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The prevention and treatment of spinal cord injury are focused upon the maintenance of spinal cord blood flow, yet no technology exists to monitor spinal cord ischemia. We recently demonstrated continuous monitoring of spinal cord ischemia with diffuse correlation and optical spectroscopies using an optical probe. Prior to clinical translation of this technology, it is critically important to demonstrate the safety profile of spinal cord exposure to the required light. To our knowledge, this is the first report of in situ safety testing of such a monitor. We expose the spinal cord to laser light utilizing a custom fiber-optic epidural probe in a survival surgery model (11 adult Dorset sheep). We compare the tissue illumination from our instrument with the American National Standards Institute maximum permissible exposures. We experimentally evaluate neurological and pathological outcomes of the irradiated sheep associated with prolonged exposure to the laser source and evaluate heating in ex vivo spinal cord samples. Spinal cord tissue was exposed to light levels at [Formula: see text] the maximum permissible exposure for the eye and [Formula: see text] for the skin. Multidisciplinary testing revealed no functional neurological sequelae, histopathologic evidence of laser-related injury to the spinal cord, or significant temperature changes in ex vivo samples. Low tissue irradiance and the lack of neurological, pathological, and temperature changes upon prolonged exposure to the laser source offer evidence that spinal cord tissues can be monitored safely with near-infrared optical probes placed within the epidural space.

Helicases are a class of nucleic acid (NA) motors that catalyze NTP-dependent unwinding of NA duplexes into single strands, a reaction essential to all areas of NA metabolism. In the last decade, single-molecule (sm) technology has proven to be highly useful in revealing mechanistic insight into helicase activity that is not always detectable via ensemble assays. A combination of methods based on fluorescence, optical and magnetic tweezers, and flow-induced DNA stretching has enabled the study of helicase conformational dynamics, force generation, step size, pausing, reversal and repetitive behaviors during translocation and unwinding by helicases working alone and as part of multiprotein complexes. The contributions of these sm investigations to our understanding of helicase mechanism and function will be discussed.

Wereportmeasurementsofgellingrigidrodnetworkscomposedofasemidilute dispersionof surfactant-stabilizedcarbonnanotubes.Microrheologyisemployedtofollowtherheologicalevolutionofthe suspensionfrom asemidilute solutionofunbondedtubestoabondedgel network withfiniteyield stress. The rheological data at various time intervals during gelation is readily collapsed onto a single time-cure superposition master curve. A theoretical model based on the crossing probability of rods confined to finite volumes is developed to account for network elasticity. Model predictions compare well with computer simulation and experiments as a function of nanotube volume fraction 1 and cure time.

Photodynamic therapy (PDT) can lead to the creation of heterogeneous, response-limiting hypoxia during illumination, which may be controlled in part through illumination fluence rate. In the present report we consider (1) regional differences in hypoxia, vascular response, and cell kill as a function of tumor depth and (2) the role of fluence rate as a mediator of depth-dependent regional intratumor heterogeneity. Intradermal RIF murine tumors were treated with Photofrin PDT using surface illumination at an irradiance of 75 or 38 mW cm(-2). Regional heterogeneity in tumor response was examined through comparison of effects in the surface vs. base of tumors, i.e. along a plane parallel to the skin surface and perpendicular to the incident illumination. 75 mW cm(-2) PDT created significantly greater hypoxia in tumor bases relative to their surfaces. Increased hypoxia in the tumor base could not be attributed to regional differences in Photofrin concentration nor effects of fluence rate distribution on photochemical oxygen consumption, but significant depth-dependent heterogeneity in vascular responses and cytotoxic response were detected. At a lower fluence rate of 38 mW cm(-2), no detectable regional differences in hypoxia or cytotoxic responses were apparent, and heterogeneity in vascular response was significantly less than that during 75 mW cm(-2) PDT. This research suggests that the benefits of low-fluence-rate PDT are mediated in part by a reduction in intratumor heterogeneity in hypoxic, vascular and cytotoxic responses.

When the packing fraction is increased sufficiently, loose particulates jam to form a rigid solid in which the constituents are no longer free to move. In typical granular materials and foams, the thermal energy is too small to produce structural rearrangements. However, because thermal motion becomes relevant when the particles are small enough, it is imperative to understand the behaviour as the temperature is increased. Zhang et al. use both colloidal experiments and computer simulations to investigate the overlap distance between neighbouring particles beyond the zero-temperature limit. They find that this quantity evolves in an unusual manner, retaining a vestige of its zero-temperature behaviour. When the packing fraction is increased sufficiently, loose particulates jam together to form a rigid solid in which the constituents are no longer free to move. Although in typical granular materials and foams the thermal energy is too small to produce structural rearrangements, thermal motion becomes relevant when the particles are small enough. Here, colloidal experiments and computer simulations are used to investigate the overlap distance between neighbouring particles beyond the zero-temperature limit, revealing some surprising behaviour. When the packing fraction is increased sufficiently, loose particulates jam to form a rigid solid in which the constituents are no longer free to move. In typical granular materials and foams, the thermal energy is too small to produce structural rearrangements. In this zero-temperature (T = 0) limit, multiple diverging1,2,3,4,5,6,7,8 and vanishing2,9,10 length scales characterize the approach to a sharp jamming transition. However, because thermal motion becomes relevant when the particles are small enough, it is imperative to understand how these length scales evolve as the temperature is increased. Here we used both colloidal experiments and computer simulations to progress beyond the zero-temperature limit to track one of the key parameters—the overlap distance between neighbouring particles—which vanishes at the T = 0 jamming transition. We find that this structural feature retains a vestige of its T = 0 behaviour and evolves in an unusual manner, which has masked its appearance until now. It is evident as a function of packing fraction at fixed temperature, but not as a function of temperature at fixed packing fraction or pressure. Our results conclusively demonstrate that length scales associated with the T = 0 jamming transition persist in thermal systems, not only in simulations but also in laboratory experiments.

