Two-photon Microscopy

Advances in optical designs are enabling the development of miniature microscopes that can examine tissue in situ for early anatomic and molecular indicators of disease, in real time, and at cellular resolution. These new devices will lead to major changes in how diseases are detected and managed, driving a shift from today's diagnostic paradigm of biopsy followed by histopathology and recommended therapy, to non-invasive point-of-care diagnosis with possible same-session definitive treatment. This shift may have major implications for the training requirements of future physicians to enable them to interpret real-time in vivo microscopic data, and will also shape the emerging fields of telepathology and telemedicine. Implementation of new technologies into clinical practice is a complex process that requires bridging gaps between clinicians, engineers and scientists. This article provides a forward-looking discussion of these issues, with a focus on malignant and pre-malignant lesions, by first highlighting some of the clinical areas where point-of-care in vivo microscopy could address unmet needs, and then by reviewing the technological challenges that are being addressed, or need to be addressed, for in vivo microscopy to become a standard clinical tool.

Basal dendrites of layer 5 cortical pyramidal neurons exhibit Na+ and N-methyl-D-aspartate receptor (NMDAR) regenerative spikes and are uniquely poised to influence somatic output. Nevertheless, due to technical limitations, how multibranch basal dendritic integration shapes and enables multiplexed barcoding of synaptic streams remains poorly mapped. Here, we combine 3D two-photon holographic transmitter uncaging, whole-cell dynamic clamp, and biophysical modeling to reveal how synchronously activated synapses (distributed and clustered) across multiple basal dendritic branches are multiplexed under quiescent and in vivo-like conditions. While dendritic regenerative Na+ spikes promote millisecond somatic spike precision, distributed synaptic inputs and NMDAR spikes regulate gain. These concomitantly occurring dendritic nonlinearities enable multiplexed information transfer amid an ongoing noisy background, including under back-propagating voltage resets, by barcoding the axo-somatic spike structure. Our results unveil a multibranch dendritic integration framework in which dendritic nonlinearities are critical for multiplexing different spatial-temporal synaptic input patterns, enabling optimal feature binding.

Histopathologic analysis of biopsy specimens obtained via white light endoscopy (WLE) is the gold standard for the diagnosis of several mucosal diseases in the upper gastrointestinal (GI) tract. However, this standard of care entails a series of critical shortcomings such as missing depth information, high costs, time inefficiency, low-resolution imaging in vivo, high sampling variability, missing intrinsic tissue-specific contrast, and anesthesia related risk. In the quest for a diagnostic technology to replace the current standard of care, in vivo optical endomicroscopy has emerged as a promising alternative. This paper tells the story of a cluster of optical microscopy-based modalities invented, further developed, or first-validated in the laboratory of Dr. Guillermo J. Tearney (Tearney Lab) at the Wellman Center for Photomedicine of Massachusetts General Hospital over the past two decades, that combined lead to a novel method for diagnosis of eosinophilic esophagitis (EoE). Rath...

We demonstrate, for the first time to our knowledge, the utility of coherent Rayleigh scattering (CRS) for temperature measurements in low-density gases and weakly ionized plasmas by measuring the translational temperature of neutral argon in a glow discharge. By analysis of the near-Gaussian spectral profile of the CRS signal, we determine temperatures with an uncertainty of #3%. We also investigate the intensity range over which this simple Gaussian analysis can be used for temperature measurements and discuss its potential for gas diagnostics.

The endothelial glycocalyx is an integral component of the brain vascular barrier. Visualizing its
structure in vivo is essential to understand its physiological and pathophysiological mechanisms.
Here, we present a surgical protocol for chronic cranial window implantation in mice, alongside
the use of multiphoton microscopy tools to image the cortical vasculature.We describe steps for
cranial window implantation, intravenous injection of fluorescent markers, and intravital imaging.
We then detail a technique to quantify glycocalyx thickness using Imaris image analysis software.

Sensory Adaptation (SA) is a prominent aspect of how neurons respond to sensory signals, ubiquitous across species and modalities. However, SA depends on the activation state of the brain and the extent to which SA is expressed in awake, behaving animals during active sensation remains unclear.
Here, we addressed this question by training head-fixed mice to detect an object using their whiskers and recording neuronal activity from barrel cortex whilst simultaneously imaging the whiskers in 3D.
We found that neuronal responses decreased during the course of whisker object touch sequences and that this was due to two factors. First, a motor effect, whereby, during a sequence of touches, later touches were mechanically weaker than early ones. Second, a sensory encoding effect, whereby neuronal tuning to touch became progressively less sensitive during the course of a touch sequence.
The sensory encoding effect was whisker-specific. These results show that SA does occur during active whisker sensing and suggest that SA is fundamental to sensation during natural behaviour.

