Label-free imaging of bulk arterial tissue is demonstrated using a multimodal nonlinear optical microscope based on a photonic crystal fiber and a single femtosecond oscillator operating at 800 nm. Colocalized imaging of extracellular elastin fibers, fibrillar collagen, and lipid-rich structures within aortic tissue obtained from atherosclerosis-prone myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits is demonstrated through two-photon excited fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering, respectively. These images are shown to differentiate healthy arterial wall, early atherosclerotic lesions, and advanced plaques. Clear pathological changes are observed in the extracellular matrix of the arterial wall and correlated with progression of atherosclerotic disease as represented by the age of the WHHLMI rabbits.

In this paper, we demonstrate that the optical microangiography (OAMG) is capable of depth-resolved imaging of directional blood perfusion within both retinal and choroid in the posterior segment of human eye. The study uses an OMAG system operating at 840 nm with an imaging speed at 27,000 A-scans per second. Sequentially registered multiple OMAG projection maps of small areas (~1 mm×1 mm) are combined to provide directional blood flow images for a larger field of view. It takes ~3.7 sec to image a small area (1×1 mm²), and ~2.5 min for a larger field (3×3 mm²). Finally, we show superior performance of OMAG in providing functional images of capillary level microcirculation at different depths of retina and choroid that correlate well with the standard retinal pathology.

The electrical properties of axons critically influence the nature of communication between neurons. However, due to their small size, direct measurement of membrane potential dynamics in intact and complex mammalian axons has been a challenge. Furthermore, quantitative optical measurements of axonal membrane potential dynamics have not been available. To characterize the basic principles of somatic voltage signal propagation in intact axonal arbors, second-harmonic-generation (SHG) imaging is applied to cultured mouse hippocampal neurons. When FM4-64 is applied extracellularly to dissociated neurons, whole axonal arbors are visualized by SHG imaging. Upon action potential generation by somatic current injection, nonattenuating action potentials are recorded in intact axonal arbors. Interestingly, however, both current- and voltage-clamp recordings suggest that nonregenerative subthreshold somatic voltage changes at the soma are poorly conveyed to these axonal sites. These results reveal the nature of membrane potential dynamics of cultured hippocampal neurons, and further show the possibility of SHG imaging in physiological investigations of axons.

Two-photon luminescence (TPL) from gold nanorods shows considerable potential in biological imaging. We study the imaging of gold nanorods in Madin-Darby canine kidney (MDCK) cells using fluorescence lifetime imaging microscopy (FLIM). FLIM provides images with better contrast and sensitivity than intensity imaging. The characteristic fluorescence lifetime of gold nanorods is found to be less than 100 ps, which can be used to distinguish gold nanorods from other fluorescent labels and endogenous fluorophores in lifetime imaging.

We present the light scattering of individual Plasmodium falciparum-parasitized human red blood cells (Pf-RBCs), and demonstrate progressive alterations to the scattering signal arising from the development of malaria-inducing parasites. By selectively imaging the electric fields using quantitative phase microscopy and a Fourier transform light scattering technique, we calculate the light scattering maps of individual Pf-RBCs. We show that the onset and progression of pathological states of the Pf-RBCs can be clearly identified by the static scattering maps. Progressive changes to the biophysical properties of the Pf-RBC membrane are captured from dynamic light scattering.

Fluorescence image-guided surgery (FIGS) is an emerging technique in oncology, neurology, and cardiology. To adapt intraoperative imaging for various surgical applications, increasingly flexible and compact FIGS instruments are necessary. We present a compact, portable FIGS system and demonstrate its use in cardiovascular mapping in a preclinical model of myocardial ischemia. Our system uses fiber optic delivery of laser diode excitation, custom optics with high collection efficiency, and compact consumer-grade cameras as a low-cost and compact alternative to open surgical FIGS systems. Dramatic size and weight reduction increases flexibility and access, and allows for handheld use or unobtrusive positioning over the surgical field.

Serotonin is both a ubiquitous neurotransmitter in the central nervous system and an important immunomodulator involved in various immune responses. The ability to unambiguously detect serotonin is therefore imperative in biomedical research. However, detection of serotonin and related indoles using immunohistochemistry has been largely limited by their small molecular size and the resultant uncertainty in antibody specificity. Here we show that surface-enhanced Raman spectroscopy (SERS) can be used to detect and distinguish serotonin from its various closely related precursors and metabolites. Compared with traditional antibody-based methods, SERS is highly specific and capable of real-time detection. We also quantify the relative concentration of serotonin against a background of other indoles using SERS. We expect this optical detection method to directly benefit a variety of immune and nervous systems studies involving serotonin.

Förster resonance energy transfer (FRET) microscopy is commonly used to monitor protein interactions with filter-based imaging systems, which require spectral bleedthrough (or cross talk) correction to accurately measure energy transfer efficiency (E). The double-label (donor+acceptor) specimen is excited with the donor wavelength, the acceptor emission provided the uncorrected FRET signal and the donor emission (the donor channel) represents the quenched donor (qD), the basis for the E calculation. Our results indicate this is not the most accurate determination of the quenched donor signal as it fails to consider the donor spectral bleedthrough (DSBT) signals in the qD for the E calculation, which our new model addresses, leading to a more accurate E result. This refinement improves E comparisons made with lifetime and spectral FRET imaging microscopy as shown here using several genetic (FRET standard) constructs, where cerulean and venus fluorescent proteins are tethered by different amino acid linkers.

This PDF file contains the editorial “Special Section Guest Editorial: Photons Plus Ultrasound: Imaging and Sensing” for JBO Vol. 15 Issue 02

We present a new-generation optical-resolution confocal photoacoustic microscope, consisting of a 0.25-numerical aperture optical microscope objective and a 75-MHz center-frequency spherically focused ultrasonic transducer. Experiments verified that this microscope has a transverse resolution of 2 μm, which is the highest to our knowledge among all photoacoustic imaging systems. In situ imaging of mouse ears shows the feasibility of resolving individual red blood cells in microvessels using the current system.

With a refined ultrasound-array-based real-time photoacoustic microscopy (UA-PAM) system, we demonstrate the feasibility of noninvasive in vivo imaging of human pulsatile dynamics. The system, capable of real-time B-scan imaging at 50 Hz and high-speed 3-D imaging, is validated by imaging the subcutaneous microvasculature in rats and humans. After the validation, a human artery around the palm-wrist area is imaged, and its pulsatile dynamics, including the arterial pulsatile motion and changes in hemoglobin concentration, is monitored with 20-ms B-scan imaging temporal resolution. To our knowledge, this is the first demonstration of real-time photoacoustic imaging of human physiological dynamics. Our results show that UA-PAM can potentially enable many new possibilities for studying functional and physiological dynamics in both preclinical and clinical imaging settings.

We propose a new method to measure transverse flow velocity based on photoacoustic Doppler bandwidth broadening, which is determined by the geometry of the probe-beam and the velocity of the transverse flow. By exploiting pulsed laser excitation and raster motor scanning, three-dimensional structure and flow velocity can be imaged simultaneously. In addition, the flow direction can be determined with bidirectional scanning. In a flowing suspension of red-dyed microspheres (diameter: 6 μm), transverse flow speeds ranging from 0 to 2.5 mm/s as well as flow direction were measured. A cross-sectional flow image was also obtained with the tube laid in a zigzag pattern.

We present our findings from a real-time laser optoacoustic imaging system (LOIS). The system utilizes a Q-switched Nd:YAG laser; a standard 128-channel ultrasonic linear array probe; custom electronics and custom software to collect, process, and display optoacoustic (OA) images at 10 Hz. We propose that this system be used during preoperative mapping of forearm vessels for hemodialysis treatment. To demonstrate the real-time imaging capabilities of the system, we show OA images of forearm vessels in a volunteer and compare our results to ultrasound images of the same region. Our OA images show blood vessels in high contrast. Manipulations with the probe enable us to locate and track arteries and veins of a forearm in real time. We also demonstrate the ability to combine a series of OA image slices into a volume for spatial representation of the vascular network. Finally, we use frame-by-frame analysis of the recorded OA video to measure dynamic changes of the crossection of the ulnar artery.

For photoacoustic imaging, usually point-like detectors are used. As a special sensing technology for photoacoustic imaging, integrating detectors have been investigated that integrate the acoustic pressure over an area or line that is larger than the imaged object. Different kinds of optical fiber-based detectors are compared regarding their sensitivity and resolution in three-dimensional photoacoustic tomography. In the same type of interferometer, polymer optical fibers yielded much higher sensitivity than glass fibers. Fabry-Pérot glass-fiber interferometers in turn gave higher sensitivity than Mach-Zehnder-type interferometers. Regarding imaging resolution, the single-mode glass fiber showed the best performance. Last, three-dimensional images of phantoms and insects using a glass-fiber-based Fabry-Pérot interferometer as integrating line detector are presented.

