generated to compare with the normal disability glare curve. This model has high potential for investigating visual performance in ordinary lighting and display conditions and under the influence of glare sources.

This study investigates a method using novel hybrid diffuse optical spectroscopies [near-infrared spectroscopy (NIRS) and diffuse correlation spectroscopy (DCS)] to obtain continuous, noninvasive measurement of absolute blood flow (BF), blood oxygenation, and oxygen consumption rate (vO_{) in exercising skeletal muscle. Healthy subjects (n = 9) performed a handgrip exercise to increase BF and O2 in forearm flexor muscles, while a hybrid optical probe on the skin surface directly monitored oxy-, deoxy-, and total hemoglobin concentrations ([HbO2], [Hb], and THC), tissue oxygen saturation (StO2), relative BF (rBF), and relative oxygen consumption rate (rvO2). The rBF and rO2 signals were calibrated with absolute baseline BF and vO2 obtained through venous and arterial occlusions, respectively. Known problems with muscle-fiber motion artifacts in optical measurements during exercise were mitigated using a novel gating algorithm that determined muscle contraction status based on control signals from a dynamometer. Results were consistent with previous findings in the literature. This study supports the application of NIRS/DCS technology to quantitatively evaluate hemodynamic and metabolic parameters in exercising skeletal muscle and holds promise for improving diagnosis and treatment evaluation for patients suffering from diseases affecting skeletal muscle and advancing fundamental understanding of muscle and exercise physiology.}

We investigate the effect of tissue fluid on the measurement of complex refractive index (RI) of animal tissue. A new model is proposed and verified through experimental results of simulation samples made of glycerol and methyl-red-doped poly(methyl methacrylate). Coupled with polarized optical reflectance measurements performed on several kinds of animal muscle tissues, RIs were resolved using the new model. We find that the tissue fluid existing at the prism-sample interface is unavoidable. We also find that with a change of proportion of the tissue fluid, the RI of muscle tissue can still be measured using the new model.

We report the first demonstration of deep ultraviolet (DUV) Raman imaging of a cell. Nucleotide distributions in a HeLa cell were observed without any labeling at 257 nm excitation with resonant bands attributable to guanine and adenine. Obtained images represent DNA localization at nucleoli in the nucleus and RNA distribution in the cytoplasm. The presented technique extends the potential of Raman microscopy as a tool to selectively probe nucleic acids in a cell with high sensitivity due to resonance.

Second-harmonic-generation (SHG) imaging of mouse ovaries ex vivo was used to detect collagen structure changes accompanying ovarian cancer development. Dosing with 4-vinylcyclohexene diepoxide and 7,12-dimethylbenz[a]anthracene resulted in histologically confirmed cases of normal, benign abnormality, dysplasia, and carcinoma. Parameters for each SHG image were calculated using the Fourier transform matrix and gray-level co-occurrence matrix (GLCM). Cancer versus normal and cancer versus all other diagnoses showed the greatest separation using the parameters derived from power in the highest-frequency region and GLCM energy. Mixed effects models showed that these parameters were significantly different between cancer and normal (P<0.008). Images were classified with a support vector machine, using 25% of the data for training and 75% for testing. Utilizing all images with signal greater than the noise level, cancer versus not-cancer specimens were classified with 81.2% sensitivity and 80.0% specificity, and cancer versus normal specimens were classified with 77.8% sensitivity and 79.3% specificity. Utilizing only images with greater than of 75% of the field of view containing signal improved sensitivity and specificity for cancer versus normal to 81.5% and 81.1%. These results suggest that using SHG to visualize collagen structure in ovaries could help with early cancer detection.

The use of upconverting lanthanide nanoparticles in fast-scanning microscopy is hindered by a long luminescence decay time, which greatly blurs images acquired in a nondescanned mode. We demonstrate herein an image processing method based on Richardson-Lucy deconvolution that mitigates the detrimental effects of their luminescence lifetime. This technique generates images with lateral resolution on par with the system's performance, ∼ 1.2  μm, while maintaining an axial resolution of 5 μm or better at a scan rate comparable with traditional two-photon microscopy. Remarkably, this can be accomplished with near infrared excitation power densities of 850  W/cm², several orders of magnitude below those used in two-photon imaging with molecular fluorophores. By way of illustration, we introduce the use of lipids to coat and functionalize these nanoparticles, rendering them water dispersible and readily conjugated to biologically relevant ligands, in this case epidermal growth factor receptor antibody. This deconvolution technique combined with the functionalized nanoparticles will enable three-dimensional functional tissue imaging at exceptionally low excitation power densities.

We present a quadriwave lateral shearing interferometer used as a wavefront sensor and mounted on a commercial non-modified transmission white-light microscope as a quantitative phase imaging technique. The setup is designed to simultaneously make measurements with both quantitative transmission phase and fluorescence modes: phase enables enhanced contrasted visualization of the cell structure including intracellular organelles, while fluorescence allows a complete and precise identification of each component. After the characterization of the phase measurement reliability and sensitivity on calibrated samples, we use these two imaging modes to measure the characteristic optical path difference between subcellular elements (mitochondria, actin fibers, and vesicles) and cell medium, and demonstrate that phase-only information should be sufficient to identify some organelles without any labeling, like lysosomes. Proof of principle results show that the technique could be used either as a qualitative tool for the control of cells before an experiment, or for quantitative studies on morphology, behavior, and dynamics of cells or cellular components.