Although electrophysiologists have been recording intracellular neural activity routinely ever since the ground-breaking work of Hodgkin and Huxley, and extracellular multichannel electrodes have also been used frequently and extensively, a practical experimental method to track changes in membrane potential along a complete single neuron is still lacking. Instead of obtaining multiple intracellular measurements on the same neuron, we propose an alternative method by combining single-channel somatic patch-clamp and multichannel extracellular potential recordings. In this work, we show that it is possible to reconstruct the complete spatiotemporal distribution of the membrane potential of a single neuron with the spatial resolution of an extracellular probe during action potential generation. Moreover, the reconstruction of the membrane potential allows us to distinguish between the two major but previously hidden components of the current source density (CSD) distribution: the resistive and the capacitive currents. This distinction provides a clue to the clear interpretation of the CSD analysis, because the resistive component corresponds to transmembrane ionic currents (all the synaptic, voltage-sensitive and passive currents), whereas capacitive currents are considered to be the main contributors of counter-currents. We validate our model-based reconstruction approach on simulations and demonstrate its application to experimental data obtained in vitro via paired extracellular and intracellular recordings from a single pyramidal cell of the rat hippocampus. In perspective, the estimation of the spatial distribution of resistive membrane currents makes it possible to distiguish between active and passive sinks and sources of the CSD map and the localization of the synaptic input currents, which make the neuron fire.

The bio-guided fractionation of an ethanol extract of Baccharis genistelloides (L) Baker, a plant that traditionally is used to treat diabetes, yielded (+)-curcuphenol (1) as agonist of GPR40, a cell surface receptor for free fatty acids. This paper describes the agonistic activity of 1 as well as of a series of semi-synthetic and synthetic analogues.

The bio-guided fractionation of an ethanol extract of Baccharis genistelloides (L) Baker, a plant that traditionally is used to treat diabetes, yielded (+)-curcuphenol (1) as agonist of GPR40, a cell surface receptor for free fatty acids. This paper describes the agonistic activity of 1 as well as of a series of semi-synthetic and synthetic analogues.

Second Harmonic Generation Microscopy (SHG) is generally acknowledged as a powerful tool for the label-free 3D visualization of tissues and advanced materials, with one of its most popular applications being collagen imaging. Although the great need, progress in superresolved SHG imaging lags behind the developments reported over the past years in fluorescencebased optical nanoscopy. In this work, we quantitatively show on collagenous tissues that by combining SHG imaging with re-scan microscopy resolutions that surpass the diffraction limit with ~1.4x become available. Besides Re-scan Second Harmonic Generation Microscopy (rSHG), we demonstrate as well super-resolved Re-scan Two-Photon Excited Fluorescence Microscopy (rTPEF). These two techniques are implemented by modifying a Re-scan Confocal Microscope (RCM), retaining its initial function, resulting thus in a multimodal rSHG/rTPEF/RCM system. Given the simplicity and flexibility of re-scan microscopy, we consider that the reported results are likely to augment the number and nature of applications relying on super-resolved non-linear optical imaging.

intact animal to the cellular, subcellular, and molecular levels. It is published 12 times a year (monthly) by the American lymphatics, including experimental and theoretical studies of cardiovascular function at all levels of organization ranging from the publishes original investigations on the physiology of the heart, blood vessels, and

University-Molecules, as opposed to atoms, present confounding possibilities of nuclear and electronic motion upon strong field ionization. The dynamics and fragmentation patterns in response to the laser field are structure sensitive; therefore, a molecule cannot simply be treated as a "bag of atoms" during field induced ionization. We consider here to what extent molecules retain their molecular identity and properties under strong laser fields. Using time-of-flight mass spectrometry in conjunction with pump-probe techniques we study the dynamical behavior of these molecules, monitoring ion yield modulation caused by intramolecular motions post ionization. The delay scans show that among positional isomers the variations in relative energies, amounting to only a few hundred meVs, influence the dynamical behavior of the molecules despite their having experienced such high fields (V/Å). Ab initio calculations were performed to predict dynamics along with single and multiphoton resonances in the neutral and ionic states. We propose that single electron ionization occurs within an optical cycle with the electron carrying away essentially all of the energy, leaving behind little internal energy in the cation. Evidence for this observation comes from coherent vibrational motion governed by the potential energy surface of the ground state of the cation. Subsequent fragmentation of the cation takes place as a result of further photon absorption modulated by one-and two-photon resonances, which provide sufficient energy to overcome the dissociation energy.