A device for three-dimensional (3-D) photoacoustic tomography with resolution in the range of tens of micrometers is presented that uses a light beam for interferometric detection of acoustic waves. Reconstruction of the 3-D initial pressure distribution from the signals representing line integrals of the acoustic field is a two-step process. It uses an inversion of 2-D wave propagation to obtain line projections of the initial pressure distribution and the inverse Radon transform. The light beam, propagating freely in a water bath, is scanned either in an πarc- or box-shaped curve around the object. Simulations are performed to compare the two scanning procedures. The projection images are obtained either using the filtered back projection algorithm for the -arc scanning mode or the frequency domain algorithm for the box scanning mode. While the former algorithm provides slightly better image quality, the latter is about 20 times faster. The ability of the photoacoustic tomography device to create 3-D images with constant resolution throughout the reconstruction volume is demonstrated experimentally using a human hair phantom. These measurements revealed a 3-D resolution below 100 µm. In a second experiment, 3-D imaging of an isolated mouse heart is demonstrated to show the applicability for preclinical and biological research.

Previous research correcting for variable speed of sound in photoacoustic tomography (PAT) based on a generalized radon transform (GRT) model assumes first-order geometrical acoustics (GA) approximation. In the GRT model, the pressure is related to the optical absorption, in an acoustically inhomogeneous medium, through integration over nonspherical isochronous surfaces. Previous research based on the GRT model assumes that the path taken by acoustic rays is linear and neglects amplitude perturbations to the measured pressure. We have derived a higher-order GA expression that takes into account the first-order effect in the amplitude of the measured signal and higher-order perturbation to the travel times. The higher-order perturbation to travel time incorporates the effect of ray bending. Incorrect travel times can lead to image distortion and blurring. These corrections are expected to impact image quality and quantitative PAT. We have previously shown that travel-time corrections in 2-D suggest that perceivable differences in the isochronous surfaces can be seen when the second-order travel-time perturbations are taken into account with a 10% speed-of-sound variation. In this work, we develop iterative image reconstruction algorithms that incorporate this higher-order GA approximation assuming that the speed of sound map is known. We evaluate the effect of higher-order GA approximation on image quality and accuracy.

The ability to visualize and track temporarily or permanently implanted metal devices is important in many applications ranging from diagnosis to therapy. Specifically, reliable imaging of metal needles is required in today's clinical settings. Currently, ultrasound is utilized to image a needle inserted into tissue in real time. However, the diagnostic value and tracking ability of these images depends highly on the orientation of the needle, and also its proximity to regions of interest in the tissue. We examine the use of photoacoustic imaging combined with current ultrasound imaging methods to obtain high-contrast images of commonly used needles in the body. Experiments were performed using 21 G and 30 G needles inserted into ex vivo porcine tissue and tissue-mimicking phantoms. The needles and surrounding tissue were imaged using an ultrasound imaging system interfaced with the pulsed laser source necessary for photoacoustic imaging. The results suggest that photoacoustic imaging, combined with ultrasound imaging, is capable of real-time, high-contrast, and high-spatial-resolution visualization of metal implants within anatomical landmarks of the background tissue.

Optoacoustic (OA) tomography has demonstrated utility in identifying blood-rich malignancies in breast tissue. We describe the development and characterization of a laser OA imaging system for the prostate (LOIS-P). The system consists of a fiber-coupled Q-switched laser operating at 757 nm, a commercial 128-channel ultrasonic probe, a digital signal processor, and software that uses the filtered radial back-projection algorithm for image reconstruction. The system is used to reconstruct OA images of a blood-rich lesion induced in vivo in a canine prostate. OA images obtained in vivo are compared to images acquired using ultrasound, the current gold standard for guiding biopsy of the prostate. Although key structural features such as the urethra could be identified with both imaging techniques, a bloody lesion representing a highly vascularized tumor could only be clearly identified in OA images. The advantages and limitations of both forward and backward illumination modes are also evaluated by collecting OA images of phantoms simulating blood vessels within tissue. System resolution is estimated to be 0.2 mm in the radial direction of the acoustic array. The minimum detectable pressure signal is 1.83 Pa. Our results encourage further development toward a dual-modality OA/ultrasonic system for prostate imaging and image-guided biopsy.

The data acquisition speed in photoacoustic computed tomography (PACT) is limited by the laser repetition rate and the number of parallel ultrasound detecting channels. Reconstructing an image with fewer measurements can effectively accelerate the data acquisition and reduce the system cost. We adapt compressed sensing (CS) for the reconstruction in PACT. CS-based PACT is implemented as a nonlinear conjugate gradient descent algorithm and tested with both phantom and in vivo experiments.

We have developed an integrated photoacoustic imaging (PAI) and high-intensity focused ultrasound (HIFU) system for solid tumor treatments. A single-element, spherically focused ultrasonic transducer, with a central frequency of 5 MHz, was used to induce HIFU lesions in soft tissue. The same ultrasonic transducer was also used as a detector during PAI to guide HIFU ablation. The use of same transducer for PAI and HIFU can reduce the requirement on acoustic windows during the imaging-guided therapy, as well as ensuring the correct alignment between the therapeutic beam and the planned treatment volume. During an experiment, targeted soft tissue was first imaged by PAI. The resulted image was used to plan the subsequent HIFU ablation. After the HIFU ablation, targeted soft tissue was imaged again by PAI to evaluate the effectiveness of treatments. Good contrast was obtained between photoacoustic images before and after HIFU ablation. In conclusion, our results demonstrated that PAI technology may potentially be integrated with HIFU ablation for image-guided therapy.

A 3-D optoacoustic imaging system was used to visualize thermal lesions produced in vivo using high-intensity focused ultrasound (HIFU). A 7.5-MHz, surgical, focused transducer with a radius of curvature of 35 mm and an aperture diameter of 23 mm was used to generate HIFU. A pulsed laser, which could operate at 755 nm and 1064 nm, was used to illuminate excised tissue and mice using a bifurcated fiber bundle resulting in two wide beams of light. Tomographic images were obtained while the specimens were rotated within a sphere outlined by a concave arc-shaped array of 64 piezo-composite transducers. These images were then combined to reconstruct 3-D volume images (voxel resolution 0.5 mm), which were acquired before and after HIFU exposure. In vivo optoacoustic images acquired at 1064 nm provided visualization of HIFU lesions. The lesion was indicated by a negative optoacoustic contrast. The molecular nature of such contrast may possibly be associated with reduction of the optical absorption due to reduced concentration of blood, tissue dehydration, denaturation of proteins and porphyrins, and reduction of thermoacoustic efficiency in the thermally treated tissue. These preliminary results demonstrate the potential of optoacoustic imaging to assess and monitor the progress of HIFU therapy.

A new, freely available third party MATLAB toolbox for the simulation and reconstruction of photoacoustic wave fields is described. The toolbox, named k-Wave, is designed to make realistic photoacoustic modeling simple and fast. The forward simulations are based on a k-space pseudo-spectral time domain solution to coupled first-order acoustic equations for homogeneous or heterogeneous media in one, two, and three dimensions. The simulation functions can additionally be used as a flexible time reversal image reconstruction algorithm for an arbitrarily shaped measurement surface. A one-step image reconstruction algorithm for a planar detector geometry based on the fast Fourier transform (FFT) is also included. The architecture and use of the toolbox are described, and several novel modeling examples are given. First, the use of data interpolation is shown to considerably improve time reversal reconstructions when the measurement surface has only a sparse array of detector points. Second, by comparison with one-step, FFT-based reconstruction, time reversal is shown to be sufficiently general that it can also be used for finite-sized planar measurement surfaces. Last, the optimization of computational speed is demonstrated through parallel execution using a graphics processing unit.

We have designed a protease-sensitive imaging probe for optoacoustic imaging whose absorption spectrum changes upon cleavage by a protease of interest. The probe comprises an active site, a derivative of chlorophyll or natural photosynthetic bacteriochlorophyll that absorbs in the near infrared, conjugated to a peptide backbone specific to the protease being imaged. The uncleaved molecules tend to aggregate in dimers and trimers, causing a change in the absorption spectrum relative to that of the monomer. Upon cleavage, the probe molecules deaggregate, giving rise to a spectrum characteristic of monomers. We show using photospectrometry that the two forms of the probe have markedly different absorption spectra, which could allow for in vivo optoacoustic identification using a multiwavelength imaging strategy. Optoacoustic measurements using a narrow-band dye laser find spectral peaks in the two forms of the probe at the expected location. The optoacoustic signal from the uncleaved probe is found to be considerably weaker than that of the cleaved probe, perhaps due to poor optical-acoustic coupling in the aggregated molecules. However, ultimately, it is detection of the cleaved probe that is of the greatest import, since it reports on the protease activity of interest.

