A longstanding issue in biomedical research concerns the biophysical factors affecting the color diversity of the human irides and its connection with ocular diseases. Although the pigmentation and morphological characteristics of heavily and moderately pigmented irides have been extensively discussed in the literature, similar studies are scarce for lightly pigmented irides. We present computer experiments indicating that the spectral signature of these specimens may be directly affected by still undetectable melanin distributions in the outermost iridal layers. Our findings represent in silico evidence of this relationship which, in turn, may have implications in the investigation of the higher risk of death from metastatic ocular melanoma verified in individuals with light-colored irides.

Angularly resolved light scattering measurements made at visible wavelengths have the ability to quantify subcellular morphology, with particular sensitivity to organelles the size of mitochondria and lysosomes. We have recently reported on a lysosome-staining-based method that provides scattering contrast between stained and unstained cells, and through the use of appropriate models, we extracted a size distribution and contribution to cellular light scattering that we attributed to lysosomes. We provide an independent measurement of the lysosomal size distribution and contribution to cellular light scattering by exploiting photodynamic ablation of lysosomes and observing its effect on angularly resolved light scattering measurements. From these measurements, we conclude that lysosomes scatter approximately 14% of the light from EMT6 cells at 633 nm and that their size distribution has a mean and standard deviation of 0.8 and 0.4 μm, respectively.

Conventional embolization of cerebral aneurysms using detachable coils is time-consuming and often requires retreatment. These drawbacks have prompted the development of new methods of aneurysm occlusion. We present the fabrication and laser deployment of a shape memory (SMP) polymer expanding foam device. Data acquired in an in vitro basilar aneurysm model with and without flow showed successful treatment, with the flow rate affecting foam expansion and the temperature at the aneurysm wall.

We demonstrate the first in vivo gated 4D images of avian embryonic hearts by use of optical coherence tomography (OCT). We present a gated 4D dataset of an in vivo beating quail heart consisting of ~864,000 A-scans accumulated over multiple heartbeats. Generation of a gating trigger from a laser Doppler velocimetry (LDV) signal, collected from an outlying vitelline vessel, enabled us to gate image acquisition to the cardiac cycle. To fully characterize the genesis and mechanisms of cardiac defects, a tool capable of assessing structure and function simultaneously at early stages of development is needed, and gated OCT has the capability to become such a tool.

This work presents a pilot study to show the potential of an emerging imaging modality, near-infrared diffuse optical tomography (DOT), for the diagnosis of osteoarthritis (OA). We report quantitative absorption and scattering images of joint tissue that allow for differentiation between diseased and healthy joints. An automatic, multichannel optical imaging system is used to image finger joints from two OA patients and three healthy volunteers. 3-D optical images of the joint tissue are recovered using a finite-element-based reconstruction algorithm. The reconstructed images demonstrate differences in optical properties at the joint region (cartilage/synovial fluid) between the OA and healthy joints. Quantitative analysis from the patients and healthy volunteers also indicate that the recovered joint sizes are consistent with those from x-ray findings. The results of this pilot study show potential for quantitative imaging and diagnosis of early OA by DOT.

Raman spectroscopy on single, living epithelial cells captured in a laser trap is shown to have diagnostic power over colorectal cancer. This new single-cell technology comprises three major components: primary culture processing of human tissue samples to produce single-cell suspensions, Raman detection on singly trapped cells, and diagnoses of the cells by artificial neural network classifications. It is compared with DNA flow cytometry for similarities and differences. Its advantages over tissue Raman spectroscopy are also discussed. In the actual construction of a diagnostic model for colorectal cancer, real patient data were taken to generate a training set of 320 Raman spectra and a test set of 80. By incorporating outlier corrections to a conventional binary neural classifier, our network accomplished significantly better predictions than logistic regressions, with sensitivity improved from 77.5% to 86.3% and specificity improved from 81.3% to 86.3% for the training set and moderate improvements for the test set. Most important, the network approach enables a sensitivity map analysis to quantitate the relevance of each Raman band to the normal-to-cancer transform at the cell level. Our technique has direct clinic applications for diagnosing cancers and basic science potential in the study of cell dynamics of carcinogenesis.

The feasibility of a novel and efficient diagnostic method for liver fibrosis using Raman spectroscopy is studied. Confocal Raman spectroscopy (CRS) is utilized to monitor the molecular changes of hepatic stellate cells (HSCs) in vitro as well as in vivo activation. In vitro activation was induced by growth in uncoated plastic plates, while the in vivo activation is induced by a single intraperitoneal injection of carbon tetrachloride (CCl₄). The biochemical changes of HSCs during activation such as the loss of retinoid, the increase of α-helical protein, and the increased production of extracellular matrix proteins are observed by CRS. A user-friendly autoclassifying system is also developed to classify Raman spectra of liver injury tissues with a 90% accuracy rate. Raman spectroscopy combined with a fiber optical probe could be potentially accomplished for in vivo detection, which can lead to a novel and efficient diagnosis for liver fibrosis.