Hyperspectral imaging (HSI) is an emerging modality for various medical applications. Its spectroscopic data might be able to be used to noninvasively detect cancer. Quantitative analysis is often necessary in order to differentiate healthy from diseased tissue. We propose the use of an advanced image processing and classification method in order to analyze hyperspectral image data for prostate cancer detection. The spectral signatures were extracted and evaluated in both cancerous and normal tissue. Least squares support vector machines were developed and evaluated for classifying hyperspectral data in order to enhance the detection of cancer tissue. This method was used to detect prostate cancer in tumor-bearing mice and on pathology slides. Spatially resolved images were created to highlight the differences of the reflectance properties of cancer versus those of normal tissue. Preliminary results with 11 mice showed that the sensitivity and specificity of the hyperspectral image classification method are 92.8% to 2.0% and 96.9% to 1.3%, respectively. Therefore, this imaging method may be able to help physicians to dissect malignant regions with a safe margin and to evaluate the tumor bed after resection. This pilot study may lead to advances in the optical diagnosis of prostate cancer using HSI technology.

Existing mouse artery ex vivo perfusion models have utilized arteries such as carotid, uterine, and mesenteric arteries, but not the aorta. However, the aorta is the principal vessel analyzed for atherosclerosis studies in vivo. We have devised a mouse aorta ex vivo perfusion model that can bridge this gap. Aortas from apoE^(-/-) mice are embedded in a transparent, gas-permeable, and elastic polymer matrix [polydimethylsiloxane (PDMS)] and artificially perfused with cell culture medium under cell culture conditions. After 24 h of artificial ex vivo perfusion, no evidence of cellular apoptosis is detected. Utilizing a standard confocal microscope, it is possible to image specific receptor targeting of cells in atherosclerotic plaques during 24 h. Imaging motion artifacts are minimal due to the polymer matrix embedding. Re-embedding of the aorta enables tissue sectioning and immuno-histochemical analysis. The ex vivo data are validated by comparison with in vivo experiments. This model can save animal lives via production of multiple endpoints in a single experiment, is easy to apply, and enables straightforward comparability with pre-existing atherosclerosis in vivo data. It is suited to investigate atherosclerotic disease in particular and vascular biology in general.

SpineLab is a software tool developed for reconstructing neuronal feature skeletons from three-dimensional single- or multi-photon image stacks. These images often suffer from limited resolution and a low signal-to-noise ratio, making the extraction of morphometric information difficult. To overcome this limitation, we have developed a software tool that offers the possibility to create feature skeletons in various modes-automatically as well as with manual interaction. We have named this novel tool SpineLab. In a first step, an investigator adjusts a set of parameters for automatic analysis in an interactive manner, i.e., with online visual feedback, followed by a second step, in which the neuronal feature skeleton can be modified by hand. We validate the ability of SpineLab to reconstruct the entire dendritic tree of identified GFP-expressing neurons and evaluate the accuracy of dendritic spine detection. We report that SpineLab is capable of significantly facilitating the reconstruction of dendrites and spines. Moreover, the automatic approach appears sufficient to detect spine density changes in time-lapse imaging experiments. Taken together, we conclude that SpineLab is an ideal software tool for partially automatic reconstruction of neural cell morphology.

Detection of ultrasound (US)-modulated fluorescence in turbid media is a challenge because of the low level of fluorescent light and the weak modulation of incoherent light. A very limited number of theoretical and experimental investigations have been performed, and this is, to our knowledge, the first demonstration of pulsed US-modulated fluorescence tomography. Experimental results show that the detected signal depends on the acoustic frequency and the fluorescent target's size along the ultrasonic propagation axis. The modulation depth of the detected signal is greatest when the length of the object along the acoustic axis is an odd number of half wavelengths and is weakest when the object is an integer multiple of an acoustic wavelength. Images of a fluorescent tube embedded within a 22- by 13- by 30 mm scattering gel phantom μ_s ∼ 15  cm⁻¹, g = 0.93) with 1-, 1.5-, and 2 MHz frequency US are presented. The modulation depth of the detected signal changes by a factor of 5 depending on the relative size of the object and the frequency. The approach is also verified by some simple experiments in a nonscattering gel and using a theoretical model.

Near infrared (NIR) multispectral imaging is a novel noninvasive technique that maps and quantifies dental caries. The technique has the ability to reduce the confounding effect of stain present on teeth. The aim of this study was to develop and validate a quantitative NIR multispectral imaging system for caries detection and assessment against a histological reference standard. The proposed technique is based on spectral imaging at specific wavelengths in the range from 1000 to 1700 nm. A total of 112 extracted teeth (molars and premolars) were used and images of occlusal surfaces at different wavelengths were acquired. Three spectral reflectance images were combined to generate a quantitative lesion map of the tooth. The maximum value of the map at the corresponding histological section was used as the NIR caries score. The NIR caries score significantly correlated with the histological reference standard (Spearman's Coefficient = 0.774, p<0.01). Caries detection sensitivities and specificities of 72% and 91% for sound areas, 36% and 79% for lesions on the enamel, and 82% and 69% for lesions in dentin were found. These results suggest that NIR spectral imaging is a novel and promising method for the detection, quantification, and mapping of dental caries.