Little information exists on the role and the characteristics of perfusion changes related to periinfarct depolarization. Our aim was to visualize and monitor periinfarct flow transients (PIFTs) in Sprague-Dawley rats (n = 10) with two different laser methods in a closed-skull model of filament middle cerebral artery occlusion. A laser Doppler probe was placed over the ischemic cortex 5 mm lateral to Bregma, and a 5 x 5 mm area centered 5 mm posterior and 4 mm lateral to Bregma was thinned for laser speckle imaging. Both neurological and histological evaluations were performed at 72 hr postinjury. Mean flow during 90-min ischemia was 29% of baseline measured by laser Doppler and 36-54% by laser speckle. Flow transients occurred in all rats, the number of PIFTs being 4.6 +/- 1.8/90 min. By both methods, 95.6% of them occurred with temporal correlation. The average duration of PIFTs was also identical (162 +/- 24 and 162 +/- 34 sec, respectively). Five different morphologies of flow transients ranging from hypoperfusive to hyperemic were identified by laser speckle. The PIFTs changed their morphology dynamically over certain regions. All of the animals showed an infarct (178.5 +/- 26 mm(3)) in the middle cerebral artery territory. Laser Doppler in itself can be a reliable method for counting/detecting PIFTs, but laser speckle is capable of monitoring the dynamic changes in PIFT morphology over the penumbral and periischemic cortex.

Bloom syndrome (BS) is a rare genetic disorder characterized by genomic instability and a high predisposition to cancer. The gene defective in BS, BLM, encodes a member of the RecQ family of 3'-5' DNA helicases, and is proposed to function in recombinational repair during DNA replication. Here, we have utilized single-molecule fluorescence resonance energy transfer microscopy to examine the behaviour of BLM on forked DNA substrates. Strikingly, BLM unwound individual DNA molecules in a repetitive manner, unwinding a short length of duplex DNA followed by rapid reannealing and reinitiation of unwinding in several successions. Our results show that a monomeric BLM can 'measure' how many base pairs it has unwound, and once it has unwound a critical length, it reverses the unwinding reaction through strand switching and translocating on the opposing strand. Repetitive unwinding persisted even in the presence of hRPA, and interaction between wild-type BLM and hRPA was necessary for unwinding reinitiation on hRPA-coated DNA. The reported activities may facilitate BLM processing of stalled replication forks and illegitimately formed recombination intermediates.

Geometric frustration arises when lattice structure prevents simultaneous minimization of local interactions. It leads to highly degenerate ground states and, subsequently, complex phases of matter such as water ice, spin ice and frustrated magnetic materials. Here we report a simple geometrically frustrated system composed of closely packed colloidal spheres confined between parallel walls. Diameter-tunable microgel spheres are self-assembled into a buckled triangular lattice with either up or down displacements analogous to an antiferromagnetic Ising model on a triangular lattice. Experiment and theory reveal single-particle dynamics governed by in-plane lattice distortions that partially relieve frustration and produce ground-states with zigzagging stripes and subextensive entropy, rather than the more random configurations and extensive entropy of the antiferromagnetic Ising model. This tunable soft matter system provides an uncharted arena in which the dynamics of frustration, thermal excitations and defects can be directly visualized., (*) These authors contributed equally to this work. Main Text: 6 pages, 5 figures. Supplementary Information and Online Movies available at http://www.physics.upenn.edu/~yair/frustration.html

The ability to induce and sustain angiogenesis is regarded as one of the hallmarks of cancer growth and metastasis. Marked advances in understanding vascular physiology and mechanisms of angiogenesis have led to the development of vascularly-targeted anti-cancer therapies. Two broad subclasses of agents are in clinical use currently: those with anti-angiogenic activity (i.e. those that target angiogenic pathways) and those that have direct cytotoxic activity against proliferating endothelial cells (i.e. vascular disruptive agents (VDAs)). This article reviews the various imaging modalities, including magnetic resonance imaging, computed tomography, photon emission imaging (positron emission tomography and single photon emission computed tomography), ultrasound and optical (near-infrared absorption and scattering) techniques that have been used in an attempt to assess the status of tumor neovasculature in vivo. In each case, we describe the basic imaging methodology, methods for image quantification, technical standardization and reproducibility, and current and potential clinical applications for monitoring the status of tumor neovasculature before and during treatment with vascular targeted agents.