An expanding body of literature suggests Raman spectroscopy is a promising tool for skin cancer diagnosis and in-vivo tumor border demarcation. The development of an in-vivo diagnostic tool is, however, hampered by the fact that construction of fiber optic probes suitable for Raman spectroscopy in the so-called fingerprint region is complicated. In contrast, the use of the high wave-number region allows for fiber optic probes with a very simple design. We investigate whether high wave-number Raman spectroscopy (2800 to 3125 cm^-1) is able to provide sufficient information for noninvasive discrimination between basal cell carcinoma (BCC) and noninvolved skin. Using a simple fiber optic probe, Raman spectra are obtained from 19 BCC biopsy specimens and 9 biopsy specimens of perilesional skin. A linear discriminant analysis (LDA)-based tissue classification model is developed, which discriminates between BCC and noninvolved skin with high accuracy. This is a crucial step in the development of clinical dermatological applications based on fiber optic Raman spectroscopy.

We examine the application of an improved noncontact and noninvasive Raman spectroscopic technique in measuring medicines at therapeutic concentrations in a model system mimicking the anterior chamber of the eye. A 90-deg laser Raman scattering geometry is employed to reduce the direct exposure of the basic cordial ocular tissues to the laser beam and increase the signal-to-noise ratio of the spectra. The technique is applied to a commercially available artificial anterior chamber (AAC) fitted with corneas of porcine eyes. Specific Raman signatures of ciprofloxacin (Ciproxin^®), a fluoroquinolone based antibiotic, have been resolved. Last, a partial least-squares (PLS) chemometric algorithm has been developed to predict the concentration of ciprofloxacin in AAC over the range from 0 to 1 mg/mL with a correlation coefficient R²=98.4% and an RMS error of prediction equal to 41 μg/mL.

More than 60 million people in the United States and 23 million people in Mexico probably are infected with the Toxoplasma parasite, but very few have symptoms because the immune system usually keeps the parasite from causing illness. However, for people whose immune system is compromised, the consequences can be fatal. Toxoplasmosis is detected indirectly by different serological tests, where the sample requires a previous preparation. We analyze the feasibility to use Raman spectroscopy and principal component analysis (PCA) as an alternative method to detect the presence or absence of antibodies IgG (immunoglobulin G), IgM (immunoglobulin M), and IgA (immunoglobulin A), against Toxoplasma gondii, in a simple and fast way, in samples of human colostrum from a group of volunteers who were in contact with the parasite and others who were not in contact with the parasite.

An effective cancer control strategy requires improved early detection methods, patient-specific drug selection, and the ability to assess response to targeted therapeutics. Recently, plasmon resonance coupling between closely spaced metal nanoparticles has been used to develop ultrasensitive bioanalytical assays in vitro. We demonstrate the first in vivo application of plasmon coupling for molecular imaging of carcinogenesis. We describe molecular-specific gold bioconjugates to image epidermal growth factor receptor (EGFR); these conjugates can be delivered topically and imaged noninvasively in real time. We show that labeling with gold bioconjugates gives information on the overexpression and nanoscale spatial relationship of EGF receptors in cell membranes, both of which are altered in neoplasia. EGFR-mediated aggregation of gold nanoparticles in neoplastic cells results in more than a 100-nm color shift and a contrast ratio of more than tenfold in images of normal and precancerous epithelium in vivo, dramatically increasing contrast beyond values reported previously for antibody-targeted fluorescent dyes.

Optical coherence tomography (OCT) is an emerging medical imaging technology that enables high-resolution, noninvasive, cross-sectional imaging of microstructure in biological tissues in situ and in real time. When combined with small-diameter catheters or needle probes, OCT offers a practical tool for the minimally invasive imaging of living tissue morphology. We evaluate the ability of OCT to image normal kidneys and discriminate pathological changes in kidney structure. Both control and experimental preserved rat kidneys were examined ex vivo by using a high-resolution OCT imaging system equipped with a laser light source at 1.3-μm wavelength. This system has a resolution of 3.3 μm (depth) by 6 µm (transverse). OCT imaging produced cross-sectional and en face images that revealed the sizes and shapes of the uriniferous tubules and renal corpuscles. OCT data revealed significant changes in the uriniferous tubules of kidneys preserved following an ischemic or toxic (i.e., mercuric chloride) insult. OCT data was also rendered to produce informative three-dimensional (3-D) images of uriniferous tubules and renal corpuscles. The foregoing observations suggest that OCT can be a useful non-excisional, real-time modality for imaging pathological changes in donor kidney morphology prior to transplantation.