Identification of abnormal pathology in situ remains one of the challenges of medicine. The interpretation of tissue conditions relies mainly on optical assessment, which can be difficult due to inadequate visual differences or poor color delineation. We propose a methodology to identify regions of abnormal tissue in a targeted area based on red, green, blue (RGB) shift analysis employing a simple CCD color camera and light-emitting diode illumination in a whole-field-imaging scheme. The concept involves analysis of RGB components in an image with respect to a reference set of RGB values under different illumination wavelengths. The magnitude of the gray value shift is estimated by calculating the Euclidean distance between their normalized RGB coordinates. The shift values obtained using these concepts are thereafter used to construct pseudo-colored images with high contrast, enabling easy identification of abnormal areas in the tissue. Images processed from experiments conducted with excised Wistar rat colon sample (lightly doped with Alexafluor 488) and with simulated tumor (cancer cell pellet placed on colon) showed clear localization of tumor region. This proposed approach and methodology is expected to find potential applications for the in vivo diagnosis of disease.

This study was designed to evaluate hemodynamic and electrophysiological motor cortex responses to voluntary finger pinching in humans, with simultaneous recording of near-infrared spectroscopy (NIRS) signals and movement-related cortical potentials (MRCP). Six healthy, right-handed subjects performed 100 trials of voluntary right-thumb index-finger pinching with about a 10-second interval at their own pace. Throughout the session, 48 regions over the bilateral motor cortex were assessed by NIRS, while MRCP and electromyogram (EMG) were simultaneously monitored. MRCP started 1536±58  ms before EMG onset and peaked 127±24  ms after EMG onset. NIRS data showed bilateral prefrontal cortex at 0.5±0.1  s before EMG onset and bilateral dorsal premotor cortex activations at 0.6±0.1  s before EMG onset. The hand area of the sensorimotor cortex was activated left-dominantly, seen obviously peaked at 3.7±0.2  s after EMG onset. The comparison between MRCP and NIRS results raised the possibility that the vascular response to neural activity occurs within 4 s with a voluntary pinch task. These results indicate that our technique allows detailed study of the motor control. Our method is a promising strategy for event-related motor control and neurovascular coupling studies.

With the increasing use of small animals for human disease studies, small-animal whole-body molecular imaging plays an important role in biomedical research. Currently, none of the existing imaging modalities can provide both anatomical and glucose molecular information, leading to higher costs of building dual-modality systems. Even with image co-registration, the spatial resolution of the molecular imaging modality is not improved. Utilizing a ring-shaped confocal photoacoustic computed tomography system, we demonstrate, for the first time, that both anatomy and glucose uptake can be imaged in a single modality. Anatomy was imaged with the endogenous hemoglobin contrast, and glucose metabolism was imaged with a near-infrared dye-labeled 2-deoxyglucose.

Detection of multiple fluorophores in conditions of low signal represents a limiting factor for the application of in vivo optical imaging techniques in immunology where fluorescent labels report for different functional characteristics. A noninvasive in vivo Multi-Spectral Normalized Epifluorescence Laser scanning (M-SNELS) method was developed for the simultaneous and quantitative detection of multiple fluorophores in low signal to noise ratios and used to follow T-cell activation and clonal expansion. Colocalized DsRed- and GFP-labeled T cells were followed in tandem during the mounting of an immune response. Spectral unmixing was used to distinguish the overlapping fluorescent emissions representative of the two distinct cell populations and longitudinal data reported the discrete pattern of antigen-driven proliferation. Retrieved values were validated both in vitro and in vivo with flow cytometry and significant correlation between all methodologies was achieved. Noninvasive M-SNELS successfully quantified two colocalized fluorescent populations and provides a valid alternative imaging approach to traditional invasive methods for detecting T cell dynamics.

Optimal treatment of skin cancer before it metastasizes critically depends on early diagnosis and treatment. Imaging spectroscopy and polarized remittance have been utilized in the past for diagnostic purposes, but valuable information can be also obtained from the analysis of skin roughness. For this purpose, we have developed an out-of-plane hemispherical Stokes imaging polarimeter designed to monitor potential skin neoplasia based on a roughness assessment of the epidermis. The system was utilized to study the rough surface scattering for wax samples and human skin. The scattering by rough skin-simulating phantoms showed behavior that is reasonably described by a facet scattering model. Clinical tests were conducted on patients grouped as follows: benign nevi, melanocytic nevus, melanoma, and normal skin. Images were captured and analyzed, and polarization properties are presented in terms of the principal angle of the polarization ellipse and the degree of polarization. In the former case, there is separation between different groups of patients for some incidence azimuth angles. In the latter, separation between different skin samples for various incidence azimuth angles is observed.

Reversible oxidation is emerging as an important regulatory mechanism in protein tyrosine phosphorylation. Generation of hydrogen peroxide (H₂O₂), upon growth factor stimulation, is hypothesized to inhibit activity of protein tyrosine phosphatases (PTPs). This ensures that protein tyrosine kinases can elevate the steady-state level of protein tyrosine phosphorylation, which then allows propagation of the tyrosine phosphorylation signal. However, the effects of H₂O₂ on the kinetics of tyrosine phosphorylation signaling remain poorly understood, especially in living cells. Therefore, we used a genetically encoded Src kinase-specific biosensor based on fluorescence resonance energy transfer (FRET) to image the kinetics of the Src-mediated tyrosine phosphorylation signaling (Src signaling) induced by epidermal growth factor (EGF). We examined the kinetics under increased and decreased H₂O₂ levels. Through a straightforward, quantitative analysis method which characterized the signaling kinetics, we demonstrated that H₂O₂ modulated the amplitude and duration of the signal by inhibiting PTPs' activity. Our evidence also suggested the effect of H₂O₂ on Src activation is mediated by H₂O₂-dependent inhibition of PTPs. Furthermore, we provide evidence showing global elevation of intracellular H₂O₂ level attenuates EGF-induced Src signaling.