Diffuse optical imaging (DOI) is a noninvasive optical technique that employs near-infrared (NIR) light to quantitatively characterize the optical properties of thick tissues. Although NIR methods were first applied to breast transillumination (also called diaphanography) nearly 80 years ago, quantitative DOI methods employing time- or frequency-domain photon migration technologies have only recently been used for breast imaging (i.e., since the mid-1990s). In this review, the state of the art in DOI for breast cancer is outlined and a multi-institutional Network for Translational Research in Optical Imaging (NTROI) is described, which has been formed by the National Cancer Institute to advance diffuse optical spectroscopy and imaging (DOSI) for the purpose of improving breast cancer detection and clinical management. DOSI employs broadband technology both in near-infrared spectral and temporal signal domains in order to separate absorption from scattering and quantify uptake of multiple molecular probes based on absorption or fluorescence contrast. Additional dimensionality in the data is provided by integrating and co-registering the functional information of DOSI with x-ray mammography and magnetic resonance imaging (MRI), which provide structural information or vascular flow information, respectively. Factors affecting DOSI performance, such as intrinsic and extrinsic contrast mechanisms, quantitation of biochemical components, image formation/visualization, and multimodality co-registration are under investigation in the ongoing research NTROI sites. One of the goals is to develop standardized DOSI platforms that can be used as stand-alone devices or in conjunction with MRI, mammography, or ultrasound. This broad-based, multidisciplinary effort is expected to provide new insight regarding the origins of breast disease and practical approaches for addressing several key challenges in breast cancer, including: Detecting disease in mammographically dense tissue, distinguishing between malignant and benign lesions, and understanding the impact of neoadjuvant chemotherapies.

After complete cerebral ischemia, the postischemic blood flow response to functional activation is severely attenuated for several hours. However, little is known about the spatial and temporal extent of the blood flow response in the acute postischemic period after incomplete cerebral ischemia. To investigate the relative cerebral blood flow (rCBF) response in the somatosensory cortex of rat to controlled vibrissae stimulation after transient incomplete ischemia (15-min bilateral common carotid artery occlusion + hypotension), we employed laser speckle imaging combined with statistical parametric mapping. We found that the ischemic insult had a significant impact on the baseline blood flow ( P 0.1). However, the time when rCBF response reached its maximum was significantly delayed ( P < 0.0001) from 2.4 ± 0.2 secs before ischemia to 3.6 ± 0.1 secs at 20 mins into reperfusion ( P < 0.001); the delay was reduced gradually to 2.9 ± 0.2 secs after 3 h, which was still significantly greater than that observed before the insult ( P = 0.04).

We report the first optical recordings of action potentials, in single trials, from one or a few (approximately 1-2 microm) mammalian nerve terminals in an intact in vitro preparation, the mouse neurohypophysis. The measurements used two-photon excitation along the "blue" edge of the two-photon absorption spectrum of di-3-ANEPPDHQ (a fluorescent voltage-sensitive naphthyl styryl-pyridinium dye), and epifluorescence detection, a configuration that is critical for noninvasive recording of electrical activity from intact brains. Single-trial recordings of action potentials exhibited signal-to-noise ratios of approximately 5:1 and fractional fluorescence changes of up to approximately 10%. This method, by virtue of its optical sectioning capability, deep tissue penetration, and efficient epifluorescence detection, offers clear advantages over linear, as well as other nonlinear optical techniques used to monitor voltage changes in localized neuronal regions, and provides an alternative to invasive electrode arrays for studying neuronal systems in vivo.

We acquire and compare three-dimensional tomographic breast images of three females with suspicious masses using diffuse optical tomography (DOT) and positron emission tomography (PET). Co-registration of DOT and PET images was facilitated by a mutual information maximization algorithm. We also compared DOT and whole-body PET images of 14 patients with breast abnormalities. Positive correlations were found between total hemoglobin concentration and tissue scattering measured by DOT, and fluorodeoxyglucose (18F-FDG) uptake. In light of these observations, we suggest potential benefits of combining both PET and DOT for characterization of breast lesions.

We quantified the effect of anesthesia on cerebral oxygen saturation and blood flow in infants with congenital heart disease. We find that both ScO2 and CBF decrease after induction of anesthesia independent of cardiac diagnosis.

Extracorporeal membrane oxygenation therapy mechanically circulates and oxygenates blood. We assess cerebral blood flow during perturbation of ECMO flow using diffuse optical and correlation spectroscopies. We observe both regulated and passive flow.

We applied a novel pressure modulation algorithm to isolate cerebral blood flow signals from extracerebral artifacts during optical monitoring of scalp hyperemia in a healthy adult. This was achieved without a priori anatomical information.

Study objectives Children with obstructive sleep apnea syndrome (OSAS) often experience periods of hypercapnia during sleep, a potent stimulator of cerebral blood flow (CBF). Considering this hypercapnia exposure during sleep, it is possible that children with OSAS have abnormal CBF responses to hypercapnia even during wakefulness. Therefore, we hypothesized that children with OSAS have blunted CBF response to hypercapnia during wakefulness, compared to snorers and controls. Methods CBF changes during hypercapnic ventilatory response (HCVR) were tested in children with OSAS, snorers, and healthy controls using diffuse correlation spectroscopy (DCS). Peak CBF changes with respect to pre-hypercapnic baseline were measured for each group. The study was conducted at an academic pediatric sleep center. Results Twelve children with OSAS (aged 10.1 ± 2.5 [mean ± standard deviation] y, obstructive apnea hypopnea index [AHI] = 9.4 [5.1-15.4] [median, interquartile range] events/hour), eight snorers (11 ± 3 y, 0.5 [0-1.3] events/hour), and 10 controls (11.4 ± 2.6 y, 0.3 [0.2-0.4] events/hour) were studied. The fractional CBF change during hypercapnia, normalized to the change in end-tidal carbon dioxide, was significantly higher in controls (9 ± 1.8 %/mmHg) compared to OSAS (7.1 ± 1.5, P = 0.023) and snorers (6.7 ± 1.9, P = 0.025). Conclusions Children with OSAS and snorers have blunted CBF response to hypercapnia during wakefulness compared to controls. Noninvasive DCS blood flow measurements of hypercapnic reactivity offer insights into physiopathology of OSAS in children, which could lead to further understanding about the central nervous system complications of OSAS.