We report the recent technical improvements in our microelectromechanical systems (MEMS)-based spectral-domain endoscopic OCT (SDEOCT) and applications for in vivo bladder imaging diagnosis. With the technical advances in MEMS mirror fabrication and endoscopic light coupling methods, the new SDEOCT system is able to visualize morphological details of the urinary bladder with high image fidelity close to bench-top OCT (e.g., 10 μm/12 μm axial/lateral resolutions, gap;108 dB dynamic range) at a fourfold to eightfold improved frame rate. An in vivo animal study based on a porcine acute inflammation model following protamine sulfate instillation is performed to further evaluate the utility of SDEOCT system to delineate bladder morphology and inflammatory lesions as well as to detect subsurface blood flow. In addition, a preliminary clinical study is performed to identify the morphological features pertinent to bladder cancer diagnosis, including loss of boundary or image contrast between urothelium and the underlying layers, heterogeneous patterns in the cancerous urothelium, and margin between normal and bladder cancers. The results of a human study (91% sensitivity, 80% specificity) suggest that SDEOCT enables a high-resolution cross-sectional image of human bladder structures to detect transitional cell carcinomas (TCC); however, due to reduced imaging depth of SDEOCT in cancerous lesions, staging of bladder cancers may be limited to T1 to T2a (prior to muscle invasion).

We have developed a new descanned parallel (32-fold) pinhole and photomultiplier detection array for multifocal multiphoton microscopy that effectively reduces the blurring effect originating from scattered fluorescence photons in strongly scattering biological media. With this method, we achieve a fourfold improvement in photon statistics for detecting ballistic photons and an increase in spatial resolution by 21% in the lateral and 35% in the axial direction compared to single-beam non-descanned multiphoton microscopy. The new detection concept has been applied to plant leaves and pollen grains to verify the improvements in imaging quality.

Efficient image cytometry of a conventional microscope slide means rapid acquisition and analysis of 20 gigapixels of image data (at 0.3-μm sampling). The voluminous data motivate increased acquisition speed to enable many biomedical applications. Continuous-motion time-delay-and-integrate (TDI) scanning has the potential to speed image acquisition while retaining sensitivity, but the challenge of implementing high-resolution autofocus operating simultaneously with acquisition has limited its adoption. We develop a dynamic autofocus system for this need using: 1. a "volume camera," consisting of nine fiber optic imaging conduits to charge-coupled device (CCD) sensors, that acquires images in parallel from different focal planes, 2. an array of mixed analog-digital processing circuits that measure the high spatial frequencies of the multiple image streams to create focus indices, and 3. a software system that reads and analyzes the focus data streams and calculates best focus for closed feedback loop control. Our system updates autofocus at 56 Hz (or once every 21 µm of stage travel) to collect sharply focused images sampled at 0.3×0.3 μm2/pixel at a stage speed of 2.3 mm/s. The system, tested by focusing in phase contrast and imaging long fluorescence strips, achieves high-performance closed-loop image-content-based autofocus in continuous scanning for the first time.

We report a new two-channel fluorescence microscopy technique for surface-generated fluorescence. The realized fluorescence microscope allows high resolution imaging of aqueous samples. The core element of the instrument is a parabolic mirror objective that is used to collect the fluorescence at large surface angles above the critical angle of the water/glass interface. An aspheric lens, incorporated into the solid parabolic element, is used for diffraction-limited laser focusing and for collecting fluorescence at low angles with respect to the optical axis. By separated collection of the fluorescence emitted into supercritical and subcritical angles, two detection volumes strongly differing in their axial resolution are generated at the surface of a glass cover slip. The collection of supercritical angle fluorescence (SAF) results in a strict surface confinement of the detection volume, whereas collecting below the critical angle allows gathering the fluorescence emitted several microns deep inside the sample. Consequently, the signals from surface-bound and unbound diffusing fluorescent molecules can be obtained simultaneously.

Human serum albumin (HSA) complexation with quercetin, a flavonoid commonly present in human diet, was monitored by means of fluorescence decays of the single HSA tryptophan - Trp214. Data analysis based on fitting to multiexponential functions and determining the lifetime distributions revealed a high sensitivity of tryptophan fluorescence to binding quercetin. Results are discussed in terms of the rotamer model for tryptophan, HSA-quercetin complexation and potential HSA to quercetin energy transfer. Evidence for quercetin stabilising tryptophan rotamers in HSA is presented.