There is a high demand for noninvasive imaging techniques for wound assessment. In vivo reflectance confocal laser scanning microscopy (CLSM) represents an innovative optical technique for noninvasive evaluation of normal and diseased skin in vivo at near cellular resolution. This study was designed to test the feasibility of CLSM for noninvasive analysis of cutaneous wound healing in 15 patients (7 male/8 female), including acute and chronic, superficial and deep dermal skin wounds. A commercially available CLSM system was used for the assessment of wound bed and wound margins in order to obtain descriptive cellular and morphological parameters of cutaneous wound repair noninvasively and over time. CLSM was able to visualize features of cutaneous wound repair in epidermal and superficial dermal wounds, including aspects of inflammation, neovascularisation, and tissue remodelling in vivo. Limitations include the lack of mechanic fixation of the optical system on moist surfaces restricting the analysis of chronic skin wounds to the wound margins, as well as a limited optical resolution in areas of significant slough formation. By describing CLSM features of cutaneous inflammation, vascularisation, and epithelialisation, the findings of this study support the role of CLSM in modern wound research and management.

As molecular imaging moves towards lower detection limits, the elimination of endogenous background signals becomes imperative. We present a facile background-suppression technique that specifically segregates the signal from surface-enhanced Raman scattering (SERS)-active nanoparticles (NPs) from the tissue autofluorescence background in vivo. SERS NPs have extremely narrow spectral peaks that do not overlap significantly with endogenous Raman signals. This can be exploited, using specific narrow-band filters, to image picomolar (pM) concentrations of NPs against a broad tissue autofluorescence background in wide-field mode, with short integration times that compare favorably with point-by-point mapping typically used in SERS imaging. This advance will facilitate the potential applications of SERS NPs as contrast agents in wide-field multiplexed biomarker-targeted imaging in vivo.

Quantitative analysis of optical coherence tomography data can be strongly hampered by speckle. Here, we introduce a new method to reduce speckle, which leverages from Fourier-domain configurations and operates on individual axial scans. By subdividing the digitized spectrum into a number of distinct narrower windows, each with a different center frequency, several independent speckle patterns result. These can be averaged to yield a lower-resolution image with strongly reduced speckle. The full resolution image remains available for human interpretation; the low resolution version can be used for parametric imaging or quantitative analysis. We demonstrate this technique using intravascular optical frequency domain imaging data acquired in vivo.

An ultrathin scanning fiber endoscope, originally developed for cancer diagnosis, was used to image dental occlusal surfaces as well as shallow artificially induced enamel erosions from human extracted teeth (n = 40). Enhanced image resolution of occlusal surfaces was obtained using a short-wavelength 405-nm illumination laser. In addition, artificial erosions of varying depths were also imaged with 405-, 404-, 532-, and 635-nm illumination lasers. Laser-induced autofluorescence images of the teeth using 405-nm illumination were also obtained. Contrast between sound and eroded enamel was quantitatively computed for each imaging modality. For shallow erosions, the image contrast with respect to sound enamel was greatest for the 405-nm reflected image. It was also determined that the increased contrast was in large part due to volume scattering with a smaller component from surface scattering. Furthermore, images obtained with a shallow penetration depth illumination laser (405 nm) provided the greatest detail of surface enamel topography since the reflected light does not contain contributions from light reflected from greater depths within the enamel tissue. Multilayered Monte Carlo simulations were also performed to confirm the experimental results.

We present a concept for quantitative characterization of a functional state of an individual eukaryotic cell based on interference imaging. The informative parameters of the phase images of quiescent and mitogen-activated T lymphocytes included the phase thickness, phase volume, the area, and the size of organelles. These parameters were obtained without a special hypothesis about cell structure. Combinations of these parameters generated a "phase portrait" of the cell. A simplified spherical multilayer optic model of a T lymphocyte was used to calculate the refractivity profile, to identify structural elements of the image with the organelles, and to interpret the parameters of the phase portrait. The values of phase image parameters underwent characteristic changes in the course of mitogenic stimulation of T cells; thereby, the functional state of individual cells can be described using these parameters. Because the values of the components of the phase portrait are measured in absolute units, it is possible to compare the parameters of images obtained with different interference microscopes. Thus, the analysis of phase portraits provides a new and perspective approach for quantitative, real-time analysis of subcellular structure and physiologic state of an individual cell.

Non-mydriatic retinal imaging is an important tool for diagnosis and progression assessment of ophthalmic diseases. Because it does not require pharmacological dilation of the patient's pupil, it is essential for screening programs performed by non-medical personnel. A typical camera is equipped with a manual focusing mechanism to compensate for the refractive errors in the eye. However, manual focusing is error prone, especially when performed by inexperienced photographers. In this work, we propose a new and robust focus measure based on a calculation of image anisotropy which, in turn, is evaluated from the directional variance of the normalized discrete cosine transform. Simulation and experimental results demonstrate the effectiveness of the proposed focus measure.