Due to their optical absorption properties, metallic nanoparticles are excellent photoacoustic imaging contrast agents. A silver nanosystem is presented here as a potential contrast agent for photoacoustic imaging and image-guided therapy. Currently, the nanosystem consists of a porous silver layer deposited on the surface of spherical silica cores ranging in diameter from 180 to 520 nm. The porous nature of the silver layer will allow for release of drugs or other therapeutic agents encapsulated in the core in future applications. In their current PEGylated form, the silver nanosystem is shown to be nontoxic in vitro at concentrations of silver up to 2 mg/ml. Furthermore, the near-infrared absorbance properties of the nanosystem are demonstrated by measuring strong, concentration-dependent photoacoustic signal from the silver nanosystem embedded in an ex vivo tissue sample. Our study suggests that silver nanosystems can be used as multifunctional agents capable of augmenting image-guided therapy techniques.

We investigate the saturation effect, which describes the violation of the linearity between the measured photoacoustic amplitude and the object's optical absorption coefficient in functional photoacoustic imaging when the optical absorption in the object increases. We model the optical energy deposition and photoacoustic signal generation and detection in a semi-infinite optical absorbing object. Experiments are carried out by measuring photoacoustic signals generated from an ink-filled plastic tube. The saturation effect is studied by varying the optical absorption coefficient in the model and the ink concentration in the photoacoustic experiments. By changing the center frequency of the ultrasonic detector, the requirement to minimize the saturation effect in functional photoacoustic imaging is established.

A photoacoustic imaging system that incorporates a commercial ultrasonic camera for real-time imaging of two-dimensional (2-D) projection planes in tissue at video rate (30 Hz) is presented. The system uses a Q-switched frequency-doubled Nd:YAG pulsed laser for photoacoustic generation. The ultrasonic camera consists of a 2-D 12×12 mm CCD chip with 120×120 piezoelectric sensing elements used for detecting the photoacoustic pressure distribution radiated from the target. An ultrasonic lens system is placed in front of the chip to collect the incoming photoacoustic waves, providing the ability for focusing and imaging at different depths. Compared with other existing photoacoustic imaging techniques, the camera-based system is attractive because it is relatively inexpensive and compact, and it can be tailored for real-time clinical imaging applications. Experimental results detailing the real-time photoacoustic imaging of rubber strings and buried absorbing targets in chicken breast tissue are presented, and the spatial resolution of the system is quantified.

We report experimentally observed ultrasound-modulated fluorescence (UMF) from a submillimeter tube filled with rhodamine B aqueous solution. The tube was submerged in water and a scattering medium. Based on the measured data, we find that the UMF signals might be generated from three mechanisms: modulation of the excitation light, modulation of the emission light, and modulation of the properties of fluorophore. In addition, a linear relationship between the UMF and the drive voltage applied to the ultrasound transducer is found.

Current innovations in optical imaging, measurement techniques, and data analysis algorithms express the need for reliable testing and comparison methods. We present the design and characterization of silicone elastomer-based optical phantoms. Absorption is included by adding a green dye and scattering by adding TiO₂ or SiO₂ particles. Optical coherence tomography measurements demonstrate a linear dependence of the attenuation coefficient with scatterer concentration in the absence of absorbers. Optical transmission spectroscopy of the nonscattering absorbing phantoms shows a linear concentration dependent absorption coefficient. Both types of samples are stable over a period of 6 months. Confocal microscopy of the samples demonstrates a homogeneous distribution of the scatterers, albeit with some clustering. Based on layers with thicknesses as small as 50 μm, we make multifaceted structures resembling flow channels, (wavy) skin-like structures, and a layered and curved phantom resembling the human retina. Finally, we demonstrate the ability to incorporate gold nanoparticles within the phantoms. In conclusion, our phantoms are easy to make, are based on affordable materials, exhibit well-defined and controllable thickness, refractive index, absorption, and scattering coefficients, are homogeneous, and allow the incorporation of novel types of nanoparticle contrast agents. We believe our phantoms fulfill many of the requirements for an "ideal" tissue phantom, and will be particularly suited for novel optical coherence tomography applications.

We deal with light diffusion in mismatched N-layered turbid media having a finite or an infinitely thick Nth layer. We focus on time-resolved light propagation in both the frequency and time domains. Based on our results for the steady-state domain, solutions of the N-layered diffusion equations in the frequency and time domains are obtained by applying the Fourier transform technique. Different methods for calculation of the inverse Fourier transform are studied to validate the solutions, showing relative differences typically smaller than 10^-6. The solutions are compared to Monte Carlo simulations, revealing good agreement. Finally, by applying the Laplace and Fourier transforms we derive a fast (≈ 1 ms) and accurate analytical solution for the time domain reflectance from a two-layered turbid medium having equal reduced scattering coefficients and refractive indices in both layers.

We deal with light diffusion in N-layered turbid media. The steady-state diffusion equation is solved for N-layered turbid media having a finite or an infinitely thick N'th layer. Different refractive indices are considered in the layers. The Fourier transform formalism is applied to derive analytical solutions of the fluence rate in Fourier space. The inverse Fourier transform is calculated using four different methods to test their performance and accuracy. Further, to avoid numerical errors, approximate formulas in Fourier space are derived. Fast solutions for calculation of the spatially resolved reflectance and transmittance from the N-layered turbid media (≈10 ms) with small relative differences (<10^-7) are found. Additionally, the solutions of the diffusion equation are compared to Monte Carlo simulations for turbid media having up to 20 layers.

We describe the amplitude and resolution trends of the signals acquired by turbidity suppression through optical phase conjugation (TSOPC) with samples that span the ballistic and diffusive scattering regimes. In these experiments, the light field scattered through a turbid material is written into a hologram, and a time-reversed copy of the light field is played back through the sample. In this manner, the wavefront originally incident on the sample is reconstructed. We examine a range of scattering samples including chicken breast tissue sections of increasing thickness and polyacrylamide tissue-mimicking phantoms with increasing scattering coefficients. Our results indicate that only a small portion of the scattered wavefront (<0.02%) must be collected to reconstruct a TSOPC signal. Provided the sample is highly scattering, all essential angular information is contained within such small portions of the scattered wavefront due to randomization by scattering. A model is fitted to our results, describing the dependence of the TSOPC signal on other measurable values within the system and shedding light on the efficiency of the phase conjugation process. Our results describe the highest level of scattering that has been phase conjugated in biological tissues to date.

A quantitative colposcopic imaging system for the diagnosis of early cervical cancer is evaluated in a clinical study. This imaging technology based on 3-D active stereo vision and motion tracking extracts diagnostic information from the kinetics of acetowhitening process measured from the cervix of human subjects in vivo. Acetowhitening kinetics measured from 137 cervical sites of 57 subjects are analyzed and classified using multivariate statistical algorithms. Cross-validation methods are used to evaluate the performance of the diagnostic algorithms. The results show that an algorithm for screening precancer produced 95% sensitivity (SE) and 96% specificity (SP) for discriminating normal and human papillomavirus (HPV)-infected tissues from cervical intraepithelial neoplasia (CIN) lesions. For a diagnostic algorithm, 91% SE and 90% SP are achieved for discriminating normal tissue, HPV infected tissue, and low-grade CIN lesions from high-grade CIN lesions. The results demonstrate that the quantitative colposcopic imaging system could provide objective screening and diagnostic information for early detection of cervical cancer.

Atrial fibrillation (AF) is the most common irregular heart rhythm and the mortality rate for patients with AF is approximately twice the mortality rate for patients with normal sinus rhythm (NSR). Some research has indicated that myocardial fibrosis plays an important role in predisposing patients to AF. Therefore, realizing the relationship between myocardial collagen fibrosis and AF is significant. Second-harmonic generation (SHG) is an optically nonlinear coherent process to image the collagen network. We perform SHG microscopic imaging of the collagen fibers in the human atrial myocardium. Utilizing the SHG images, we can identify the differences in morphology and the arrangement of collagen fibers between NSR and AF tissues. We also quantify the arrangement of the collagen fibers using Fourier transform images and calculating the values of angle entropy. We indicate that SHG imaging, a nondestructive and reproducible method to analyze the arrangement of collagen fibers, can provide explicit information about the relationship between myocardial fibrosis and AF.

Scanning laser image correlation (SLIC) is an optical correlation technique for measuring the fluid velocity of particles suspended in a liquid. This technique combines laser scanning of an arbitrary pattern with pair cross-correlation between any two points in the pattern. SLIC overcomes many of the limitations of other optical correlation techniques for flow measurement, such as laser speckle, spatial temporal image correlation spectroscopy, and two-foci methods. One of the main advantages of SLIC is that the concept can be applied to measurements on a range of scales through simple zooming or modifications in the instrumentation. Additionally, SLIC is relatively insensitive to instrument noise through the use of correlation analysis and is insensitive to background. SLIC can provide detailed information about the direction and pattern of flow. SLIC has potential applications ranging from microfluidics to blood flow measurements.