We report on a study designed to assess variability among three different fluorescence spectroscopy devices, four fiber optic probes, and three sets of optical calibration standards to better understand the reproducibility of measurements and interdevice comparisons of fluorescence spectroscopic data intended for clinical diagnostic use. Multiple measurements are acquired from all sets of standards using each combination of spectrometer, fiber optic probe, and optical standard. Data are processed using standard calibration methods to remove instrument-dependant responses. Processed spectra are analyzed using an analysis of variance to assess the percent variance explained by each factor that was statistically significant. Analysis of processed data confirms statistically significant differences among the spectrometers and fiber optic probes. However, no differences are found when varying calibration standards or measurement date and time. The spectrometers and fiber optic probes are significant sources of variability, but appropriate data processing substantially reduces these effects. Studies of inter- and intradevice variability are important methodological issues for optical device trials and must be included in the quality assurance studies for the clinical trial design.

Large phase II trials of fluorescence and reflectance spectroscopy using a fiber optic probe in the screening and diagnostic settings for detecting cervical neoplasia have been conducted. We present accrual and histopathology data, instrumentation, data processing, and the preliminary results of interdevice consistencies throughout the progression of a trial. Patients were recruited for either a screening trial (no history of abnormal Papanicolaou smears) or a diagnostic trial (a history of abnormal Papanicolaou smears). Colposcopy identified normal and abnormal squamous, columnar, and transformation zone areas that were subsequently measured with the fiber probe and biopsied. In the course of the clinical trial, two generations of spectrometers (FastEEM2 and FastEEM3) were designed and utilized as optical instrumentation for in vivo spectroscopic fluorescence and reflectance measurements. Data processing of fluorescence and reflectance data is explained in detail and a preliminary analysis of the variability across each device and probe combination is explored. One thousand patients were recruited in the screening trial and 850 patients were recruited in the diagnostic trial. Three clinical sites attracted a diverse range of patients of different ages, ethnicities, and menopausal status. The fully processed results clearly show that consistencies exist across all device and probe combinations throughout the diagnostic trial. Based on the stratification of the data, the results also show identifiable differences in mean intensity between normal and high-grade tissue diagnosis, pre- and postmenopausal status, and squamous and columnar tissue type. The mean intensity values of stratified data show consistent separation across each ofthe device and probe combinations. By analyzing trial spectra, we provide more evidence that biographical variables such as menopausal status as well as tissue type and diagnosis significantly affect the data. (PARTIAL ABSTRACT)

Optical imaging is unique among in vivo imaging methods because it is possible to simultaneously resolve two or more probes emitting at different wavelengths of light. We employed two near-infrared (NIR) fluorescent optical probes, each labeled with a different protein, to simultaneously evaluate the pharmacokinetics of each probe. Dynamic optical imaging was performed in live mice after the coinjection of bovine serum albumin (BSA) and galactosamine-conjugated bovine serum albumin (GmSA) labeled with either Cy5.5 or Cy7 NIR dyes. The pharmacokinetics of BSA and GmSA were independently and simultaneously visualized. Next, two-color dynamic imaging of biotinylated BSA (b-BSA) and BSA labeled with Cy5.5 or Cy7 was performed before and after an avidin "chase." Following avidin injection, fluorescently labeled b-BSA rapidly accumulated in the liver, while minimal liver uptake of BSA was noted. Thus, multicolor dynamic contrast-enhanced optical imaging can be performed to noninvasively track the pharmacokinetics of different proteins. This imaging technique can be applied to a wide variety of optically labeled proteins in order to simultaneously track their biodistribution.

Cameleons are genetically encoded fluorescence resonance energy transfer (FRET)–based Ca²⁺ indicators. Attempts to use cameleons to detect neural activity in vertebrate systems have been largely frustrated by the small FRET signal, in contradistinction to the higher signals seen in Drosophila and Caenorhabditis elegans. We have developed a statistical optimization method capable of detecting small ratiometric signals in noisy imaging data, called statistical optimization for the analysis of ratiometric signals. Using this method, we can detect and estimate anticorrelated ratiometric signals with subcellular resolution in cultured, dissociated zebrafish spinal neurons expressing cameleon or loaded with fluo-4 and fura-red. This method may make it possible to use yellow cameleons for measuring neural activity at high resolution in transgenic animals.

We perform combined magnetic resonance and bioluminescence imaging of live mice for the purpose of improving the accuracy of bioluminescence tomography. The imaging is performed on three live nude mice in which tritium-powered light sources are surgically implanted. High-resolution magnetic resonance images and multispectral, multiview bioluminescence images are acquired in the same session. An anatomical model is constructed by segmenting the magnetic resonance images for all major tissues. The model is subsequently registered with nonlinear transformations to the 3-D light exittance (exiting intensity) surface map generated from the luminescence images. A Monte Carlo algorithm, along with a set of tissue optical properties obtained from in vivo measurements, is used to solve the forward problem. The measured and simulated light exittance images are found to differ by a factor of up to 2. The greatest cause of this moderate discrepancy is traced to the small errors in source positioning, and to a lesser extent to the optical properties used for the tissues. Discarding the anatomy and using a homogeneous model leads to a marginally worse agreement between the simulated and measured data.