We demonstrate spatially-dense diffuse optical tomographic (DOT) reconstructions of breast cancer utilizing two forms of structural priors: MR images from a concurrent DOT-MRI instrument and fringe-projection profilometry in an optical-only system.

We investigate the feasibility of constructing an instrument, which can help critical care physicians by real-time monitoring/measuring absolute cerebral blood flow (CBF) and cerebral metabolic rate for oxygen (CMRO2) to facilitate fast diagnosis of Ischemia.

We demonstrate the bedside measurement & characterization of cerebrovascular autoregulation (CVAR) dynamics from 10 healthy volunteers, using a new diffuse correlation spectroscopy (DCS) instrument for high-speed measurement of cerebral blood flow.

We report instrumentation & methods for depth sensitive imaging of tumor fluorescence in an intraoperative setting with spatial frequency domain imaging. We show preliminary reconstruction results, with simulation, using an analytic inversion method.

Supine postures are utilized to increase cerebral blood flow following ischemic stroke. We find supine cerebral blood flow is lower than sitting flow in 25% of subjects and that postural changes increase supine blood flow.

Acetylation is one of the most abundant histone modifications found in nucleosomes. Although such modifications are thought to function mainly in recognition, acetylation is known to produce nucleosome structural alterations. These could be of functional significance in vivo. Here, the basic features of mouse mammary tumor virus (MMTV) promoter nucleosomal arrays reconstituted with highly acetylated histones prepared from butyrate-treated HeLa cells are characterized by atomic force microscopy. Results are compared to previous results obtained with hypoacetylated MMTV and hyper- or hypoacetylated 5S rDNA arrays. MMTV arrays containing highly acetylated histones show diminished intramolecular compaction compared to hypoacetylated MMTV arrays and no tendency for cooperativity in nucleosome occupation. Both features have been suggested to reflect histone tail-mediated internucleosomal interactions; these observations are consistent with that suggestion. 5S arrays show qualitatively similar behavior. Two other effects of acetylation show stronger DNA template dependence. Nucleosome salt stability is diminished in highly acetylated compared to hypoacetylated MMTV arrays, but nucleosome (histone) loading tendencies are unaffected by acetylation. However, highly acetylated histones show reduced loading tendencies on 5S templates (vs hypoacetylated), but 5S nucleosome salt stabilities are unaffected by acetylation. ATP-dependent nucleosome remodeling by human Swi-Snf is similar on hyper- and hypoacetylated MMTV arrays.

Aerogels are ultralight, highly porous materials typicallyfabricated by subjecting a wet-gel precursor to critical-point-drying (CPD) or lyophilization (freeze-drying) in order toremove background liquid without collapsing the network.Microscopically, aerogels are composed of tenuous networksof clustered nanoparticles, and the materials often haveunique properties, including very high strength-to-weight andsurface-area-to-volume ratios. To date most aerogels are fab-ricated from silica

Introduction: Neonates with critical congenital heart disease (CHD) exhibit a high prevalence of white matter injury (WMI). Recent work in neonates with hypoplastic left heart syndrome has shown that a longer wait from birth to surgery was associated with lower cerebral tissue oxygen saturations (StO2) on the morning of surgery and an increased risk for postoperative white matter injury (WMI). Understanding the daily preoperative changes in cerebral physiology during this vulnerable period may lead to new therapeutic algorithms aimed at prevention of WMI. Methods: Term neonates with critical CHD were recruited for this study. Frequency domain diffuse optical spectroscopy was employed to noninvasively quantify StO2. Daily StO2 measurements were made from day of recruitment until the day of surgery. Results: We studied 37 neonates with critical CHD. Operations were performed at 1-8 days of life. Non-elective reasons for timing of surgery resulted only in earlier surgery in this cohort. The subjects were placed in 2 groups depending on if they had a normal arch (N=20) or obstructed arch (N=17). In a linear mixed-effects model, StO2 decreased as a function of time from birth but was not specific to diagnostic group. Conclusions: Observed longitudinal daily decline in StO2 from birth until surgery supports our earlier findings and extends them to other groups of CHD. These results suggest that reported increases in risk for WMI with time-to-surgery could be due to mismatched oxygen delivery to metabolic demand. Therapeutic interventions such as increasing cerebral blood flow or decreasing cerebral oxygen demand may be considered when earlier surgery is not possible.

This study introduces liquid crystal (LC) Janus droplets. We describe a process for the preparation of these droplets, which consist of nematic LC and polymer compartments. The process employs solvent-induced phase separation in emulsion droplets generated by microfluidics. The droplet morphology was systematically investigated and demonstrated to be sensitive to the surfactant concentration in the background phase, the compartment volume ratio, and the possible coalescence of multiple Janus droplets. Interestingly, the combination of a polymer and an anisotropic LC introduces new functionalities into Janus droplets, and these properties lead to unusual dynamical behaviors. The different densities and solubilities of the two compartments produce gravity-induced alignment, tumbling, and directional self-propelled motion of Janus droplets. LC Janus droplets with remarkable optical properties and dynamical behaviors thus offer new avenues for applications of Janus colloids and active soft matter.