Two-color nonlinear absorption imaging has been previously demonstrated with endogenous contrast of hemoglobin and melanin in turbid media using transmission-mode detection and a dual-laser technology approach. For clinical applications, it would be generally preferable to use backscattering mode detection and a simpler single-laser technology. We demonstrate that imaging in backscattering mode in turbid media using nonlinear absorption can be obtained with as little as 1-mW average power per beam with a single laser source. Images have been achieved with a detector receiving backscattered light at a 45-deg angle relative to the incoming beams' direction. We obtain images of capillary tube phantoms with resolution as high as 20 μm and penetration depth up to 0.9 mm for a 300-μm tube at SNR ~1 in calibrated scattering solutions. Simulation results of the backscattering and detection process using nonimaging optics are demonstrated. A Monte Carlo-based method shows that the nonlinear signal drops exponentially as the depth increases, which agrees well with our experimental results. Simulation also shows that with our current detection method, only 2% of the signal is typically collected with a 5-mm-radius detector.

Fluorescence lifetime imaging (FLi) could potentially improve exogenous near-infrared (NIR) fluorescence imaging, because it offers the capability of discriminating a signal of interest from background, provides real-time monitoring of a chemical environment, and permits the use of several different fluorescent dyes having the same emission wavelength. We present a high-power, LED-based, NIR light source for the clinical translation of wide-field (larger than 5 cm in diameter) FLi at frequencies up to 35 MHz. Lifetime imaging of indocyanine green (ICG), IRDye 800-CW, and 3,3-diethylthiatricarbocyanine iodide (DTTCI) was performed over a large field of view (10 cm by 7.5 cm) using the LED light source. For comparison, a laser diode light source was employed as a gold standard. Experiments were performed both on the bench by diluting the fluorescent dyes in various chemical environments in Eppendorf tubes, and in vivo by injecting the fluorescent dyes mixed in Matrigel subcutaneously into CD-1 mice. Last, measured fluorescence lifetimes obtained using the LED and the laser diode sources were compared with those obtained using a state-of-the-art time-domain imaging system and with those previously described in the literature. On average, lifetime values obtained using the LED and the laser diode light sources were consistent, exhibiting a mean difference of 3% from the expected values and a coefficient of variation of 12%. Taken together, our study offers an alternative to laser diodes for clinical translation of FLi and explores the use of relatively low frequency modulation for in vivo imaging.

Despite the unique brain imaging capabilities and advantages of functional near-infrared spectroscopy (fNIRS), including portability and comprehensive hemodynamic measurement, widespread acceptance in the neuroimaging community has been hampered by low spatial resolution and image localization errors. While recent technical developments such as high-density diffuse optical tomography (HD-DOT) have, in principle, been shown to have superior in silico image quality, the majority of optical imaging studies are still conducted with sparse fNIRS arrays, perhaps partially because the performance increases of HD-DOT appear incremental. Without a quantitative comparative analysis between HD-DOT and fNIRS, using both simulation and in vivo neuroimaging, the implications of the new HD-DOT technology have been difficult to judge. We present a quantitative comparison of HD-DOT and two commonly used fNIRS geometries using (1) standard metrics of image quality, (2) simulated brain mapping tasks, and (3) in vivo visual cortex mapping results in adult humans. The results show that better resolution and lower positional errors are achieved with HD-DOT and that these improvements provide a substantial advancement in neuroimaging capability. In particular, we demonstrate that HD-DOT enables detailed phase-encoded retinotopic mapping, while sparse arrays are limited to imaging individual block-design visual stimuli.

A multiphoton microscope incorporating a Hartmann-Shack (HS) wavefront sensor to control the ultrafast laser beam's wavefront aberrations has been developed. This instrument allowed us to investigate the impact of the laser beam aberrations on two-photon autofluorescence imaging of human retinal tissues. We demonstrated that nonlinear microscopy images are improved when laser beam aberrations are minimized by realigning the laser system cavity while wavefront controlling. Nonlinear signals from several human retinal anatomical features have been detected for the first time, without the need of fixation or staining procedures. Beyond the improved image quality, this approach reduces the required excitation power levels, minimizing the side effects of phototoxicity within the imaged sample. In particular, this may be important to study the physiology and function of the healthy and diseased retina.

Noninvasive detection of skin microcirculation is very significant for clinical diagnosis and therapy of peripheral vascular disease. In this study, an optical clearing method enables a laser speckle contrast imaging (LSCI) technique to image dermal blood flow through intact rat skin. The optical clearing effect of rat skin in vitro caused by a mixture of PEG-400 and Thiazone is evaluated by a commercially available spectrophotometer with an integrating sphere. A LSCI system is applied to image the dermal blood flow of in vivo rat skin after topical treatment of the agents. The results show that the reduced scattering coefficient (630 nm) is down to 60% of the initial value after 40 min of topical treatment of the mixture on skin in vitro. In vivo experimental results show that a 12-min treatment of the mixture on the epidermis can make the skin transparent and dermal vessels can be observed, while treatment with pure PEG-400 cannot. The process of skin optical clearing lasts, and the skin can be recovered by treatment of saline solution. Because optical clearing decreases the temporal contrast, dermal blood flow can be obtained by the laser speckle contrast imaging technique.

A new spot centroid detection algorithm for a Shack-Hartmann wavefront sensor (SHWFS) is experimentally investigated. The algorithm is a kind of dynamic tracking algorithm that tracks and calculates the corresponding spot centroid of the current spot map based on the spot centroid of the previous spot map, according to the strong correlation of the wavefront slope and the centroid of the corresponding spot between temporally adjacent SHWFS measurements. That is, for adjacent measurements, the spot centroid movement will usually fall within some range. Using the algorithm, the dynamic range of an SHWFS can be expanded by a factor of three in the measurement of tilt aberration compared with the conventional algorithm, more than 1.3 times in the measurement of defocus aberration, and more than 2 times in the measurement of the mixture of spherical aberration plus coma aberration. The algorithm is applied in our SHWFS to measure the distorted wavefront of the human eye. The experimental results of the adaptive optics (AO) system for retina imaging are presented to prove its feasibility for highly aberrated eyes.

The neurodevelopmental outcome of neonatal intensive care unit (NICU) infants is a major clinical concern with many infants displaying neurobehavioral deficits in childhood. Functional neuroimaging may provide early recognition of neural deficits in high-risk infants. Near-infrared spectroscopy (NIRS) has the advantage of providing functional neuroimaging in infants at the bedside. However, limitations in traditional NIRS have included contamination from superficial vascular dynamics in the scalp. Furthermore, controversy exists over the nature of normal vascular, responses in infants. To address these issues, we extend the use of novel high-density NIRS arrays with multiple source-detector distances and a superficial signal regression technique to infants. Evaluations of healthy term-born infants within the first three days of life are performed without sedation using a visual stimulus. We find that the regression technique significantly improves brain activation signal quality. Furthermore, in six out of eight infants, both oxy- and total hemoglobin increases while deoxyhemoglobin decreases, suggesting that, at term, the neurovascular coupling in the visual cortex is similar to that found in healthy adults. These results demonstrate the feasibility of using high-density NIRS arrays in infants to improve signal quality through superficial signal regression, and provide a foundation for further development of high-density NIRS as a clinical tool.

We present high-resolution wide-field imaging of retinal and choroidal blood perfusion with optical microangiography (OMAG) technology. Based on spatial frequency analysis, OMAG is capable of visualizing the vascular perfusion map down to capillary-level resolution. An OMAG system operating at 840 nm is used with an A-scan rate of 27,000 Hz, axial resolution of 8 µm, and sensitivity of 98 dB. To achieve wide-field imaging, we capture 16 optical coherence tomography (OCT) 3-D datasets in a sequential order, which together provide an area of ~7.4×7.4 mm² at the posterior segment of the human eye. For each of these datasets, the bulk tissue motion artifacts are eliminated by applying a phase compensation method based on histogram estimation of bulk motion phases, while the displacements occurring between adjacent B-frames are compensated for by 2-D cross correlation between two adjacent OMAG flow images. The depth-resolved capability of OMAG imaging also provides volumetric information on the ocular circulations. Finally, we compare the clinical fluorescein angiography and indocyanine green angiography imaging results with the OMAG results of blood perfusion map within the retina and choroid, and show excellent agreement between these modalities.