in gas phase polyatomic molecules has been a hot topic for quite some time. Here we explore the time resolved dynamics of acetophenone and its methyl substituted isomer when excited by intense 800nm femtosecond pump and probe pulses. The parent ion yield show 500 fs modulations that die down within 3ps. Similar modulations having the same timescales in the parent ion yield are also observed for the p-methyl isomer. The o-methyl isomer however shows longer 1ps modulations. Interestingly enough no oscillations are observed for the meta isomer. Quantum chemical calculations at the CASSCF/6-311G level of theory predicts that upon excitation the neutral ground state is planar and the energy spacing between the levels is very small. Preliminary calculations also predict torsional motion coupled to electronic modulations on the D 0 state and further calculations are being performed to ascertain the involvement of the D 1 and D 2 states. This could help us better understand the electronic effect of substitution on a benzene ring.

organic molecules and biomolecules has been widely investigated and understood over the years through various spectroscopic techniques. We have recently studied the nonlinear optical effects of excitation of IR144 in methanol, at room temperature using chirped femtosecond pulses and have found that the characteristic relaxation times between excited and fluorescent states of this system ranges from 20-40 fs. Here we have used a tunable broadband source coupled with a phase and amplitude pulse shaper to investigate the effects of pulse duration and excitation wavelength on the rate of Dynamic Stokes Shift of laser dyes. We take advantage of the relation between chirp in frequency domain and the chirp in time domain to calculate the rate of Dynamic Stokes Shift. The effect of changing the excitation wavelength is even more interesting. Negatively detuned (from absorption maxima) pulses do not induce any change in rate of DSS, however, positively detuned pulses induce an exponential rise in the rate of DSS. These observations give additional experimental background knowledge for deeper understanding of the dynamics of the excited state of large organic molecules in solution.

Editorial on the Research Topic Modern Tools for Time-Resolved Luminescence Biosensing and Imaging The time evolution of luminescence decay, often described by the parameter of luminescence lifetime assuming first-order kinetics, is among the key characteristics of a luminescence substance. This makes time-resolved luminescence measurement a powerful tool for identifying substances of interest, especially in biological samples where differentiation based on luminescence colors may be inadequate or ineffective [1-3]. Moreover, for quantitative luminescence intensity measurements, the concentration in biological samples is hard to control, and typically needs a calibration, whereas the luminescence decay is largely independent of the luminophore concentration. The kinetics of luminescence decay is also influenced by the environment, enabling various sensing applications of time-resolved luminescence in biology and medicine [4, 5]. In this multidisciplinary Research Topic, a collection of six Original Research, three Brief Research Report, one Technology and Code, and three Review articles are presented, featuring some of the latest advances in time-resolved luminescence instruments, probes, data processing, interpretation, and applications for biosensing and imaging. Depending on the timescale of the luminescence decay, which varies from nanoseconds for typical fluorophores to seconds and longer for some phosphors, time-resolved luminescence measurement encompasses a diverse range of techniques, probes and devices suited for different temporal resolution. Hirvonen and Suhling provide a detailed overview of the fast timing underpinning fluorescence lifetime imaging (FLIM)-a technique which celebrated its 30th anniversary recently [6], and highlight a few recent examples showcasing applications in life sciences. Meanwhile, Deng et al. survey techniques for measuring long-lived emission, with particular focus on methods using mechanical choppers. Special attention is paid to automatic synchronization based on the same chopper acting as both the pulse generator and the detection shutter, with detailed discussion on their advantages, challenges, and future development prospects. Generally speaking, time-resolved measurement takes longer than steady-state measurement to perform in order to capture the time evolution at the cost of analytical throughput. Bitton et al. review the recent advancements in microscopy and flow cytometry for high-throughput lifetime measurements, and provide their outlook on future developments integrating machine learning. On the other hand, regardless of the temporal resolution, common principles and methods are applicable to time-resolved luminescence measurement, which can be performed either in the time domain or in the frequency domain [7]. On the former, Ziniuk et al. implement a rapid lifetime determination technique for lifetime imaging of Er-doped nanoparticles emitting shortwave infrared luminescence, which has the potential for in vivo bioimaging. Li et al. compare the outputs of several