Introduction Non-invasive diffuse optical tomography (DOT) and diffuse correlation spectroscopy (DCS) can detect and characterize breast cancer and predict tumor responses to neoadjuvant chemotherapy, even in patients with radiographically dense breasts. However, the relationship between measured optical parameters and pathological biomarker information needs to be further studied to connect information from optics to traditional clinical cancer biology. Thus we investigate how optically measured physiological parameters in malignant tumors such as oxy-, deoxy-hemoglobin concentration, tissue blood oxygenation, and metabolic rate of oxygen correlate with microscopic histopathological biomarkers from the same malignant tumors, e.g., Ki67 proliferation markers, CD34 stained vasculature markers and nuclear morphology. Methods In this pilot study, we investigate correlations of macroscopic physiological parameters of malignant tumors measured by diffuse optical technologies with microscopic histopathological biomarkers of the same tumors, i.e., the Ki67 proliferation marker, the CD34 stained vascular properties marker, and nuclear morphology. Results The tumor-to-normal relative ratio of Ki67-positive nuclei is positively correlated with DOT-measured relative tissue blood oxygen saturation (R = 0.89, p-value: 0.001), and lower tumor-to-normal deoxy-hemoglobin concentration is associated with higher expression level of Ki67 nuclei (p-value: 0.01). In a subset of the Ki67-negative group (defined by the 15 % threshold), an inverse correlation between Ki67 expression level and mammary metabolic rate of oxygen was observed (R = −0.95, p-value: 0.014). Further, CD34 stained mean-vessel-area in tumor is positively correlated with tumor-to-normal total-hemoglobin and oxy-hemoglobin concentration. Finally, we find that cell nuclei tend to have more elongated shapes in less oxygenated DOT-measured environments. Conclusions Collectively, the pilot data are consistent with the notion that increased blood is supplied to breast cancers, and it also suggests that less conversion of oxy- to deoxy-hemoglobin occurs in more proliferative cancers. Overall, the observations corroborate expectations that macroscopic measurements of breast cancer physiology using DOT and DCS can reveal microscopic pathological properties of breast cancer and hold potential to complement pathological biomarker information. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0578-z) contains supplementary material, which is available to authorized users.

Recently there has been a surge in interest in using video-microscopy techniques to infer the local mechanical properties of disordered solids. One common approach is to minimize the difference between particle vibrational displacements in a local coarse-graining volume and the displacements that would result from a best-fit affine deformation. Effective moduli are then be inferred under the assumption that the components of this best-fit affine deformation tensor have a Boltzmann distribution. In this paper, we combine theoretical arguments with experimental and simulation data to demonstrate that the above does not reveal information about the true elastic moduli of jammed packings and colloidal glasses., 12 pages, 8 figures

Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ∼16. Ten animals showed no residual tumor cells in the wound bed (mean SBR1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

The modified Beer-Lambert law is among the most widely used approaches for analysis of near-infrared spectroscopy (NIRS) reflectance signals for measurements of tissue blood volume and oxygenation. Briefly, the modified Beer-Lambert paradigm is a scheme to derive changes in tissue optical properties based on continuous-wave (CW) diffuse optical intensity measurements. In its simplest form, the scheme relates differential changes in light transmission (in any geometry) to differential changes in tissue absorption. Here we extend this paradigm to the measurement of tissue blood flow by diffuse correlation spectroscopy (DCS). In the new approach, differential changes of the intensity temporal auto-correlation function at a single delay-time are related to differential changes in blood flow. The key theoretical results for measurement of blood flow changes in any tissue geometry are derived, and we demonstrate the new method to monitor cerebral blood flow in a pig under conditions wherein the semi-infinite geometry approximation is fairly good. Specifically, the drug dinitrophenol was injected in the pig to induce a gradual 200% increase in cerebral blood flow, as measured with MRI velocity flow mapping and by DCS. The modified Beer-Lambert law for flow accurately recovered these flow changes using only a single delay-time in the intensity auto-correlation function curve. The scheme offers increased DCS measurement speed of blood flow. Further, the same techniques using the modified Beer-Lambert law to filter out superficial tissue effects in NIRS measurements of deep tissues can be applied to the DCS modified Beer-Lambert law for blood flow monitoring of deep tissues.

Surgery is the most effective treatment strategy for solid tumors. Intraoperative imaging of tumors helps detect tumor margins and establish the most appropriate surgical margins. Endoscopic surgery is a standard of care procedure for the resection of tumors, and is applicable for a wide range of solid tumors. While several imaging methodologies can be used for intraoperative imaging, optical imaging is promising for clinical application because it can detect microscopic disease, is minimally invasive, is inexpensive, does not require advance training for surgeons and can provide real-time images. Fluorescence from an injected contrast agent (Indo-cyanine green, ICG) has been effectively used for the identification of tumors in humans. In this study, we adapt a commercially available endoscope for intraoperative imaging of solid tumors. Our instrument utilizes light from a near-infrared 780nm LED to illuminate the surgical field of view and two CCD cameras for imaging the reflected fluorescence as well as the background tissue. We show that our instrument can simultaneously image fluorescence from the tumor as well as the background tissue. We characterize our instrument in tissue simulating phantoms, with tumor simulating ‘targets’.