To evaluate if clinical fluorescence imaging of IRDye800CW is feasible on our fluorescence optical mammography system by estimating detection limits assessed by breast-cancer-simulating phantom experiments. Phantoms (2.1  cm³, 0.9  cm³) with IRDye800CW concentrations of 0.5 to 120 nM were suspended in a 550  cm³ measurement cup containing 507 surface-mounted source and detector fibers. The cup was filled with optical matching fluid containing IRDye800CW concentrations of 0, 5, 10, or 20 nM. Tomographic fluorescence images were acquired by exciting IRDye800CW at 730 nm; wavelengths above 750 nm were filtered. Signal intensities were calculated over a volume of interest corresponding to the size and location of the phantom in the reconstructed images. Correlations (R²) were calculated, and detection limits with associated upper 95% prediction interval were estimated. Between-day reproducibility was assessed with intraclass correlation coefficients (ICC). Fluorescent intensities were strongly correlated with phantom IRDye800CW concentrations (R²∶0.983 to 0.999). IRDye800CW detection limits ranged from 0.14 to 2.46 nM (upper 95% prediction limit 4.63 to 18.63 nM). ICC ranged from 0.88 to 1.00. The estimated detection limits for IRDye800CW were in the low-nanomolar range. These results support the start of clinical trials to evaluate the fluorescence optical mammography system using IRDye800CW labeled breast cancer targeting ligands.

The relation between protein structural alterations and tissue dysfunction is a major concern as protein fibrillation and/or aggregation due to structural alterations has been reported in many disease states. In the current study, Fourier transform infrared microspectroscopic imaging has been used to investigate diabetes-induced changes on protein secondary structure and macromolecular content in streptozotocin-induced diabetic rat liver. Protein secondary structural alterations were predicted using neural network approach utilizing the amide I region. Moreover, the role of selenium in the recovery of diabetes-induced alterations on macromolecular content and protein secondary structure was also studied. The results revealed that diabetes induced a decrease in lipid to protein and glycogen to protein ratios in diabetic livers. Significant alterations in protein secondary structure were observed with a decrease in α-helical and an increase in β-sheet content. Both doses of selenium restored diabetes-induced changes in lipid to protein and glycogen to protein ratios. However, low-dose selenium supplementation was not sufficient to recover the effects of diabetes on protein secondary structure, while a higher dose of selenium fully restored diabetes-induced alterations in protein structure.

We present a new integrated micro-endoscopy system combining label-free, fiber-based, real-time C-scan optical-resolution photoacoustic microscopy (F-OR-PAM) and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the F-OR-PAM sub-system is able to reach a resolution of ~ 7 μm. The fluorescence subsystem, which does not require any mechanical scanning, consists of a 447.5-nm-centered diode laser as the light source, an objective lens, and a CCD camera. Proflavine is used as the fluorescent contrast agent by topical application. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single-mode fibers. The absorption of proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural information given by F-OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping visualize angiogenesis and the effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.

Waveguide evanescent field fluorescence microscopy (WEFF) is a novel microscopy technology that allows imaging of a cell's plasma membrane in the vicinity of a glass substrate with high axial resolution, low background and little photobleaching. Time-lapse imaging can be performed to investigate changes in cell morphology in the presence or absence of chemical agents. WEFF microscopy provides a method to investigate plasma membranes of living cells and allows a comparison to simplified model membranes immobilized on planar substrates. The interaction of the nonionic detergent Triton X-100 with plasma membranes of osteoblasts in an aqueous environment was investigated. Solubilization of the membranes very close to the waveguide surface was visualized and related to the three-stage solubilisation model proposed for liposomes and supported lipid bilayers. Findings for the plasma membranes of cells are in excellent agreement with results reported for these artificial model systems.

Glaucoma is a blinding disease for which intraocular pressure (IOP) is the only treatable risk factor. The mean IOP is regulated through the aqueous outflow system, which contains the trabecular meshwork (TM). Considerable evidence indicates that trabecular tissue movement regulates the aqueous outflow and becomes abnormal during glaucoma; however, such motion has thus far escaped detection. The purpose of this study is to describe anovel use of a phase-sensitive optical coherence tomography (PhS-OCT) method to assess pulse-dependent TM movement. For this study, we used enucleated monkey eyes, each mounted in an anterior segment holder. A perfusion system was used to control the mean IOP as well as to provide IOP sinusoidal transients (amplitude 3 mmHg, frequency 1 pulse/second) in all experiments. Measurements were carried out at seven graded mean IOPs (5, 8, 10, 20, 30, 40, and 50 mm Hg). We demonstrate that PhS-OCT is sensitive enough to image/visualize TM movement synchronous with the pulse-induced IOP transients, providing quantitative measurements of dynamic parameters such as velocity, displacement, and strain rate that are important for assessing the biomechanical compliance of the TM. We find that the largest TM displacement is in the area closest to Schlemm's canal (SC) endothelium. While maintaining constant ocular pulse amplitude, an increase of mean IOP results in a decrease of TM displacement and mean size of the SC. These results demonstrate that the PhS-OCT is a useful imaging technique capable of assessing functional properties necessary to maintain IOP in a healthy range, offering a new diagnostic alternative for glaucoma.