A time-resolved optical instrument allowing for noninvasive assessment of cerebral oxygenation is presented. The instrument is equipped with picosecond diode lasers, fast photodetectors, and time-correlated single photon counting electronics. This technology enables depth-resolved estimation of changes in absorption and, in consequence, assessment of changes in hemoglobin concentrations in the brain cortex. Changes in oxyhemoglobin (HbO₂) and deoxyhemoglobin (Hb) can be evaluated selectively in extra- and intracerebral tissue compartments using the moments of distributions of times of flight of photons measured at two wavelengths in the near-infrared region. The combination of the data acquired from multiple sources and detectors located on the surface of the head with the depth-resolved analysis, based on the moments, enables imaging of cortex oxygenation. Results of the tests on physical phantoms as well as in vivo validation of the instrument during the motor stimulation experiment are presented.

Previous work describing a resilient method for measuring oxyhemoglobin saturation using the blue-green spectral shift was performed using cell free hemoglobin solutions. Hemoglobin solution and whole blood sample spectra measured under similar conditions in a spectrophotometer are used here to begin evaluating the impact of cellular scattering on this method. The blue-green spectral shift with changing oxyhemoglobin saturation was preserved in these blood samples and the blue-green spectral shift was relatively unaffected by physiological changes in blood pH (6.6, 7.1, and 7.4), path length through blood (100 and 200 μm), and blood hematocrit (19 to 48%). The packaging of hemoglobin in red blood cells leads to a decreased apparent path length through hemoglobin, and an overall decrease in scattering loss with increasing wavelength from 450 to 850 nm. The negative slope of the scattering loss in the 476 to 516-nm range leads to a +3.0 nm shift in the oxyhemoglobin saturation calibration line when the blue-green spectral minimum in these blood samples was compared to cell free hemoglobin. Further research is needed to fully evaluate the blue green spectral shift method in cellular systems including in vivo testing.

We determine the impact of artificial light scatter on quantitative, noninvasive assessment of retinal arteriolar hemodynamics. One eye from each of 10 healthy young subjects between the ages of 18 and 30 (23.6±3.4) is randomly selected. To simulate light scatter, cells comprising a plastic collar and two plano lenses are filled with solutions of differing concentration of polystyrene microspheres (Polysciences Inc., USA). We prepare 0.002, 0.004, 0.006, and 0.008% microsphere concentrations as well as distilled water only. The Canon laser blood flowmeter (CLBF) is used to noninvasively assess retinal arteriolar blood flow. After a preliminary screening to confirm subject eligibility, seven arteriolar blood flow measurements are taken by randomly placing the cells between the instrument objective lens and the subjects' cornea. To achieve a baseline, subjects are first imaged with no cell in place. Both low- and high-intensity CLBF laser settings are assessed. Our light scatter model results in an artifactual increase of retinal arteriolar diameter (p<0.0001) and thereby increased retinal blood flow (p<0.0001). The 0.006 and 0.008% microsphere concentrations produce significantly higher diameter and flow values than baseline. Centerline blood velocity, however, is not affected by light scatter. Retinal arteriolar diameter values are significantly less with the high-intensity laser than with the low-intensity laser (p=0.0007). Densitometry assessment of vessel diameter is increasingly impacted as the magnitude of artificial light scatter increases; this effect can be partially negated by increasing laser intensity. A cataract is an inevitable consequence of aging and, therefore, care must be exercised in the interpretation of studies of retinal vessel diameter that use similar densitometry techniques.

We measure the tumor vascular response to varying irradiance rates during photodynamic therapy (PDT) in a Dunning rat prostate model with interstitial Doppler optical coherence tomography (IS-DOCT). Rats are given a photosensitizer drug, Photofrin, and the tumors are exposed to light (635 nm) with irradiance rates ranging from 8 to 133 mW/cm² for 25 min, corresponding to total irradiance of 12 to 200 J/cm² (measured at surface). The vascular index computed from IS-DOCT results shows the irradiance rate and total irradiance dependent microvascular shutdown in the tumor tissue during PDT. While faster rates of vascular shutdown were associated with increasing PDT irradiance rate and total irradiance, a threshold effect was observed as irradiance rates above 66 mW/cm² (surface), where no further increase in vascular shutdown rate was detected. The maximum post-treatment vascular shutdown (81%) without immediate microcirculatory recovery was reached with the PDT condition of 33 mW/cm² and 50 J/cm². Control groups without Photofrin show no significant microvascular changes. Microvascular shutdown occurs at different rates and shows correlation with PDT total irradiance and irradiance rates. These dependencies may play an important role in PDT treatment planning, feedback control for treatment optimization, and post-treatment assessment.