Deeper insight into the molecular pathways that orchestrate skeletal myogenesis should enhance our understanding of, and ability to treat, human skeletal muscle disease. It is now widely appreciated that nutrients, such as molecular oxygen (O2), modulate skeletal muscle formation. During early stages of development and regeneration, skeletal muscle progenitors reside in low O2 environments before local blood vessels and differentiated muscle form. Moreover, low O2 availability (hypoxia) impedes progenitor-dependent myogenesis in vitro through multiple mechanisms, including activation of hypoxia inducible factor 1α (HIF1α). However, whether HIF1α regulates skeletal myogenesis in vivo is not known. Here, we explored the role of HIF1α during murine skeletal muscle development and regeneration. Our results demonstrate that HIF1α is dispensable during embryonic and fetal myogenesis. However, HIF1α negatively regulates adult muscle regeneration after ischemic injury, implying that it coordinates adult myogenesis with nutrient availability in vivo. Analyses of Hif1a mutant muscle and Hif1a-depleted muscle progenitors further suggest that HIF1α represses myogenesis through inhibition of canonical Wnt signaling. Our data provide the first evidence that HIF1α regulates skeletal myogenesis in vivo and establish a novel link between HIF and Wnt signaling in this context.

Purpose Local recurrence of rectal cancer remains a significant clinical problem despite multi-modality therapy. Photodynamic Therapy (PDT) is a cancer treatment which generates tumor kill through the production of singlet oxygen in cells containing a photosensitizing drug when exposed to laser light of a specific wavelength. PDT is a promising modality for prevention of local recurrence of rectal cancer for several reasons: tumor cells may selectively retain photosensitizer at higher levels than normal tissues, the pelvis after mesorectal excision is a fixed space amenable to intra-operative illumination, and PDT can generate toxicity in tissues up to 1 cm thick. This study evaluated the safety, tissue penetration of 730 nm light, normal tissue toxicity and surgical outcome in a dog model of rectal resection after motexafin lutetium-mediated photodynamic therapy. Methods Ten mixed breed dogs were used. Eight dogs underwent proctectomy and low rectal end to end stapled anastomosis. Six dogs received the photosensitizing agent motexafin lutetium (MLu, Pharmacyclics, Inc., Sunnyvale, CA) of 2 mg/kg preoperatively and underwent subsequent pelvic illumination of the transected distal rectum of 730 nm light with light doses ranging from 0.5 J/cm 2 to 10 J/cm 2 three hours after drug delivery. Two dogs received light, but no drug, and underwent proctectomy and low-rectal stapled anastomosis. Two dogs underwent midline laparotomy and pelvic illumination. Light penetration in tissues was determined for small bowel, rectum, pelvic sidewall, and skin. Clinical outcomes were recorded. Animals were sacrificed at 14 days and histological evaluation was performed. Results All dogs recovered uneventfully. No dog suffered an anastomotic leak. Severe tissue toxicity was not seen. Histological findings at necropsy revealed mild enteritis in all dogs. The excitation light penetration depths were 0.46 ± 0.18, 0.46 ± 0.15, and 0.69 ± 0.39 cm, respectively, for rectum, small bowel, and peritoneum in dogs that had received MLu. For control dogs without photosensitizer MLu, the optical penetration depths were longer: 0.92 ± 0.63, 0.67 ± 0.10, and 1.1 ± 0.80 cm for rectum, small bowel, and peritoneum, respectively. Conclusion Low rectal stapled anastomosis is safe when performed with MLu-mediated pelvic PDT in a dog model. Significant tissue penetration of 730 nm light into the rectum and pelvic sidewall was revealed without generation of significant toxicity or histological sequelae. Penetration depths of 730 nm light in pelvic tissue suggest that microscopic residual disease of less than 5 mm are likely to be treated adequately with MLu-mediated PDT. This approach merits further investigation as an adjuvant to total mesorectal excision and chemoradiation for rectal cancer.

Being able to follow assembly/disassembly reactions of biomolecular complexes directly at the single molecule level would be very useful. Here, we use an AFM technique that can simultaneously obtain topographic images and identify the locations of a specific type of protein within those images to monitor the histone H2A component of nucleosomes acted on by human Swi–Snf, an ATP-dependent nucleosome remodeling complex. Activation of remodeling results in significant H2A release from nucleosomes, based on recognition imaging and nucleosome height changes, and changes in the recognition patterns of H2A associated directly with hSwi–Snf complexes.