Significance: The current gold standard for monitoring small intestinal transplant (IT) rejection is endoscopic visual assessment and biopsy of suspicious lesions; however, these lesions are only superficially visualized by endoscopy. Invasive biopsies provide a coarse sampling of tissue health without depicting the true presence and extent of any pathology. Optical coherence tomography (OCT) presents a potential alternative approach with significant advantages over traditional white-light endoscopy. Aim: The aim of our investigation was to evaluate OCT performance in distinguishing clinically relevant morphological features associated with IT graft failure. Approach: OCT was applied to evaluate the small bowel tissues of two rhesus macaques that had undergone IT of the ileum. The traditional assessment from routine histological observation was compared with OCT captured using a handheld surgical probe during the days post-transplant and subsequently was compared with histophaology. Results: The reported OCT system was capable of identifying major biological landmarks in healthy intestinal tissue. Following IT, one nonhuman primate (NHP) model suffered a severe graft ischemia, and the second NHP graft failed due to acute cellular rejection. OCT images show visual evidence of correspondence with histological signs of IT rejection. Conclusions: Results suggest that OCT imaging has significant potential to reveal morphological changes associated with IT rejection and to improve patient outcomes overall.

We introduce a novel system for geometrically accurate, continuous, live, volumetric middle ear optical coherence tomography imaging over a 10.9 × 30° × 30° field of view (FOV) from a handheld imaging probe. The system employs a discretized spiral scanning (DC-SC) pattern to rapidly collect volumetric data and applies real-time scan conversion and lateral angular distortion correction to reduce geometric inaccuracies to below the system's lateral resolution over 92% of the FOV. We validate the geometric accuracy of the resulting images through comparison with co-registered micro-computed tomography (micro-CT) volumes of a phantom target and a cadaveric middle ear. The system's real-time volumetric imaging capabilities are assessed by imaging the ear of a healthy subject while performing dynamic pressurization of the middle ear in a Valsalva maneuver.

Cephalotocin is a bioactivity-regulating peptide expressed in octopus (Octopus vulgaris). The peptide sequence of cephalotocin is very similar to the peptide sequence of mammalian vasopressin, and cephalotocin has been proposed to mainly activate arginine vasopressin 1b receptor (Avpr1b) in the brain. However, the effects of cephalotocin on mammalian behavior have not been studied. In the current study, cephalotocin significantly reduced both the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) recorded from not only cultured neuronal cells from postnatal Sprague-Dawley (SD) rats but also hippocampal slices from 4-week-old male C57BL/6 mice. Intraperitoneal (IP) injection did not affect the open field behaviors of C57BL/6 mice. Cephalotocin was directly infused into the hippocampus because the normalized Avpr1b staining intensity divided by the DAPI staining intensity indicated that Avpr1b expression tended to be high in the hippocampus. A hippocampal infusion of 1 mg/kg cephalotocin via an implanted cannula exerted an anti-stress effect, significantly reducing the immobility time in the tail suspension test (TST). The present results provide evidence that the effects of cephalotocin on the activity of hippocampal neurons are related to ameliorating stress, suggesting that cephalotocin may be developed as an anti-stress biomodulator that functions by affecting the brain.

The laser confocal scanning microscopy combined with time-correlated single photon counting imaging technique to obtain fluorescence intensity and fluorescence lifetime images for fluorescence resonance energy transfer measurement is reported. Both the fluorescence lifetime imaging microscopy (FLIM) and intensity images show inhomogeneous cyan fluorescent protein and yellow fluorescent protein (CFP /YFP) expression or inhomogeneous energy transfer between CFP and YFP over whole cell. The results presented in this work show that FLIM could be a potential method to reveal the structure-function behavior of NAD(P)H dehydrogenase complexes in living cell.