(PDT). This technique has been extensively validated in tissue phantoms; however, validation in patients has been limited. This pilot study compares blood oxygenation and photosensitizer tissue uptake measured by multiwavelength DRS with ex vivo assays of the hypoxia marker, 2-(2-nitroimida-zol-1[H]-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5), and the photosensitizer (motexafin lutetium, MLu) from tissues at the same tumor site of three tumors in two patients with intra-abdominal cancers. Similar in vivo and ex vivo measurements of MLu concentration are carried out in murine radiation-induced fibrosarcoma (RIF) tumors (n=9). The selection of optimal DRS wavelength range and source-detector separations is discussed and implemented, and the association between in vivo and ex vivo measurements is examined. The results demonstrate a negative correlation between blood oxygen saturation (StO2) and EF5 binding, consistent with published relationships between EF5 binding and electrode measured pO₂, and between electrode measured pO₂ and StO₂. A tight correspondence is observed between in vivo DRS and ex vivo measured MLu concentration in the RIF tumors; similar data are positively correlated in the human intraperitoneal tumors. These results further demonstrate the potential of in vivo DRS measurements in clinical PDT.

Many current light diffusers for photodynamic therapy are inflexible, and the applied light dose is difficult to adjust during treatment, especially on complex body surfaces. A thin and flexible luminous textile is developed using plastic optical fibers as a light distributor. The textile diffuser is evaluated for flexibility, irradiance, brightness distribution, and temperature rise with a 652-nm laser set to 100 mW. The bending force of the textile diffuser resembles a defined optical film. On the textile surface, an average output power of 3.6±0.6 mW/cm² is measured, corresponding to a transmission rate of 40±3.8% on an area of 11 cm². Aluminum backing enhances the irradiance to the face (treatment side). The measured brightness distribution seems to lie within a range similar to other photodynamic therapy (PDT) devices. A power setting of 100 mW increases the temperature of the textile diffuser surface of up to 27°C, and 1 W raises the temperature above 40°C. Results confirm that the flexible textile diffuser supplies suitable radiation for low fluence rate photodynamic therapy on an area of several cm².

Complete blood vessel occlusion is required for the treatment of age-related macular degeneration (AMD). AMD is the leading cause of blindness in developed countries and current treatment regimes have potential to cause collateral damage, or do not remove pre-existing unwanted vasculature. It has been proposed that two-photon excitation (TPE) photodynamic therapy (PDT) can be applied to cause local blood vessel occlusion without damaging surrounding retinal tissues. The in ovo chicken chorioallantoic membrane (CAM) is used as the model for vascularization in the wet form of AMD; novel techniques for the utilization of the CAM are reported. Complete occlusion of CAM vessels ~15 μm in diameter is achieved using the clinically approved photosensitizer Verteporfin (Visudyne^®, QLT, Incorporated, Vancouver, British Columbia, Canada) and TPE activation. The average and peak irradiances used for treatment are 3.3×10⁶ W/cm² and 3.7×10¹¹ W/cm², respectively. A total fluence of 1.1×10⁸ J/cm² is the dosage required for successful occlusion, and it is expected that for optimal conditions it will be much less. These results are the first proof-of-principle evidence in the literature that indicate TPE-PDT can be used to occlude small blood vessels. Further investigation will help determine the utility of TPE-PDT for treating wet AMD, perhaps through targeting feeder vessels.

Although the benefits of topical sensitizer administration have been confirmed for photodynamic therapy (PDT), ALA-induced protoporphyrin IX is the only sensitizer clinically used with this administration route. Unfortunately, ALA-PDT results in poor treatment response for thicker lesions. Here, selectivity and depth distribution of the highly potent sensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC), supplied in a novel liposome formulation was investigated following topical administration for 4 and 6 h in a murine skin tumor model. Extraction data indicated an average [± standard deviation (SD)] mTHPC concentration within lesions of 6.0(±3.1) ng/mg tissue with no significant difference (p<0.05) between 4- and 6-h application times and undetectable levels of generalized photosensitivity. Absorption spectroscopy and chemical extraction both indicated a significant selectivity between lesion and normal surrounding skin at 4 and 6 h, whereas the more sensitive fluorescence imaging setup revealed significant selectivity only for the 4-h application time. Absorption data showed a significant correlation with extraction, whereas the results from the fluorescence imaging setup did not correlate with the other methods. Our results indicate that this sensitizer formulation and administration path could be interesting for topical mTHPC-PDT, decreasing the effects of extended skin photosensitivity associated with systemic mTHPC administration.