Purpose: A previous phase I trial of i.p. photodynamic therapy established the maximally tolerated dose of Photofrin (Axcan Pharma, Birmingham, AL)-mediated photodynamic therapy and showed encouraging efficacy. The primary objectives of this phase II study were to determine the efficacy and toxicities of i.p. photodynamic therapy in patients with peritoneal carcinomatosis and sarcomatosis. Experimental Design: Patients received Photofrin 2.5 mg/kg i.v. 48 hours before debulking surgery. Intraoperative laser light was delivered to the peritoneal surfaces of the abdomen and pelvis. The outcomes of interest were (a) complete response, (b) failure-free survival time, and (c) overall survival time. Photosensitizer levels in tumor and normal tissues were measured. Results: One hundred patients were enrolled into one of three strata (33 ovarian, 37 gastrointestinal, and 30 sarcoma). Twenty-nine patients did not receive light treatment. All 100 patients had progressed by the time of statistical analysis. The median failure-free survival and overall survival by strata were ovarian, 2.1 and 20.1 months; gastrointestinal cancers, 1.8 and 11.1 months; sarcoma, 3.7 and 21.9 months. Substantial fluid shifts were observed postoperatively, and the major toxicities were related to volume overload. Two patients died in the immediate postoperative period from bleeding, sepsis, adult respiratory distress syndrome, and cardiac ischemia. Conclusions: Intraperitoneal Photofrin-mediated photodynamic therapy is feasible but does not lead to significant objective complete responses or long-term tumor control. Heterogeneity in photosensitizer uptake and tumor oxygenation, lack of tumor specificity for photosensitizer uptake, and the heterogeneity in tissue optical properties may account for the lack of efficacy observed.

One of the most important brain rhythms is that which generates involuntary breathing movements. The lower brain stem contains neural circuitry for respiratory rhythm generation in mammals. To date, microsectioning and selective lesioning studies have revealed anatomical regions necessary for respiratory rhythmogenesis. Although respiratory neurons distributed within these regions can be identified by their firing patterns in different phases of the respiratory cycle, conventional electrophysiology techniques have limited the study of spatial organization within this network. Optical imaging techniques offer the potential for monitoring the spatiotemporal activity of large groups of neurons simultaneously. Using high-speed voltage-sensitive dye imaging and spatial correlation analysis in an arterially perfused in situ preparation of the juvenile rat, we determined the spatial distribution of respiratory neuronal activity in a region of the ventrolateral respiratory group containing the pre-Bötzinger complex (pBC) during spontaneous eupneic breathing. While distinctly pre- and postinspiratory-related responses were spatially localizable on length scales less than 100 μm, we found the studied area on whole exhibited a spatial mixture of phase-spanning and postinspiratory-related activity. Additionally, optical recordings revealed significant widespread hyperpolarization, suggesting inhibition in the same region during expiration. This finding is consistent with the hypothesis that inhibitory neurons play a crucial role in the inspiration-expiration phase transition in the pBC. To our knowledge this is the first optical imaging of a near fully intact in situ preparation that exhibits both eupneic respiratory activity and functional reflexes.

Background and Objectives: Interstitial photodynamic therapy (PDT) is an emerging modality for the treatment of solid organ disease. Our group at the University of Pennsylvania has performed extensive studies that demonstrate the feasibility of interstitial PDT for prostate cancer. Our preclinical and clinical experience is herein detailed. StudyDesign/MaterialsandMethods: We have treated 16 canines in preclinical studies, and 16 human subjects in a Phase I study, using motexafin lutetium-mediated PDT for recurrent prostate adenocarcinoma. Dosimetry of light fluence, drug level and oxygen distribution for these patients were performed. Results: We demonstrate the safe and comprehensive treatment of the prostate using PDT. However, there is significant variability in the dose distribution and the subsequent tissue necrosis throughout the prostate. Conclusions: PDT is an attractive option for the treatment of prostate adenocarcinoma. However, the observed variation in PDT dose distribution translates into uncertain therapeutic reproducibility. Our future focus will be on the development of an integrated system that is able to both detect and compensate for dose variations in real-time, in order to deliver a consistent overall PDT dose distribution. Lasers Surg. Med. 38:427–434, 2006. 2006 Wiley-Liss, Inc.

Microscopy based on voltage-sensitive dyes has proven effective for revealing spatio-temporal patterns of neuronal activity in vivo and in vitro. Two-photon microscopy using voltage-sensitive dyes offers the possibility of wide-field visualization of membrane potential on sub-cellular length scales, hundreds of microns below the tissue surface. Very little information is available, however, about the utility of voltage-sensitive dyes for two-photon imaging purposes. Here we report on measurements of two-photon fluorescence excitation crosssections for nine voltage-sensitive dyes in a solvent, octanol, intended to simulate the membrane environment. Ultrashort light pulses from a Ti:sapphire laser were used for excitation from 790 to 960 nm, and fluorescein dye was used as a calibration standard. Overall, dyes RH795, RH421, RH414, di-8-ANEPPS, and di-8-ANEPPDHQ had the largest two-photon excitation cross-sections (∼15 × 10 −50 cm 4 s photon −1 )i n this wavelength region and are therefore potentially useful for two-photon microscopy. Interestingly, di-8-ANEPPDHQ, a chimera constructed from the potentiometric dyes RH795 and di-8-ANEPPS, exhibited larger cross-sections than either of its constituents. © 2005 Elsevier B.V. All rights reserved.