The FER-controlled pollen tube block To gain insight into how FER blocks supernumerary pollen tubes from entering the female gametophyte, we first examined when the mechanism is engaged. FER is broadly expressed, including in the sporophytic tissues of pistils 4,10 (Extended Data Fig. 1b). Pollen tubes normally exit the transmitting track one at a time (Fig. 1b, Extended Data Fig. 1a), and fewer than 5% of wild-type ovules are penetrated by multiple pollen tubes (Fig. 1c, Extended Data Fig. 1c). In fer pistils, pollen tubes

Recent interest in vascular targeting and anti-angiogenic drug treatments for cancer has stimulated fundamental research regarding the modes of action of these drugs as well as studies of the development and remodelling of the vascular network following treatment. Multiphoton fluorescence microscopy is employed for in vivo mapping of threedimensional blood vessel distribution in tumours grown in rodent dorsal skin-flap window chamber preparations. Accurate visualisation of the vasculature in three-dimensions allows us to perform dynamic experiments in thick biological specimens in vivo. Examples of in vivo imaging of tumour vasculature are given and compared to normal tissue vasculature. The dynamic responses of blood vessels to treatment with the vascular targeting drug combretastatin A4-P are presented and discussed. The implementation of time-domain imaging by reversed stop-start time-correlated single photon counting (RSS-TCSPC) is discussed as a method for feature extraction in the presence of exogenous and endogenous fluorophores. In particular, the segmentation of the vascular network is demonstrated. Additional contrast, indicative of probe environmental factors, may also be realised. We present examples of in vivo lifetime imaging as a method to elucidate the physiological processes of the tumour microenvironment.

Two-photon fluorescence and second harmonic generation microscopy have enabled functional and morphological in vivo imaging. However, in vivo applications of those techniques to living animals are limited by bulk optics on a bench top. Fortunately, growing functionality of fiber-optic devices and miniaturization of scanning mirrors stimulate the race to develop nonlinear optical endoscopy. In this paper, we report on a prototype of a nonlinear optical endoscope based on a double-clad photonic crystal fiber to improve the detection efficiency and a MEMS mirror to steer the light at the fiber tip. The miniaturized fiber-optic nonlinear microscope is characterized by rat esophagus imaging. Line profiles from the rat tail tendon and esophagus prove the potential of the technology in in vivo applications.

The protein product of the oncogene bcl-2 is a potent inhibitor of apoptotic cell death. The Bcl-2 protein has variously been reported to reside in the nuclear envelope and endoplasmic reticulum or exclusively in the inner membrane of mitochondria. We therefore undertook a detailed analysis of the intracellular distribution of Bcl-2 by immunofluorescence, immunogold electron microscopy, and subcellular fractionation in three mouse cell lines expressing a human bcl-2 transgene and measured its importation into isolated mitochondria. By these methods, the protein was localized to the nuclear envelope, the endoplasmic reticulum, and the outer mitochondrial membrane. Any proposal for the mechanism by which Bcl-2 inhibits apoptosis must therefore accommodate the fact that Bcl-2 localizes to cytoplasmic membranes facing the cytosol.

e207 vation in the neuronal cell due to femtosecond laser irradiation. The increase in fluorescence intensity was frequently observed at higher laser power and longer irradiation time of a femtosecond laser, indicating that a femtosecond laser had a potential to induce the processing of neurons due to multi-photon excitation. This method can be applied to optical stimulation of single neuron in a neuronal network without any chemical applications.

Synergids are metabolically dynamic cells of the egg apparatus and represent an important component of the female gametophyte. Besides directing the growth of the pollen tube towards the micropylar end of the embryo sac, these ephemeral structures make room for the pollen tube cytoplasm. The nature of chemotrophic substances that direct the growth of the pollen tube, the mechanism of degeneration of one of the synergids before fertilization and the molecular aspects of synergid morphogenesis have been studied in detail. Research carried out on model systems such as Arabidopsis, Brassica, Capsella, Triticum and Torenia has expanded our understanding of the molecular regulation of the pollen tube journey, its guidance and navigation in the pistil. Recently, the critical role of the central cell in fertilization and prevention of polytubey has also been thoroughly investigated. Interesting aspects that lead to degeneration of synergids, and the factors governing degeneration, including...