Precise removal of basal cell carcinomas (BCCs) with minimal damage to the surrounding normal skin is guided by the examination of frozen histology of each excision during Mohs surgery. The preparation of frozen histology is slow, requiring 20 to 45 min per excision. Confocal reflectance mosaicing may enable rapid detection of BCCs directly in surgical excisions, with minimal need for frozen histology. Soaking the excisions in acetic acid rapidly brightens nuclei and enhances BCC-to-dermis contrast. Clinically useful concentrations of acetic acid from 10 to 1% require 30 s to 5 min, respectively. A tissue fixture precisely controls the stability, flatness, tilt, and sag of the excisions, which enables mosaicing of 36×36 images to create a field of view of 12×12 mm. This simulates a 2× magnification view in light microscopes, which is routinely used by Mohs surgeons to examine frozen histology. Compared to brightfield, cross-polarization enhances contrast and detectability of BCCs in the papillary dermis but not in the reticular dermis. Comparison of mosaics to histology shows that nodular, micronodular, and superficial BCCs are easily detected. However, infiltrative and sclerosing BCCs tend to be obscured within the surrounding bright dermis. The mosaicing method currently requires 9 min, and thus may expedite Mohs surgery.

Frequency-domain photothermal radiometry (FD-PTR or PTR) is used to detect mechanical holes and demineralized enamel in the interproximal contact area of extracted human teeth. Thirty-four teeth are used in a series of experiments. Preliminary tests to detect mechanical holes created by dental burs and 37% phosphoric acid etching for 20 s on the interproximal contact points show distinct differences in the signal. Interproximal contact areas are demineralized by using a partially saturated acidic buffer system. Each sample pair is examined with PTR before and after micromachining or treating at sequential treatment periods spanning 6 h to 30 days. Dental bitewing radiographs showed no sign of demineralized lesion even for samples treated for 30 days. Microcomputer tomography (μ-CT), transverse microradiography (TMR), and scanning electron microscopy (SEM) analyses are performed. Although μ-CT and TMR measured mineral losses and lesion depths, only SEM surface images showed visible signs of treatment because of the minimal extent of the demineralization. However, the PTR amplitude increased by more than 300% after 80 h of treatment. Therefore, PTR is shown to have sufficient contrast for the detection of very early interproximal demineralized lesions. The technique further exhibits excellent signal reproducibility and consistent signal changes in the presence of interproximal demineralized lesions, attributes that could lead to PTR as a reliable probe to detect early interproximal demineralization lesions. Modulated luminescence is also measured simultaneously, but it shows a lower ability than PTR to detect these interproximal demineralized lesions.

Tissue-specific modification of treatment strategy is proposed to increase the antimicrobial activity of light-activated therapy (LAT) for root canal disinfection. Methylene blue (MB) dissolved in different formulations: water, 70% glycerol, 70% poly ethylene glycol (PEG), and a mixture of glycerol:ethanol:water (30:20:50) (MIX), is analyzed for photophysical, photochemical, and photobiological characteristics. Aggregation of MB molecules, as evident from monomer to dimer ratio, depends on the molar concentrations of MB, which is significantly higher in water compared to other formulations. MIX-based MB formulation effectively penetrates the dentinal tubules. Although, the affinity of MB for Enterococcus faecalis (gram positive) and Actinomycetes actinomycetemcomitans (gram negative) was found to be high in the water-based formulation, followed by MIX, the MIX-based formulation significantly enhanced the model substrate photooxidation and singlet oxygen generation compared to MB dissolved in other formulations. Finally, the efficacy of LAT is evaluated on biofilms produced by both organisms under in vitro and ex vivo conditions. A dual-stage approach that applies a photosensitization medium and an irradiation medium separately is tested. The MIX-based photosensitization medium in combination with dual-stage approach demonstrates thorough disinfection of the root canal with bacterial biofilms. This method will have potential application for root canal disinfection.

The determination of safe exposure levels for lasers has come from damage assessment experiments in live animals, which typically involve correlating visually identifiable damage with laser dosimetry. Studying basic mechanisms of laser damage in animal retinal systems often requires tissue sampling (animal sacrifice), making justification and animal availability problematic. We determined laser damage thresholds in cultured monolayers of a human retinal pigment epithelial (RPE) cell line. By varying exposure duration and laser wavelength, we identified conditions leading to damage by presumed photochemical or thermal mechanisms. A comparison with literature values for ocular damage thresholds validates the in vitro model. The in vitro system described will facilitate molecular and cellular approaches for understanding laser-tissue interaction.

Treatment to increase secretion of growth factors related to angiogenesis by gene transfection is a promising therapeutic solution for improving the outcome of tissue transplantation. We attempted to deliver a therapeutic vector construct carrying the human hepatocyte growth factor (hHGF) gene to skin grafts of rats using laser-induced stress waves (LISWs), with the objective of enhancing their adhesion. First we delivered the hHGF gene to rat native skin in vivo to determine the optimum gene transfer conditions. We then transferred the hHGF gene to excised rat skins, with which autografting was performed. We found that the density and uniformity of neovascularities were significantly enhanced in the grafted skins that were transfected using LISWs. These results suggest the efficacy of this method to improve the outcome of skin grafting. To our knowledge, this is the first experimental demonstration of a therapeutic efficacy based on LISW-mediated gene transfection. Since the present method can be applied not only to various types of tissues but also to bioengineered tissues, this technique has the potential to contribute to progress in transplantation medicine and future regenerative medicine.