The excellent properties of transistors, wires, and sensors made from single-walled carbon nanotubes (SWNTs) make them promising candidates for use in advanced nanoelectronic systems. Gas-phase growth procedures such as the high pressure decomposition of carbon monoxide (HiPCO) method yield large quantities of small diameter semiconducting SWNTs, which are ideal for use in nanoelectronic circuits. As-grown HiPCO material, however, commonly contains a large fraction of carbonaceous impurities that degrade properties of SWNT devices. Here we demonstrate a purification, deposition, and fabrication process that yields devices consisting of metallic and semiconducting nanotubes with electronic characteristics vastly superior to those of circuits made from raw HiPCO. Source-drain current measurements on the circuits as a function of temperature and backgate voltage are used to quantify the energy gap of semiconducting nanotubes in a field effect transistor geometry. This work demonstrates significant progress towards the goal of producing complex integrated circuits from bulk-grown SWNT material., Comment: 6 pages, 4 figures, to appear in Nature Materials

Nucleosomes are the basic units of eukaryotic chromatin structure. By restricting factor access to regulatory DNA sequences, nucleosomes significantly impact genomic processes such as transcription, and various mechanisms to alter nucleosome structure to relieve this repression have evolved. Both nucleosomes and processes that alter them are inherently dynamic in nature. Thus, studies of dynamics will be necessary to truly understand these relief mechanisms. We describe here the characteristics of a novel fluorescence resonance energy transfer-based reporter that can clearly signal the formation of a canonical nucleosome structure and follow conformational and compositional changes in that structure, both at the ensemble-average (bulk) and at the single molecule level. Labeled nucleosomes behave conformationally and thermodynamically like typical nucleosomes; thus they are relevant reporters of nucleosome behavior. Nucleosomes and free DNA are readily distinguishable at the single-molecule level. Thus, these labeled nucleosomes are well suited to studies of dynamic changes in nucleosome structure including single-molecule dynamics.

We employ diffuse optical tomography (DOT) to track treatment progress in a female subject presenting with locally advanced invasive carcinoma of the breast during neoadjuvant chemotherapy. Three-dimensional images of total hemoglobin concentration and scattering identified the tumor. Our measurements reveal tumor shrinkage during the course of chemotherapy, in reasonable agreement with magnetic resonance images of the same subject. A decrease in total hemoglobin concentration contrast between tumor and normal tissue was also observed over time. The results demonstrate the potential of DOT for measuring physiological parameters of breast lesions during chemotherapy.

In eukaryotes, genomic processes like transcription, replication, repair, and recombination typically require alterations in nucleosome structure on specific DNA regions to operate. ATP-dependent nucleosome remodeling complexes provide a major mechanism for carrying out such alterations in vivo. To learn more about the action of these important complexes, we have utilized an atomic force microscopy in situ technique that permits comparison of the same individual molecules before and after activation of a particular process, in this case nucleosome remodeling. This direct approach was used to look for changes induced by the action of the human Swi-Snf remodeling complex on individual, single-copy mouse mammary tumor virus promoter nucleosomal arrays. Using this technique, we detect a variety of changes on remodeling. Many of these changes are larger in scale than suggested from previous studies and involve a number of DNA-mediated events, including a preference for the removal of a complete turn (80 basepairs) of nucleosomal DNA. The latter result raises the possibility of an unanticipated mode of human Swi-Snf interaction with the nucleosome, namely via the 11-nm histone surface.

We present results from the AMANDA high energy neutrino telescope located at the South Pole. They include measurements of the atmospheric neutrino flux, search for UHE point sources, and diffuse sources producing electromagnetic/hadronic showers at the detector or close to it.

Subsaturated nucleosomal arrays were reconstituted on a single-copy MMTV promoter DNA fragment by salt dialysis procedures and studied by atomic force microscopy. Up to an occupation level of approximately eight nucleosomes on this 1900 bp template, salt reconstitution produces nucleosomal arrays which look very similar to comparably loaded 5S rDNA nucleosomal arrays; i.e., nucleosomes are dispersed on the DNA template. Thus, at these occupation levels, the single-copy MMTV template forms arrays suitable for biophysical analyses. A quantitative comparison of the population features of subsaturated MMTV and 5S arrays detects differences between the two: a requirement for higher histone levels to achieve a given level of nucleosome occupation on MMTV templates, indicating that nucleosome loading is thermodynamically less favorable on this template; a preference for pairwise nucleosome occupation of the MMTV (but not the 5S) template at midrange occupation levels; and an enhanced salt stability for nucleosomes on MMTV versus 5S arrays, particularly in the midrange of array occupation. When average occupation levels exceed approximately eight nucleosomes per template, MMTV arrays show a significant level of mainly intramolecular compaction; 5S arrays do not. Taken together, these results show clearly that the nature of the underlying DNA template can affect the physical properties of nucleosomal arrays. DNA sequence-directed differences in the physical properties of chromatin may have important consequences for functional processes such as gene regulation.

We show new results from both the older and newer incarnations of AMANDA (AMANDA-B10 and AMANDA-II, respectively). These results demonstrate that AMANDA is a functioning, multipurpose detector with significant physics and astrophysics reach. They include a new higher-statistics measurement of the atmospheric muon neutrino flux and preliminary results from searches for a variety of sources of ultrahigh energy neutrinos: generic point sources, gamma-ray bursters and diffuse sources producing muons in the detector, and diffuse sources producing electromagnetic or hadronic showers in or near the detector.

We report a simple process to solubilize high weight fraction single-wall carbon nanotubes in water by the nonspecific physical adsorption of sodium dodecylbenzene sulfonate. The diameter distribution of nanotubes in the dispersion, measured by atomic force microscopy, showed that even at 20 mg/mL ∼63 ± 5% of single-wall carbon nanotube bundles exfoliated into single tubes. A measure of the length distribution of the nanotubes showed that our dispersion technique reduced nanotube fragmentation.