Confocal laser endomicroscopy is an emerging methodology to perform real time optical biopsy. Fluorescence images with histology-like quality can be collected instantaneously from the epithelium of hollow organs. Currently, scanning is performed at the proximal end of probe-based instruments used routinely in the clinic, and flexibility to control the focus is limited. We demonstrate use of a parametric resonance scanner packaged in the distal end of the endomicroscope to perform high speed lateral deflections. An aperture was etched in the center of the reflector to fold the optical path. This design reduced the dimensions of the instrument to 2.4 mm diameter and 10 mm length, allowing for forward passage through the working channel of a standard medical endoscope. A compact lens assembly provides lateral and axial resolution of 1.1 and 13.6 μm, respectively. A working distance of 0 μm and field-of-view of 250 μm × 250 μm was achieved at frame rates up to 20 Hz. Excitation at 488 n...

The tumor microenvironment is recognized as playing a significant role in the behavior of tumor cells and their progression to metastasis. However, tools to manipulate the tumor microenvironment directly, and image the consequences of this manipulation with single cell resolution in real time in vivo, are lacking. We describe here a method for the direct, local manipulation of microenvironmental parameters through the use of an implantable Induction Nano Intravital Device (iNANIVID) and simultaneous in vivo visualization of the results at single-cell resolution. As a proof of concept, we deliver both a sustained dose of EGF to tumor cells while intravital imaging their chemotactic response as well as locally induce hypoxia in defined microenvironments in solid tumors.

The tumor microenvironment is recognized as playing a significant role in the behavior of tumor cells and their progression to metastasis. However, tools to manipulate the tumor microenvironment directly, and image the consequences of this manipulation with single cell resolution in real time in vivo, are lacking. We describe here a method for the direct, local manipulation of microenvironmental parameters through the use of an implantable Induction Nano Intravital Device (iNANIVID) and simultaneous in vivo visualization of the results at single-cell resolution. As a proof of concept, we deliver both a sustained dose of EGF to tumor cells while intravital imaging their chemotactic response as well as locally induce hypoxia in defined microenvironments in solid tumors.

Thin film optical elements exhibiting translational invariance, and thus robustness to optical misalignment, are crucial for rapid development of compact and integrated optical devices. In this letter, we experimentally demonstrate a beam-shaping element that generates an annular beam by spatially filtering the fundamental Gaussian mode of a laser beam. The element comprises of a one-dimensional photonic crystal cavity fabricated using sputtered thin films. The planar architecture and in-plane symmetry of the element render our beam-shaping technique translationally invariant. The generated annular beam is sensitive to the polarization direction and the wavelength of the incident laser beam. Using this property of the annular beam, we show simultaneous generation of concentric annular beams of different wavelengths. Our experimental observations show an excellent agreement with simulation results performed using finite-difference time-domain method. Such a beam-shaping element has applications in areas ranging from microscopy and medicine to semiconductor lithography and manufacturing in microelectronics industry.

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Fluorescent proteins (FPs) have revolutionized cell biology by allowing genetic tagging of specific proteins inside living cells. In conjunction with Fö rster's resonance energy transfer (FRET) measurements, FP-tagged proteins can be used to study protein-protein interactions and estimate distances between tagged proteins. FRET is mediated by weak Coulombic dipole-dipole coupling of donor and acceptor fluorophores that behave independently, with energy hopping discretely and incoherently between fluorophores. Stronger dipole-dipole coupling can mediate excitonic coupling in which excitation energy is distributed near instantaneously between coherently interacting excited states that behave as a single quantum entity. The interpretation of FP energy transfer measurements to estimate separation often assumes that donors and acceptors are very weakly coupled and therefore use a FRET mechanism. This assumption is considered reasonable as close fluorophore proximity, typically associated with strong excitonic coupling, is limited by the FP b-barrel structure. Furthermore, physiological temperatures promote rapid vibrational dephasing associated with a rapid decoherence of fluorophore-excited states. Recently, FP dephasing times that are 50 times slower than traditional organic fluorophores have been measured, raising the possibility that evolution has shaped FPs to allow stronger than expected coupling under physiological conditions. In this study, we test if excitonic coupling between FPs is possible at physiological temperatures. FRET and excitonic coupling can be distinguished by monitoring spectral changes associated with fluorophore dimerization. The weak coupling mediating FRET should not cause a change in fluorophore absorption, whereas strong excitonic coupling causes Davydov splitting. Circular dichroism spectroscopy revealed Davydov splitting when the yellow FP Venus A206 dimerizes, and a novel approach combining photon antibunching and fluorescence correlation spectroscopy was used to confirm that the two fluorophores in a Venus A206 homodimer behave as a single-photon emitter. We conclude that excitonic coupling between Venus A206 fluorophores is possible at physiological temperatures.