Angle-resolved signals of polarized light scattered by biological cells provide rich information on cell morphology. Quantitative study of these signals can lead to new methods to develop and improve high-throughput instrumentation for cell probing such as scattering-based flow cytometry. We employ a goniometer system with a photoelastic modulation scheme to determine selected Mueller matrix elements of B-cell hydrosol samples. The angular dependence of S₁₁, S₁₂, and S₃₄ is determined from the scattered light signals between 10 and 160 deg at the three wavelengths 442, 633, and 850 nm. A finite-difference, time-domain (FDTD) method and coated-sphere model are used to investigate the effect of nuclear refractive index on the angle-resolved Mueller elements at different wavelengths using the 3-D structures of selected B cells reconstructed from confocal images. With these results, we demonstrate the value of the light-scattering method in obtaining the cell morphology information.

Optical fibers can deliver light to, and collect it from, regions deep in tissue. However, reported illumination and fluorescence collection volumes adjacent to the fiber tip have been inconsistent, and systematic data on this topic are not available. Illumination and fluorescence collection profiles were characterized with high spatial resolution for different optical fibers in tissue and various fluids using two-photon flash photolysis and excitation. We confirm that illumination and fluorescence collection volumes for optical fibers are near identical. Collection volume is determined by the core dimensions and numerical aperture (NA) of the fiber and the scattering properties of the medium. For a multimode optical fiber with 100 μm core diam and NA=0.22, 80% of the total fluorescence is collected from a depth of 170 μm in tissue and 465 μm in nonscattering fluid. A semiempirical mathematical description of photon flux adjacent to the fiber tip was also developed and validated. This was used to quantify the extent of temporal blurring associated with propagation of a wavefront of altered fluorescence emission across the region addressed by fiber optic probes. We provide information that will facilitate the design of optical probes for tissue imaging or therapeutic applications.

Exhaled nitric oxide (NO) is an important biomarker in asthma and other respiratory disorders. The optical performance of a NO/CO₂ sensor employing integrated cavity output spectroscopy (ICOS) with a quantum cascade laser operating at 5.22 μm capable of real-time NO and CO₂ measurements in a single breath cycle is reported. A NO noise-equivalent concentration of 0.4 ppb within a 1-sec integration time is achieved. The off-axis ICOS sensor performance is compared to a chemiluminescent NO analyzer and a nondispersive infrared (NDIR) CO₂ absorption capnograph. Differences between the gas analyzers are assessed by the Bland-Altman method to estimate the expected variability between the gas sensors. The off-axis ICOS sensor measurements are in good agreement with the data acquired with the two commercial gas analyzers. This work demonstrates the performance characteristics and merits of mid-infrared spectroscopy for exhaled breath analysis.

Diffusing wave spectroscopy (DWS), without the use of tracer particles, has been used to study the internal dynamics of polyvinyl alcohol (PVA) phantoms, which mimic the properties of normal and malignant breast tissues. From the measured intensity autocorrelations, the mean square displacement (MSD) of phantom meshing is estimated, leading to the storage and loss moduli of the medium covering frequencies up to 10 KHz. These are verified with independent measurements from a dynamic mechanical analyzer (DMA) at low frequencies. We thus prove the usefulness of DWS to extract visco-elastic properties of the phantom and its possible application in detecting malignancy in soft tissues.

The understanding of drug delivery to organs, such as the brain, has been hampered by the inability to measure tissue drug concentrations in real time. We report an application of an optical spectroscopy technique that monitors in vivo the real-time drug concentrations in small volumes of brain tissue. This method will facilitate development of new protocols for delivery of drugs to treat brain cancers. The delivery of many anticancer drugs to the brain is limited by the presence of the blood-brain barrier (BBB). Mitoxantrone (MTX) is a water-soluble anticancer drug that poorly penetrates the BBB. It is preliminarily determined in an animal model that the brain tissue uptake of chemotherapy agents—in this demonstration, MTX—delivered intra-arterially is enhanced when the BBB is disrupted.

This PDF file contains the errata for “JBO Vol. 12 Issue 03 Paper 2756853” for JBO Vol. 12 Issue 03

This PDF file contains the errata for “JBO Vol. 12 Issue 03 Paper 2761917” for JBO Vol. 12 Issue 03

This PDF file contains the editorial “Responsible Conduct of Research” for JBO Vol. 12 Issue 03