We develop and test a new method for automatic determination of vessel wall diameters from image stacks obtained using two-photon laser scanning microscopy (TPLSM) on viable arteries in perfusion flow chambers. To this extent, a new method is proposed for estimating the parameters of a circle describing the inner diameter of the blood vessels. The new method is based on the Hough transform and the observation that three points that are not colinear uniquely define a circle. By only storing the estimated center location, the computational and memory costs of the Hough transform can be greatly reduced. We test the algorithm on 20 images and compare the result with a ground-truth established by human volunteers and a standard least-squares technique. With errors of 3 to 5%, the algorithm enables accurate estimation of the vessel diameters from image stacks containing only small parts of the vessel cross section. Combined with TPLSM imaging of anatomical vessel wall properties, potentially, the algorithm enables the correlation of structural and functional properties of large intact arteries simultaneously, without requirements for additional experiments.

We present a structured light system to dynamically measure human chest wall motion for respiratory volume estimation. Based on a projection of an encoded color pattern and a few active markers attached to the trunk, respiratory volumes are obtained by evaluating the 3-D topographic changes of the chest wall in an anatomically consistent measuring region during respiration. Three measuring setups are established: a single-sided illuminating-recording setup for standing posture, an inclined single-sided setup for supine posture, and a double-sided setup for standing posture. Results are compared with the pneumotachography and show good agreement in volume estimations [correlation coefficient: R>0.99 (P<0.001) for all setups]. The isovolume tests present small variations of the obtained volume during the isovolume maneuver (standard deviation<0.085 L for all setups). After validation by the isovolume test, an investigation of a patient with pleural effusion using the proposed method shows pulmonary functional differences between the diseased and the contralateral sides of the thorax, and subsequent improvement of this imbalance after drainage. These results demonstrate the proposed optical method is capable of not only whole respiratory volume evaluation with high accuracy, but also regional pulmonary function assessment in different chest wall behaviors, with the advantage of whole-field measurement.

Nearly real-time visualization of 3-D volumes is crucial for the use of optical coherence tomography (OCT) during microsurgery. With an ultrahigh speed spectral domain OCT coupled to a surgical microscope, on-line display of 7.2 rendered volumes at 87 megapixels per second is demonstrated. Calculating the A-scans from the spectra is done on a quad-core personal computer (PC), while dedicated software for the 3-D rendering is executed on a high performance video card. Imaging speed is practically only limited by the readout of the camera. First experiments show the feasibility of real-time 3-D OCT for guided interventions.

The main curative therapy for patients with nonsmall cell lung cancer is surgery. Despite this, the survival rate is only 50%, therefore it is important to more efficiently diagnose and predict prognosis for lung cancer patients. Raman spectroscopy is useful in the diagnosis of malignant and premalignant lesions. The aim of this study is to investigate the ability of Raman microscopy to diagnose lung cancer from surgically resected tissue sections, and predict the prognosis of these patients. Tumor tissue sections from curative resections are mapped by Raman microscopy and the spectra analzsed using multivariate techniques. Spectra from the tumor samples are also compared with their outcome data to define their prognostic significance. Using principal component analysis and random forest classification, Raman microscopy differentiates malignant from normal lung tissue. Principal component analysis of 34 tumor spectra predicts early postoperative cancer recurrence with a sensitivity of 73% and specificity of 74%. Spectral analysis reveals elevated porphyrin levels in the normal samples and more DNA in the tumor samples. Raman microscopy can be a useful technique for the diagnosis and prognosis of lung cancer patients receiving surgery, and for elucidating the biochemical properties of lung tumors.

A wide-field four-channel fluorescence imager has been developed. The instrument uses four expanded laser beams to image a large section (6 mm×9 mm). An object can be sequentially illuminated with any combination of 408-, 532-, 658-, and 784-nm lasers for arbitrary (down to 1 ms) exposure times for each laser. Just two notch filters block scattered light from all four lasers. The design approach described here offers great flexibility in treatment of objects, very good sensitivity, and a wide field of view at low cost. There appears to be no commercial instrument capable of simultaneous fluorescence imaging of a wide field of view with four-laser excitation. Some possible applications are following events such as flow and mixing in microchannel systems, the transmission of biological signals across a culture, and following simulations of biological membrane diffusion. It can also be used in DNA sequencing by synthesis to follow the progress of the photolytic removal of dye and terminator. Without utilizing its time resolution, it can be used to obtain four independent images of a single tissue section stained with four targeting agents, with each coupled to a different dye matching one of the lasers.

In conventional fluorescence or confocal microscopy, emitted light is generated not only in the focal plane but also above and below. The situation is different in multiphoton-induced fluorescence and multiphoton-induced higher harmonic generation. Here, restriction of signal generation to a single focal point permits that all emitted photons can contribute to image formation if collected, regardless of their path through the specimen. Often, the intensity of the emitted light is rather low in biological specimens. We present a method to significantly increase the fraction of photons collected by an epi (backward) detector by placing a simple mirror, an aluminum-coated coverslip, directly under the sample. Samples investigated include fluorescent test slides, collagen gels, and thin-layered, intact mouse skeletal muscles. Quantitative analysis revealed an intensity increase of second- and third-harmonic generated signal in skeletal muscle of nine- and sevenfold respectively, and of fluorescent signal in test slides of up to twofold. Our approach thus allows significant signal improvement also for situations were a forward detection is impossible, e.g., due to the anatomy of animals in intravital microscopy.

Imaging hematoxylin-and-eosin-stained cancerous histological sections with multicontrast nonlinear excitation fluorescence, second- and third-harmonic generation (THG) microscopy reveals cellular structures with extremely high image contrast. Absorption and fluorescence spectroscopy together with second hyperpolarizability measurements of the dyes shows that strong THG appears due to neutral hemalum aggregation and is subsequently enhanced by interaction with eosin. Additionally, fluorescence lifetime imaging microscopy reveals eosin fluorescence quenching by hemalums, showing better suitability of only eosin staining for fluorescence microscopy. Multicontrast nonlinear microscopy has the potential to differentiate between cancerous and healthy tissue at a single cell level.

Nondestructive optical imaging methods such as optical coherence tomography (OCT) have been proposed for characterizing engineered tissues such as collagen gels. In our study, OCT was used to image collagen gels with different seeding densities of smooth muscle cells (SMCs), including acellular gels, over a five-day period during which the gels contracted and became turbid with increased optical scattering. The gels were characterized quantitatively by their optical properties, specified by analysis of OCT data using a theoretical model. At 6 h, seeded cell density and scattering coefficient (µ_s) were correlated, with µ_s equal to 10.8 cm^-1/(10⁶ cells/mL). Seeded cell density and the scattering anisotropy (g) were uncorrelated. Over five days, the reflectivity in SMC gels gradually doubled with little change in optical attenuation, which indicated a decrease in g that increased backscatter, but only a small drop in µ_s. At five days, a subpopulation of sites on the gel showed substantially higher reflectivity (approximately a tenfold increase from the first 24 h). In summary, the increased turbidity of SMC gels that develops over time is due to a change in the structure of collagen, which affects g, and not simply due to a change in number density of collagen fibers due to contraction.

We use second harmonic generation (SHG) microscopy to assess changes in collagen structure of murine cervix during cervical remodeling of normal pregnancy and in a preterm birth model. Visual inspection of SHG images revealed substantial changes in collagen morphology throughout normal gestation. SHG images collected in both the forward and backward directions were analyzed quantitatively for changes in overall mean intensity, forward to backward intensity ratio, collagen fiber size, and porosity. Changes in mean SHG intensity and intensity ratio take place in early pregnancy, suggesting that submicroscopic changes in collagen fibril size and arrangement occur before macroscopic changes become evident. Fiber size progressively increased from early to late pregnancy, while pores between collagen fibers became larger and farther apart. Analysis of collagen features in premature cervical remodeling show that changes in collagen structure are dissimilar from normal remodeling. The ability to quantify multiple morphological features of collagen that characterize normal cervical remodeling and distinguish abnormal remodeling in preterm birth models supports future studies aimed at development of SHG endoscopic devices for clinical assessment of collagen changes during pregnancy in women and for predicting risk of preterm labor which occurs in 12.5% of all pregnancies.

Previous investigations on mammalian cells have shown that microgravity, either that experienced in space, or simulated on earth, causes severe cellular modifications that compromise tissue determination and function. The aim of this study is to investigate, in real time, the morphological changes undergone by cells experiencing simulated microgravity by using digital holographic microscopy (DHM). DHM analysis of living mouse myoblasts (C2C12) is undertaken under simulated microgravity with a random positioning machine. The DHM analysis reveals cytoskeletal alterations similar to those previously reported with conventional methods, and in agreement with conventional brightfield fluorescence microscopy a posteriori investigation. Indeed, DHM is shown to be able to noninvasively and quantitatively detect changes in actin reticular formation, as well as actin distribution, in living unstained samples. Such results were previously only obtainable with the use of labeled probes in conjunction with conventional fluorescence microscopy, with all the classically described limitations in terms of bias, bleaching, and temporal resolution.