The unsaturated cyclic [M 3 (dppm) 3 (CO)] 2+ clusters (M) Pt, Pd; dppm) Ph 2 PCH 2 PPh 2 ; such as PF 6salt) exhibit a cavity formed by the six dppm-phenyl groups placed like a picket fence above the unsaturated triangular M 3 dicationic center. Electrostatic interactions of the M 3+ units inside this cavity with the carboxylate anion RCO 2-[R) tetraphenylporphyrinatozinc(II), ZnTPP; p-phenyltritolylporphyrinatozinc(II), ZnTTPP; p-phenyltritolylporphyrinatopalladium(II), PdTTPP] form dyads for through-space triplet energy transfers. The binding constants are on the order of 20 000 M-1 in all six cases (298 K). The energy diagram built upon absorption and emission spectra at 298 and 77 K places the [Pt 3 (dppm) 3 (CO)] 2+ and [Pd 3 (dppm) 3 (CO)] 2+ as triplet energy donors, respectively, with respect to the ZnTPPCO 2-, ZnTTPPCO 2-, and PdTTPPCO 2pigments, which act as acceptors. Evidence for energy transfer is provided by the transient absorption spectra at 298 K, where triplet-triplet absorption bands of the metalloporphyrin chromophores are depicted at all time (at 298 K) with total absence of the charge-separated state in the nanosecond to microsecond time scale. Rates for energy transfer (ranging in the 10 4 s-1 time scale) are extracted from the emission lifetimes of the [Pt 3 (dppm) 3 (CO)] 2+ donor in the free chromophore and the host-guest assemblies. The emission intensity of [Pd 3 (dppm) 3 (CO)] 2+ is too weak to measure its spectrum and emission lifetime in the presence of the strongly luminescent metalloporphyrin-containing materials. For the [Pd 3-(dppm) 3 (CO)] 2+ • • • metalloporphyrin dyads, evidence for fluorescence and phosphorescence lifetime quenching of the porphyrin chromophore at 298 K is provided. These quenchings, exhibiting rates of 10 4 (triplet) and 10 8 s-1 (singlet), are attributed to a photoinduced electron transfer from the metalloporphyrin to the cluster due to the low reduction potential.

Imaging plasticity in the honeybee antennal lobe The olfactory system of the honeybee, Apis mellifera, is a well-established model for studying the anatomical bases of odor coding and olfactory memory. The bee primary olfactory centers, the antennal lobes (ALs), are organized in a fixed arrangement of spheroidal neuropil units, the glomeruli. These structures receive inputs from the bee olfactory receptor neurons (ORNs), are modulated by local interneurons (LINs), and send outputs to higher brain centers through the projection neurons (PNs). The spatio-temporal pattern of activation of the glomeruli is odor-specific, thus, imaging of intracellular calcium of the PNs may provide maps of odor representation. Such maps have been demonstrated to vary upon olfactory conditioning (i.e. through enhancement of the discrimination of a rewarded odor against an unrewarded one), but the underlying mechanisms for this plasticity are not known. The aim of this study is to investigate plastic changes following odor conditioning and, in particular, possible variations in number and morphology of the synapses involved in the ALs circuitry.

Imaging plasticity in the honeybee antennal lobe The olfactory system of the honeybee, Apis mellifera, is a well-established model for studying the anatomical bases of odor coding and olfactory memory. The bee primary olfactory centers, the antennal lobes (ALs), are organized in a fixed arrangement of spheroidal neuropil units, the glomeruli. These structures receive inputs from the bee olfactory receptor neurons (ORNs), are modulated by local interneurons (LINs), and send outputs to higher brain centers through the projection neurons (PNs). The spatio-temporal pattern of activation of the glomeruli is odor-specific, thus, imaging of intracellular calcium of the PNs may provide maps of odor representation. Such maps have been demonstrated to vary upon olfactory conditioning (i.e. through enhancement of the discrimination of a rewarded odor against an unrewarded one), but the underlying mechanisms for this plasticity are not known. The aim of this study is to investigate plastic changes following odor conditioning and, in particular, possible variations in number and morphology of the synapses involved in the ALs circuitry.