The formation of kidney stones is a common and highly studied disease, which causes intense pain and presents a high recidivism. In order to find the causes of this problem, the characterization of the main compounds is of great importance. In this sense, the analysis of the composition and structure of the stone can give key information about the urine parameters during the crystal growth. But the usual methods employed are slow, analyst dependent and the information obtained is poor. In the present work, the near infrared (NIR)-hyperspectral imaging technique was used for the analysis of 215 samples of kidney stones, including the main types usually found and their mixtures. The NIR reflectance spectra of the analyzed stones showed significant differences that were used for their classification. To do so, a method was created by the use of artificial neural networks, which showed a probability higher than 90% for right classification of the stones. The promising results, robust methodology, and the fast analytical process, without the need of an expert assistance, lead to an easy implementation at the clinical laboratories, offering the urologist a rapid diagnosis that shall contribute to minimize urolithiasis recidivism.

We investigated the potential of the pH-sensitive dye, CypHer5E, conjugated to Herceptin (pH-Her) for the sensitive detection of breast tumors in mice using noninvasive time-domain near-infrared fluorescence imaging and different methods of data analysis. First, the fluorescence properties of pH-Her were analyzed as function of pH and/or dye-to-protein ratio, and binding specificity was confirmed in cell-based assays. Subsequently, the performance of pH-Her in nude mice bearing orthotopic HER2-positive (KPL-4) and HER2-negative (MDA-MB-231) breast carcinoma xenografts was compared to that of an always-on fluorescent conjugate Alexa Fluor 647-Herceptin (Alexa-Her). Subtraction of autofluorescence and lifetime (LT)-gated image analyses were performed for background fluorescence suppression. In mice bearing HER2-positive tumors, autofluorescence subtraction together with the selective fluorescence enhancement of pH-Her solely in the tumor's acidic environment provided high contrast-to-noise ratios (CNRs). This led to an improved sensitivity of tumor detection compared to Alexa-Her. In contrast, LT-gated imaging using LTs determined in model systems did not improve tumor-detection sensitivity in vivo for either probe. In conclusion, pH-Her is suitable for sensitive in vivo monitoring of HER2-expressing breast tumors with imaging in the intensity domain and represents a promising tool for detection of weak fluorescent signals deriving from small tumors or metastases.

We present a reconstruction method to eliminate the autocorrelation noise (ACN) in optical coherence tomography (OCT). In this method, the optical fields scattered from the sample features are regarded as the response of a sparse finite impulse response (FIR) filter. Then the OCT reconstruction is formulated as one of identifying the parameters of a sparse FIR filter, which are obtained via an ℓ1 optimization with soft thresholding. The experimental results show that the proposed method can obtain OCT reconstruction results with effective attenuation of ACN.

We report on a Raman microspectroscopic characterization of the inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis (UC). Therefore, Raman maps of human colon tissue sections were analyzed by utilizing innovative chemometric approaches. First, support vector machines were applied to highlight the tissue morphology ( = Raman spectroscopic histopathology). In a second step, the biochemical tissue composition has been studied by analyzing the epithelium Raman spectra of sections of healthy control subjects (n = 11), subjects with CD (n = 14), and subjects with UC (n = 13). These three groups exhibit significantly different molecular specific Raman signatures, allowing establishment of a classifier (support-vector-machine). By utilizing this classifier it was possible to separate between healthy control patients, patients with CD, and patients with UC with an accuracy of 98.90%. The automatic design of both classification steps (visualization of the tissue morphology and molecular classification of IBD) paves the way for an objective clinical diagnosis of IBD by means of Raman spectroscopy in combination with chemometric approaches.

Three-dimensional gold nanostructures fabricated through a novel convective assembly method are treated thermally to obtain a nanoisland morphology. The new structure is proved to be adequate for the detection of bovine growth hormone, by using an immunoassay method based on the localized surface plasmon resonance band of gold. The nanoisland structures are integrated into a microfluidic device and the spectral measurements are carried out by introducing the device directly in the light beam of a ultraviolet-visible spectrophotometer. The principal motivation for this work is the need for a simple and rapid method of detection of hormone levels in milk and milk products.

We report for the first time the implementation of transnasal, image-guided Raman endoscopy to directly assess Raman spectral properties of nasopharyngeal and laryngeal tissue in vivo during clinical endoscopic examinations. A rapid 785-nm excitation Raman endoscopy system, coupled with a miniaturized fiber-optic Raman probe, was utilized for real-time, in vivo Raman measurements of different anatomical locations in the head and neck. A total of 874 high-quality in vivo Raman spectra were successfully acquired from different anatomic locations of the nasopharynx and larynx [i.e., posterior nasopharynx (PN) (n = 521), the fossa of Rosenmüller (FOR) (n = 157), and true laryngeal vocal chords (LVC) (n = 196)] in 23 normal subjects at transnasal endoscopy. Difference spectra and principal component analysis (PCA) were employed for tissue characterization, uncovering the tissue variability at the biomolecular level. The PCA-linear discriminant analysis (LDA) provides sensitivity of 77.0% and specificity of 89.2% for differentiation between PN and FOR, and sensitivity of 67.3% and specificity of 76.0% for distinguishing LVC and PN using the leave-one-subject-out, cross-validation method. This work demonstrates that transnasal, image-guided Raman endoscopy can be used to acquire in vivo Raman spectra from the nasopharynx and larynx in real time. Significant Raman spectral differences (p<0.05) identified as reflecting the distinct composition and morphology in the nasopharynx and larynx should be considered to be important parameters in the interpretation and rendering of diagnostic decision algorithms for in vivo tissue diagnosis and characterization in the head and neck.