Videokeratometers and Scheimpflug cameras permit accurate estimation of corneal surfaces. From height data it is possible to adjust analytical surfaces that will be later used for aberration calculation. Zernike polynomials are often used as adjusting polynomials, but they have shown to be not precise when describing highly irregular surfaces. We propose a combined zonal and modal method that allows an accurate reconstruction of corneal surfaces from height data, diminishing the influence of smooth areas over irregular zones and vice versa. The surface fitting error is decreased in the considered cases, mainly in the central region, which is more important optically. Therefore, the method can be established as an accurate resampling technique.

Skin cancer is the most common form of human cancer. Its early diagnosis and timely treatment is of paramount importance for dermatology and surgical oncology. In this study, we evaluate the use of reflectance and fluorescence confocal microscopy for detecting skin cancers in an in-vivo trial with B16F10 melanoma and SCCVII squamous cell carcinoma in mice. For the experiments, the mice are anesthetized, then the tumors are infiltrated with aqueous solution of methylene blue and imaged. Reflectance images are acquired at 658 nm. Fluorescence is excited at 658 nm and registered in the range between 690 and 710 nm. After imaging, the mice are sacrificed. The tumors are excised and processed for hematoxylin and eosin histopathology, which is compared to the optical images. The results of the study indicate that in-vivo reflectance images provide valuable information on vascularization of the tumor, whereas the fluorescence images mimic the structural features seen in histopathology. Simultaneous dye-enhanced reflectance and fluorescence confocal microscopy shows promise for the detection, demarcation, and noninvasive monitoring of skin cancer development.

Spectropolarimetric skin imaging is becoming an attractive technique for early detection of skin cancer. Using two liquid crystal retarders in combination with a dual-band passive spectral filter and two linear polarizers, we demonstrate the spectral and polarimetric imaging of skin tissue in the near infrared. Based on this concept, a compact prototype module has been built and is being used for clinical evaluation.

The capability of optical coherence tomography (OCT) to perform "optical biopsy" of tissues within a depth range of 1 to 2 mm with micron-scale resolution in real time makes it a promising biomedical imaging modality for dermatologic applications. Three high-speed, spectrometer-based frequency-domain OCT systems operating at 800 nm (20,000 A-scans/s), 1060 nm, and 1300 nm (both 47,000 A-scans/s) at comparable signal-to-noise ratio (SNR), SNR roll-off with scanning depth, and transverse resolution (<15 µm) were used to acquire 3-D tomograms of glabrous and hairy human skin in vivo. Images obtained using these three systems were compared in terms of penetration depth, resolution, and contrast. Normal as well as abnormal sites like moles and scar tissue were examined. In this preliminary study, skin pigmentation had little effect on penetration accomplished at three different wavelengths. The epidermis and dermal-epidermal junction could be properly delineated using OCT at 800 nm, and this wavelength offered better contrast over the other two wavelength regions. OCT at 1300 nm permits imaging of deeper dermal layers, critical for detecting deeper tumor boundaries and other deeper skin pathologies. The performance at 1060 nm compromises between the other wavelengths in terms of penetration depth and image contrast.

A compact high-power fiber-based femtosecond laser system is presented for coherent anti-Stokes Raman scattering/second-harmonic generation (CARS/SHG) microscopy, and quantitatively compared with a conventional picosecond optical parametric oscillator (OPO)-based system. While the broad spectral width of the femtosecond pulses results in 2.5 times lower image contrast and limited spectral selectivity, lipid stores, myosin, and collagen filaments in living cells can clearly be identified at 60 times lower excitation powers compared to the picosecond system. Visually the images contain the same information. Together with simple operation, small footprint, and low cost, the capabilities of this high-power all-fiber-based laser system promise a more general use of nonlinear microscopy within the biosciences.

Early detection of neoplasia in patients with Barrett's esophagus is essential to improve outcomes. The aim of this ex vivo study was to evaluate the ability of high-resolution microendoscopic imaging and quantitative image analysis to identify neoplastic lesions in patients with Barrett's esophagus. Nine patients with pathologically confirmed Barrett's esophagus underwent endoscopic examination with biopsies or endoscopic mucosal resection. Resected fresh tissue was imaged with fiber bundle microendoscopy; images were analyzed by visual interpretation or by quantitative image analysis to predict whether the imaged sites were non-neoplastic or neoplastic. The best performing pair of quantitative features were chosen based on their ability to correctly classify the data into the two groups. Predictions were compared to the gold standard of histopathology. Subjective analysis of the images by expert clinicians achieved average sensitivity and specificity of 87% and 61%, respectively. The best performing quantitative classification algorithm relied on two image textural features and achieved a sensitivity and specificity of 87% and 85%, respectively. This ex vivo pilot trial demonstrates that quantitative analysis of images obtained with a simple microendoscope system can distinguish neoplasia in Barrett's esophagus with good sensitivity and specificity when compared to histopathology and to subjective image interpretation.

A high frequency ultrasound-coupled fluorescence tomography system, primarily designed for imaging of protoporphyrin IX production in skin tumors in vivo, is demonstrated for the first time. The design couples fiber-based spectral sampling of the protoporphyrin IX fluorescence emission with high frequency ultrasound imaging, allowing thin-layer fluorescence intensities to be quantified. The system measurements are obtained by serial illumination of four linear source locations, with parallel detection at each of five interspersed detection locations, providing 20 overlapping measures of subsurface fluorescence from both superficial and deep locations in the ultrasound field. Tissue layers are defined from the segmented ultrasound images and diffusion theory used to estimate the fluorescence in these layers. The system calibration is presented with simulation and phantom validation of the system in multilayer regions. Pilot in-vivo data are also presented, showing recovery of subcutaneous tumor tissue values of protoporphyrin IX in a subcutaneous U251 tumor, which has less fluorescence than the skin.

A fluorescence confocal microscope incorporating a 1.8-mm-diam gradient-index relay lens is developed for in vivo histological guidance during resection of brain tumors. The microscope utilizes a dual-axis confocal architecture to efficiently reject out-of-focus light for high-contrast optical sectioning. A biaxial microelectromechanical system (MEMS) scanning mirror is actuated at resonance along each axis to achieve a large field of view with low-voltage waveforms. The unstable Lissajous scan, which results from actuating the orthogonal axes of the MEMS mirror at highly disparate resonance frequencies, is optimized to fully sample 500×500 pixels at two frames per second. Optically sectioned fluorescence images of brain tissues are obtained in living mice to demonstrate the utility of this microscope for image-guided resections.

In this work, a novel technique for rapid image analysis of Fourier transform infrared (FTIR) data obtained from human lymph nodes is explored. It uses the mathematical principle of orthogonality as a method to quickly and efficiently obtain tissue and pathology information from a spectral image cube. It requires less computational power and time compared to most forms of cluster analysis. The values obtained from different tissue and pathology types allows for discrimination of noncancerous from cancerous lymph nodes. It involves the calculation of the dot product between reference spectra and individual spectra from across the tissue image. These provide a measure of the correlation between individual spectra and the reference spectra, and each spectrum or pixel in the image is given a color representing the reference most closely correlating with it. The correlation maps are validated with the tissue and pathology features identified by an expert pathologist from corresponding hematoxylin and eosin stained tissue sections. Although this novel technique requires further study to properly test and validate this tool, with inclusion of more lymph node hyperspectral datasets (containing a greater variety of tissue states), it demonstrates significant clinical potential for pathology diagnosis.

We present the clinical applicability of fluorescence ratio reference standard (FRRS) to discriminate different stages of dental caries. Toward this, laser-induced autofluorescence emission spectra are recorded in vivo in the 400- to 800-nm spectral range on a miniature fiber optic spectrometer from 65 patients, with a 404-nm diode laser as the excitation source. Autofluorescence spectra of sound teeth consist of a broad emission at 500 nm that is typical of natural enamel, whereas in caries teeth additional peaks are seen at 635 and 680 nm due to emission from porphyrin compounds in oral bacteria. Scatter plots are developed to differentiate sound teeth from enamel caries, sound teeth from dentinal caries, and enamel caries from dentinal caries using the mean fluorescence intensity (FI) and ratios F500/F635 and F500/F680 measured from 25 sites of sound teeth and 65 sites of carious teeth. The sensitivity and specificity of both the FI and FRRS are determined. It is observed that a diagnostic algorithm based on FRRS scatter plots is able to discriminate enamel caries from sound teeth, dentinal caries from sound teeth, and enamel from dentinal caries with overall sensitivities of 85, 100, and 88% and specificities of 90, 100, and 77%, respectively.