Raman spectroscopy has been employed to identify differences in the biochemical constitution of malignant [basal cell carcinoma (BCC) and melanoma (MEL)] cells compared to normal skin tissues, with the goal of skin cancer diagnosis. We collected Raman spectra from compounds such as proteins, lipids, and nucleic acids, which are expected to be represented in human skin spectra, and developed a linear least-squares fitting model to estimate the contributions of these compounds to the tissue spectra. We used a set of 145 spectra from biopsy fragments of normal (30 spectra), BCC (96 spectra), and MEL (19 spectra) skin tissues, collected using a near-infrared Raman spectrometer (830 nm, 50 to 200 mW, and 20 s exposure time) coupled to a Raman probe. We applied the best-fitting model to the spectra of biochemicals and tissues, hypothesizing that the relative spectral contribution of each compound to the tissue Raman spectrum changes according to the disease. We verified that actin, collagen, elastin, and triolein were the most important biochemicals representing the spectral features of skin tissues. A classification model applied to the relative contribution of collagen III, elastin, and melanin using Euclidean distance as a discriminator could differentiate normal from BCC and MEL.

There are increased interests on using multiphoton imaging and spectroscopy for skin tissue characterization and diagnosis. However, most studies have been done with just a few excitation wavelengths. Our objective is to perform a systematic study of the two-photon fluorescence (TPF) properties of skin fluorophores, normal skin, and diseased skin tissues. A nonlinear excitation-emission-matrix (EEM) spectroscopy system with multiphoton imaging guidance was constructed. A tunable femtosecond laser was used to vary excitation wavelengths from 730 to 920 nm for EEM data acquisition. EEM measurements were performed on excised fresh normal skin tissues, seborrheic keratosis tissue samples, and skin fluorophores including: NADH, FAD, keratin, melanin, collagen, and elastin. We found that in the stratum corneum and upper epidermis of normal skin, the cells have large sizes and the TPF originates from keratin. In the lower epidermis, cells are smaller and TPF is dominated by NADH contributions. In the dermis, TPF is dominated by elastin components. The depth resolved EEM measurements also demonstrated that keratin structure has intruded into the middle sublayers of the epidermal part of the seborrheic keratosis lesion. These results suggest that the imaging guided TPF EEM spectroscopy provides useful information for the development of multiphoton clinical devices for skin disease diagnosis.

Collagen is a rich component in skin that provides skin structure integrity; however, its contribution to the absorption and scattering properties of various types of skin has not been extensively studied. We considered the contribution of the collagen to the absorption spectrum of in vivo normal skin and keloids of 12 subjects derived from our diffuse reflectance spectroscopy (DRS) system in the wavelength range from 550 to 860 nm. It was found that the collagen concentration, the hemoglobin oxygen saturation, and the reduced scattering coefficient of keloids were remarkably different from that of normal skin. Our results suggest that our DRS system could assist clinicians in understanding the functional and structural condition of keloid scars. In the future, we will evaluate the accuracy of our system in the keloid diagnosis and investigate the applicability of our system for other skin-collagen-related studies.

In the field of biomedical optics, Raman spectroscopy is a powerful tool for probing the chemical composition of biological samples. In particular, fiber Raman probes play a crucial role for in vivo and ex vivo tissue analysis. However, the high-fluorescence background typically contributed by the auto fluorescence from both a tissue sample and the fiber-probe interferes strongly with the relatively weak Raman signal. Here we demonstrate the implementation of wavelength-modulated Raman spectroscopy (WMRS) to suppress the fluorescence background while analyzing tissues using fiber Raman probes. We have observed a significant signal-to-noise ratio enhancement in the Raman bands of bone tissue, which have a relatively high fluorescence background. Implementation of WMRS in fiber-probe-based bone tissue study yielded usable Raman spectra in a relatively short acquisition time (~ 30 s), notably without any special sample preparation stage. Finally, we have validated its capability to suppress fluorescence on other tissue samples such as adipose tissue derived from four different species.

Dispersion encoded full-range (DEFR) frequency-domain optical coherence tomography (FD-OCT) and its enhanced version, fast DEFR, utilize dispersion mismatch between sample and reference arm to eliminate the ambiguity in OCT signals caused by non-complex valued spectral measurement, thereby numerically doubling the usable information content. By iteratively suppressing asymmetrically dispersed complex conjugate artifacts of OCT-signal pulses the complex valued signal can be recovered without additional measurements, thus doubling the spatial signal range to cover the full positive and negative sampling range. Previously the computational complexity and low processing speed limited application of DEFR to smaller amounts of data and did not allow for interactive operation at high resolution. We report a graphics processing unit (GPU)-based implementation of fast DEFR, which significantly improves reconstruction speed by a factor of more than 90 in respect to CPU-based processing and thereby overcomes these limitations. Implemented on a commercial low-cost GPU, a display line rate of ∼21,000  depth scans/s for 2048  samples/depth scan using 10 iterations of the fast DEFR algorithm has been achieved, sufficient for real-time visualization in situ.

An implantable, optical oxygenation and perfusion sensor to monitor liver transplants during the two-week period following the transplant procedure is currently being developed. In order to minimize the number of animal experiments required for this research, a phantom that mimics the optical, anatomical, and physiologic flow properties of liver parenchyma is being developed as well. In this work, the suitability of this phantom for liver parenchyma perfusion research was evaluated by direct comparison of phantom perfusion data with data collected from in vivo porcine studies, both using the same prototype perfusion sensor. In vitro perfusion and occlusion experiments were performed on a single-layer and on a three-layer phantom perfused with a dye solution possessing the absorption properties of oxygenated hemoglobin. While both phantoms exhibited response patterns similar to the liver parenchyma, the signal measured from the multilayer phantom was three times higher than the single layer phantom and approximately 21 percent more sensitive to in vitro changes in perfusion. Although the multilayer phantom replicated the in vivo flow patterns more closely, the data suggests that both phantoms can be used in vitro to facilitate sensor design.