Light-transmission and light-backscattering methods are commonly used to determine red-blood-cell (RBC) aggregation. Even though the results reveal good correlations between the parameters that are measured by these two methods, the methods themselves yield quite different values. The objective of this research is to investigate and delineate the characteristics of the two optical methods. We measured RBC aggregation by using a newly developed microchip-based aggregometer. An orthogonal polarization technique, wherein multiple scattering causes polarized light to be depolarized and passed through an orthogonal polarizer, was applied to the backscattering method. Our results were also compared to those of conventional aggregometers [laser-assisted optical rotational cell analyzer (LORCA)], and revealed that the backscattering method yielded higher aggregation indices than the transmission method and LORCA. However, the backscattering method with orthogonal polarization yielded the same values of aggregation indices as the transmission method. These agreements between the two methods were also found in measurements of RBC aggregability in various concentrations of dextran solutions.

A highly accurate, real-time multisensor agent monitor for biomarker detection is required for early detection of kidney diseases. Urine creatinine level can provide useful information on the status of the kidney. We prepare nanostructured surface-enhanced Raman spectroscopy (SERS) substrates without template or lithography, which provides controllable, well-organized nanostructures on the surface, for the quantitative analysis of creatinine concentration in urine. We present our work on sensitivity of the SERS substrate to urine samples collected from diabetic patients and healthy persons. We report the preparation of a new type of SERS substrate, which provides fast (<10 s), highly sensitive (creatinine concentration <0.5 μg/mL) and reproducible (<5% variation) detection of urine. Our method to analyze the creatinine level in urine is in good agreement with the enzymatic method.

An understanding of the mechanisms of drug diffusion and uptake through cellular membranes is critical for elucidating drug action and in the development of effective drug delivery systems. We study these processes for emodin, a potential anticancer drug, in live cancer cells using surface-enhanced Raman scattering. Micrometer-sized silica beads covered by nanosized silver colloids are passively embedded into the cell and used as sensors of the drug. We demonstrate that the technique offers distinct advantages: the possibility to study the kinetics of drug diffusion through the cellular membrane toward specific cell organelles, the detection of lower drug concentrations compared to fluorescence techniques, and less damage imparted on the cell.

Reactive oxygen species (ROS) produced by photodynamic therapy (PDT) is recorded in vivo using a chemiluminescence (CL)-based gated optical system. A novel approach is developed to utilize the fluorescence (FL) of the CL probe as an internal fluorescence to calibrate the observed CL on pharmacokinetics of the probe in situ. The results show that during an in vivo PDT session, the intensity of CL decreases significantly and the decaying of CL is governed by fast and slow time components. By comparing the temporal profile of FL to that of the corresponding CL, it is found that the slow component is mainly attributed to the probe pharmacokinetics, whereas the fast component is likely due to rapid oxygen consumption as a result of PDT treatment. With carefully selected criteria, it is possible to minimize the effect of probe pharmacokinetics. This significantly improves the monitoring method for practical applications.

Human leukocyte antigen (HLA) class I molecules are formed by three immunoglobulin-like domains (α1, α2, and α3) once folded by peptide and β₂-microglobulin show the presence of two α-helix streams and one β-sheet limiting the pocket for the antigenic peptide. The loss of HLA class I expression in tumors and virus-infected cells, on one hand, prevents T cell recognition, while on the other hand, it leads to natural killer (NK) cell mediated cytotoxicity. We propose the possibility of using Raman spectroscopy to measure the relative expression of HLA class I molecules at the single-cell level. Raman spectra are recorded for three cell lines (K562, T2, and T3) and monomers (HLA class I folded, unfolded and peptide+β₂-microlobulin refolded) using 830 nm laser line. Our data are consistent with the hypothesis that in the Raman spectra, ranging from 1600 to 1800 cm^-1, the intensity variation of cells associated with HLA class I molecules could be measured.

The goal of this study is to determine the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for delineation of brain tumor from surrounding normal tissue in order to assist the neurosurgeon in near-complete tumor excision. A time-domain TR-LIFS prototype apparatus (gated photomultiplier detection, fast digitizer) was used for recording tissue autofluorescence in normal cortex (NC), normal white matter (NWM), and various grades of gliomas intraoperatively. Tissue fluorescence was induced with a pulsed nitrogen laser (337 nm, 700 ps), and the intensity decay profiles were recorded in the 360- to 550-nm spectral range (10-nm interval). Histopathological analysis (hematoxylin & eosin) of the biopsy samples taken from the site of TR-LIFS measurements was used for validation of spectroscopic results. Preliminary results on 17 patients demonstrate that normal cortex (N=16) and normal white matter (N=3) show two peaks of fluorescence emission at 390 nm (lifetime=1.8±0.3 ns) and 460 nm (lifetime=0.8±0.1 ns). The 390-nm emission peak is absent in low-grade glioma (N=5; lifetime=1.1 ns) and reduced in high-grade glioma (N=9; lifetime=1.7±0.4 ns). The emission characteristics at 460 nm in all tissues correlated with the nicotinamide adenine dinucleotide fluorescence (peak: 440 to 460 nm; lifetime: 0.8 to 1.0 ns). These findings demonstrate the potential of using TR-LIFS as a tool for enhanced delineation of brain tumors during surgery. In addition, this study evaluates similarities and differences between TR-LIFS signatures of brain tumors obtained in vivo and those previously reported in ex vivo brain tumor specimens.

Intramyocardial oxygen transport was assessed during open-chest surgery in calves by diffuse reflectance spectroscopy using a small intramuscular fiber-optic probe. The sum of hemo- and myoglobin tissue fraction and oxygen saturation, the tissue fraction and oxidation of cytochrome aa3, and the tissue fraction of methemoglobin were estimated using a calibrated empirical light transport model. Increasing the oxygen content in the inhaled gas, 21%-50%-100%, in five calves (group A) gave an increasing oxygen saturation of 19±4%, 24±5%, and 28±8% (p<0.001, ANOVA repeated measures design) and mean tissue fractions of 1.6% (cytochrome aa3) and 1.1% (hemo- and myoglobin). Cardiac arrest in two calves gave an oxygen saturation lower than 5%. In two calves (group B), a left ventricular assistive device (LVAD pump) was implanted. Oxygen saturation in group B animals increased with LVAD pump speed (p<0.001, ANOVA) and with oxygen content in inhaled gas (p<0.001, ANOVA). The cytochrome aa3 oxidation level was above 96% in both group A and group B calves, including the two cases involving cardiac arrest. In conclusion, the estimated tissue fractions and oxygenation/oxidation levels of the myocardial chromophores during respiratory and hemodynamic provocations were in agreement with previously presented results, demonstrating the potential of the method.

We report on a novel acousto-optic tunable filter (AOTF)-based near-infrared (NIR) fluorescence excitation-emission matrix (EEM) spectroscopy technique for rapid multifluorophore characterization. We implement a unique light filtering module design by using cascaded AOTFs coupled with three orthogonally oriented polarizers to effectively remove the side-ripple artifacts of AOTFs as well as by using a pair of AOTFs coupled with two orthogonally oriented polarizers to improve detection efficiency for high-quality fluorescence EEM acquisitions. NIR fluorescence EEM spectroscopy (41 excitation wavelengths ranging from 550 to 950 nm in 10-nm increments; fluorescence emission from 570 to 1000 nm at 10-nm intervals) can be acquired from fluorescence dyes [e.g., diethylthiatricarbocyanine (DTTC) iodide, oxazine 750, and IR 140] within 10 s or even less, illustrating the potential of the AOTF-based NIR EEM technique developed for rapid multifluorophore analysis and characterization in biochemical and biomedical systems.

Dermally implanted luminescent sensors have been proposed for monitoring of tissue biochemistry, which has the potential to improve treatments for conditions such as diabetes and kidney failure. Effective in vivo monitoring via noninvasive transdermal measurement of emission from injected microparticles requires a matched optoelectronic system for excitation and collection of luminescence. We applied Monte Carlo modeling to predict the characteristics of output luminescence from microparticles in skin to facilitate hardware design. Three-dimensional, multiwavelength Monte Carlo simulations were used to determine the spatial and spectral distribution of the escaping luminescence for different implantation depths, excitation light source properties, particle characteristics, and particle packing density. Results indicate that the ratio of output emission to input excitation power ranged 10^&-3 to 10^-6 for sensors at the upper and lower dermal boundaries, respectively, and 95% of the escaping emission photons induced by a 10-mm-diam excitation beam were confined within an 18-mm circle. Tightly packed sensor configurations yielded higher output intensity with fewer particles, even after luminophore concentration effects were removed. Most importantly, for the visible wavelengths studied, the ability to measure spectral changes in emission due to glucose changes was not significantly affected by absorption and scattering of tissue, which supports the potential to accurately track changes in luminescence of sensor implants that respond to the biochemistry of the skin.

Splanchnic organs are particularly vulnerable to hypoperfusion. Currently, there is no technique that allows for the continuous estimation of splanchnic blood oxygen saturation (SpO₂). As a preliminary to developing a suitable splanchnic SpO₂ sensor, a new reflectance fiber optic photoplethysmographic (PPG) sensor and processing system are developed. An experimental procedure to examine the effect of fiber source detector separation distance on acquired PPG signals is carried out before finalizing the sensor design. PPG signals are acquired from four volunteers for separation distances of 1 to 8 mm. The separation range of 3 to 6 mm provides the best quality PPG signals with large amplitudes and the highest signal-to-noise ratios (SNRs). Preliminary calculation of SpO₂ shows that distances of 3 and 4 mm provide the most realistic values. Therefore, it is suggested that the separation distance in the design of a fiber optic reflectance pulse oximeter be in the range of 3 to 4 mm. Preliminary PPG signals from various splanchnic organs and the periphery are obtained from six anaesthetized patients. The normalized amplitudes of the splanchnic PPGs are, on average, approximately the same as those obtained simultaneously from the periphery. These observations suggest that fiber optic pulse oximetry may be a valid monitoring technique for splanchnic organs.

Radiation burns account for the vast majority of damage by accidental radiation exposure. They are characterized by successive and unpredictable inflammatory bursts that are preceded by a clinically latent postirradiation period. Diagnosis and prognosis of the clinical course of radiation burns have proven to be a difficult task. In a classical clinical setting, no technique can distinguish irradiated versus healthy skin during the clinically latent period, hence development of new tools is required. This work describes a noninvasive technique based on speckle phenomenon, designed to support radiation burn diagnosis and prognosis. Speckle produced by strongly scattering media contains information about their optical properties. The difficulty is to extract significant information from speckle patterns to discriminate between strongly scattering media and to characterize any change. Speckle patterns from irradiated and nonirradiated porcine skins are recorded in vivo several times after radiation exposure. A fractal approach is used in the treatment of speckle patterns. The results show that this technique allows discrimination between healthy and irradiated skin, in particular during the clinically latent period (p<0.01). Parameters extracted from speckle patterns discriminate and vary differently with radiation, which means they represent different information about skin changes.

Methods capable of quickly and inexpensively collecting genetic information are of increasing importance. We report a method of using surface-enhanced Raman spectroscopy to probe single-stranded DNA for genetic markers. This unique approach is used to analyze unmodified genes of moderate length for genetic markers by hybridizing native test oligonucleotides into a surface-enhanced Raman complex, vastly increasing detection sensitivity as compared to traditional Raman spectroscopy. The Raman complex is formed by sandwiching the test DNA between 40-nm gold nanoparticles and a photolithographically defined gold surface. With this design, we are able to collect characteristic Raman spectra about the test DNA and to detect genetic markers such as single-nucleotide polymorphisms (SNPs) and polymorphic regions. Results show that strands containing one of three different types of polymorphism can be differentiated using statistically significant trends regarding Raman intensity.

Variations in skin perfusion are easily detected by laser speckle contrast maps, but a robust interpretation of the information has been lacking. We show that multiple-exposure laser speckle methods produce the same spectral information as laser Doppler methods when applied to targets with embedded moving scatterers. This enables laser speckle measurements to be interpreted more quantitatively. We do this by using computer simulation of speckle data, and by experimental measurements on Brownian motion and skin perfusion using a laser Doppler system and a multiple-exposure laser speckle system. The power spectral density measurements of the light fluctuations derived using both techniques are exactly equivalent. Dermal perfusion can therefore be measured by laser Doppler or laser speckle contrast methods. In particular, multiexposure laser speckle can be rapidly processed to generate a full-field map of the perfusion index proportional to the concentration and mean velocity of red blood cells.

advent of automatic analyzers such as flow cytometers and impedance counters. Though these current methods have proven to be indispensible tools for physicians and researchers alike, they provide limited information on the detailed morphology of individual cells, and merely alert the operator to manually examine a blood smear by raising flags when abnormalities are detected. We demonstrate an automatic interferometry-based smear analysis technique known as diffraction phase cytometry (DPC), which is capable of providing the same information on red blood cells as is provided by current clinical analyzers, while rendering additional, currently unavailable parameters on the 2-D and 3-D morphology of individual red blood cells. To validate the utility of our technique in a clinical setting, we present a comparison between tests generated from 32 patients by a state of the art clinical impedance counter and DPC.

Light emitted from a femtosecond laser is capable of plasma-induced ablation of various materials. We tested the feasibility of utilizing femtosecond-pulsed laser radiation (λ=800 nm, 140 fs, 0.9 mJ/pulse) for ablation of urinary calculi. Ablation craters were observed in human calculi of greater than 90% calcium oxalate monohydrate (COM), cystine (CYST), or magnesium ammonium phosphate hexahydrate (MAPH). Largest crater volumes were achieved on CYST stones, among the most difficult stones to fragment using Holmium:YAG (Ho:YAG) lithotripsy. Diameter of debris was characterized using optical microscopy and found to be less than 20 µm, substantially smaller than that produced by long-pulsed Ho:YAG ablation. Stone retropulsion, monitored by a high-speed camera system with a spatial resolution of 15 µm, was negligible for stones with mass as small as 0.06 g. Peak shock wave pressures were less than 2 bars, measured by a polyvinylidene fluoride (PVDF) needle hydrophone. Ablation dynamics were visualized and characterized with pump-probe imaging and fast flash photography and correlated to shock wave pressures. Because femtosecond-pulsed laser ablates urinary calculi of soft and hard compositions, with micron-sized debris, negligible stone retropulsion, and small shock wave pressures, we conclude that the approach is a promising candidate technique for lithotripsy.

The human hepatoma HepaRG cell line is an in vitro cell model that is becoming an important tool in drug metabolism, hepatotoxicity, genotoxicity, and enzyme induction studies. The cells are highly proliferative during their undifferentiated state but once committed, they differentiate into two distinctly different cell types, namely, hepatocyte-like and biliary epithelial-like cells. The presence of the latter in the cell culture is considered to be a drawback of the cell model. Since the proliferating undifferentiated HepaRG cells have a bipotent character, the only way to improve the content ratio of hepatic versus biliary cells of differentiated HepaRG cells is to eradicate biliary cells in situ, in a way that free surface space does not become available and thus no transdifferentiation can occur. Spatially selective photodynamic therapy has proven to be effective for that purpose. First, all the cells were administered aminolevulinic acid (δ-ALA) to stimulate the synthesis of protoporphyrin IX (PpIX), a naturally occurring photosensitizer. Then, the biliary cells were automatically identified and outlined by bright-field image processing. Last, UV light patterns were projected onto the epithelial cells alone by a spatial light modulation device connected to an optical microscope; therefore, only these cells were destroyed by photodynamic therapy.

The calculation of corneal asphericity as a 3-D fit renders more accurate results when it is based on the corneal wavefront aberrations rather than on the corneal topography of the principal meridians. A more accurate prediction could be obtained for hyperopic treatments compared to myopic treatments. We evaluate a method to calculate corneal asphericity and asphericity changes after refractive surgery. Sixty eyes of 15 consecutive myopic patients and 15 consecutive hyperopic patients (n=30 each) are retrospectively evaluated. Preoperative and 3-month-postoperative topographic and corneal wavefront analyses are performed using corneal topography. Ablations are performed using a laser with an aberration-free profile. Topographic changes in asphericity and corneal aberrations are evaluated for a 6-mm corneal diameter. The induction of corneal spherical aberrations and asphericity changes correlates with the achieved defocus correction. Preoperatively as well as postoperatively, asphericity calculated from the topography meridians correlates with asphericity calculated from the corneal wavefront in myopic and hyperopic treatments. A stronger correlation between postoperative asphericity and the ideally expected/predicted asphericity is obtained based on aberration-free assumptions calculated from corneal wavefront values rather than from the meridians. In hyperopic treatments, a better correlation can be obtained compared to the correlation in myopic treatments. Corneal asphericity calculated from corneal wavefront aberrations represents a 3-D fit of the corneal surface; asphericity calculated from the main topographic meridians represents a 2-D fit of the principal corneal meridians. Postoperative corneal asphericity can be calculated from corneal wavefront aberrations with higher fidelity than from corneal topography of the principal meridians. Hyperopic treatments show a greater accuracy than myopic treatments.

This PDF file contains the errata for “JBO Vol. 15 Issue 02 Paper JBO ERR-MAR2010-1” for JBO Vol. 15 Issue 02

This PDF file contains the errata for “JBO Vol. 15 Issue 02 Paper JBO ERR-MAR2010-2” for JBO Vol. 15 Issue 02