Near-infrared spectroscopy (NIRS) is a noninvasive technique evaluating microvascular function. The aim of this study was to assess the reproducibility of NIRS parameters during reactive hyperemia induced by a 5 min brachial artery occlusion. Twenty-four healthy young males (mean 34±8 years old) had two microvascular function evaluations by NIRS over a 7 to 30-day period (mean 16±10 days). Intra-subject and inter-observer reproducibility were evaluated with intraclass correlation coefficient (ICC), coefficient of variation (CV), and standard error of measurement (SEM%) for every parameter. Mean NIRS parameters did not differ between both evaluations. Reproducibility was greatest for muscle oxygen consumption (ICC: 0.84; CV: 6.51%; SEM: 7.11%), time to basal O2Hb (ICC: 0.63, CV: 20.04%, SEM 27.22%), time to maximal O2Hb (ICC: 0.71; CV: 15.61%; SEM: 19.27%), peak of O2Hb (ICC: 0.63, CV: 6.68%, SEM 8.53%), time to maximal tHb (ICC: 0.73, CV: 19,61%, SEM 24.56%) and area under the O2Hb and tHb curves (ICC: 0.68, CV: 16.15%, SEM 22.93% and ICC: 0.62, CV: 18.59%, SEM 26.64%, respectively). Moreover, inter-observer reproducibility ranged from excellent to perfect (ICC from 0.85 to 1.00) for every parameter. NIRS parameters during reactive hyperemia are highly reproducible which enables their repeated measurement to study microvascular function in healthy subjects.

Nonobtrusive pulse rate measurement using a webcam is considered. We demonstrate how state-of-the-art algorithms based on independent component analysis suffer from a sorting problem which hinders their performance, and propose a novel algorithm based on constrained independent component analysis to improve performance. We present how the proposed algorithm extracts a photoplethysmography signal and resolves the sorting problem. In addition, we perform a comparative study between the proposed algorithm and state-of-the-art algorithms over 45 video streams using a finger probe oxymeter for reference measurements. The proposed algorithm provides improved accuracy: the root mean square error is decreased from 20.6 and 9.5 beats per minute (bpm) for existing algorithms to 3.5 bpm for the proposed algorithm. An error of 3.5 bpm is within the inaccuracy expected from the reference measurements. This implies that the proposed algorithm provided performance of equal accuracy to the finger probe oximeter.

An inverse Monte Carlo based model has been developed to extract intrinsic fluorescence from turbid media. The goal of this work was to experimentally validate the model to extract intrinsic fluorescence of three biologically meaningful fluorophores related to metabolism from turbid media containing absorbers and scatterers. Experimental studies were first carried out on tissue-mimicking phantoms that contained individual fluorophores and their combinations, across multiple absorption, scattering, and fluorophore concentrations. The model was then tested in a murine tumor model to determine both the kinetics of fluorophore uptake as well as overall tissue fluorophore concentration through extraction of the intrinsic fluorescence of an exogenous contrast agent that reports on glucose uptake. Results show the model can be used to recover the true intrinsic fluorescence spectrum with high accuracy (R² = 0.988) as well as accurately compute fluorophore concentration in both single and multiple fluorophores phantoms when appropriate calibration standards are available. In the murine tumor, the model-corrected intrinsic fluorescence could be used to differentiate drug dose injections between different groups. A strong linear correlation was observed between the extracted intrinsic fluorescence intensity and injected drug dose, compared with the distorted turbid tissue fluorescence.

In order to model the thermal effect of laser exposure of the iris during laser corneal surgery, we simulated the temperature increase in porcine cadaver iris. The simulation data for the 60 kHz FS60 Laser showed that the temperature increased up to 1.23°C and 2.45°C (at laser pulse energy 1 and 2 µJ, respectively) by the 24 second procedure time. Calculated temperature profiles show good agreement with data obtained from ex vivo experiments using porcine cadaver iris. Simulation results of different types of femtosecond lasers indicate that the Laser in situ keratomileusis procedure does not present a safety hazard to the iris.

Photodynamic therapy (PDT) is a photochemical modality approved for cancer treatment. PDT has demonstrated efficacy in early stage lung cancer and esophageal cancer. The accumulation of photosensitizers in cancer cells is necessary to enhance the therapeutic benefits of PDT; however, photosensitizers have low uptake efficiency. To overcome this limitation, a drug delivery system, such as the hemagglutinating virus of Japan envelope (HVJ-E) vector, is required. In this study, the combination of PDT and HVJ-E was investigated for enhancing the efficacy of PDT. The photosensitizers that were evaluated included 5-aminolaevulinic acid (5-ALA), protoporphyrin IX (PPIX), and HVJ-PPIX. The uptake of the photosensitizers as increased twenty-fold with the addition of HVJ-E. The cytotoxicity of conventional 5-ALA was enhanced by the addition of HVJ-E vector. In conclusion, HVJ-E vector improved the uptake of photosensitizers and the PDT effect.

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