A paracrine interaction between epidermal growth factor (EGF)-secreting tumor-associated macrophages (TAMs) and colony-stimulating factor 1 (CSF-1)-secreting breast carcinoma cells promotes invasion and metastasis. Here, we show that mice deficient in the hematopoietic-cell-specific Wiskott-Aldrich syndrome protein (WASp) are unable to support TAM-dependent carcinoma cell invasion and metastasis in both orthotopic and transgenic models of mammary tumorigenesis. Motility and invasion defects of tumor cells were recapitulated ex vivo upon coculture with WASp À/À macrophages. Mechanistically, WASp is required for macrophages to migrate toward CSF-1-producing carcinoma cells, as well as for the release of EGF through metalloprotease-dependent shedding of EGF from the cell surface of macrophages. Our findings suggest that WASp acts to support both the migration of TAMs and the production of EGF, which in concert promote breast tumor metastasis.

The high rate of alcohol use among emergency department (ED) patients makes the ED setting an obvious target for increased screening and interventions. However, interventions to change alcohol behavior may be applied inappropriately if a patient's motivation to change is not factored in. Purpose of the Study: To identify correlates of readiness to change problem drinking among a sample of ED patients. Method: This study analyzes secondary data obtained from an earlier ED study conducted at the King/Drew Medical Center between August 2001 and December 2001 (9 am to 6 pm, 5 days a week). This study used a sample of 295 ED patients who scored positive for an alcohol problem based on CAGE (score $ 1). Results: 68% African American; 32% Latino; 80% male; 46% less than high school diploma; 85% not married; 72% no health insurance; 85% no primary care health provider. Whereas 12% of the patients were ''not ready'' to change their drinking behaviors, 47% and 41% were ''unsure'' and ''ready,'' respectively. Using multiple regression analysis revealed that among a list of four potential predictors (illicit drug use, tobacco use, exposure to violence, and having a primary care doctor),and six controlling variables (gender, ethnicity, education, marital status, insurance, ability to afford medication), reporting use of illicit drug(s), reporting exposure to violence, being female, and being married contributed significantly to the likelihood of changing one's level of readiness to change alcohol behaviors (p # .05). Conclusion: Recognizing ED patients' level of motivation to change problem drinking and factors that move one toward a higher level of readiness can help researchers improve the receptiveness and effectiveness of alcohol behavior interventions.

The Xenopus tadpole has been used for decades to study neural development. Every stage of neural development, from neural induction and neurogenesis to axon guidance and circuit refinement has been described in this particular model. This review discusses the unique experimental attributes of the Xenopus tadpole that make it a powerful model for studying how neural circuits form and function, and highlights the especially innovative ways this model is currently being used to identify and characterize the pathophysiology associated with neurological disorders. Focus is on three key stages of neural development that collectively are known loci for many major neurological disorders: (1) neurogenesis, (2) differentiation, and (3) circuit wiring. Keywords Neural development Á Xenopus tadpole Á Vertebrate Á Experimental model Á Neurodevelopmental disorders Á Review This article is part of the Topical Collection on Xenopus as a Model for Pathobiology.

Background and purpose: To quantify the late dose-related responses of the rat cervical spinal cord to Xray irradiations by an array of microbeams or by a single millimeter beam. Materials and methods: Necks of anesthetized rats were irradiated transversely by an 11 mm wide array of 52 parallel, 35 lm wide, vertical X-ray microbeams, separated by 210 lm intervals between centers. Comparison was made with rats irradiated with a 1.35 mm wide single beam of similar X-rays. Rats were killed when paresis developed, or up to 383 days post irradiation (dpi). Results: Microbeam peak/valley doses of %357/12.7 Gy to 715/25.4 Gy to an 11 mm long segment of the spinal cord, or single beam doses of %146-454 Gy to a 1.35 mm long segment caused foreleg paresis and histopathologically verified spinal cord damage; rats exposed to peak/valley doses up to 253/9 Gy were paresis-free at 383 dpi. Conclusions: Whereas microbeam radiation therapy [MRT] for malignant gliomas implanted in rat brains can be safe, palliative or curative, the high tolerance of normal rat spinal cords to similar microbeam exposures justifies testing MRT for autochthonous malignancies in the central nervous system of larger animals with a view to subsequent clinical applications.

Measurements of neuronal activity across brain areas are important for understanding the neural correlates of cognitive and motor processes such as attention, decision-making and action selection. However, techniques that allow cellular resolution measurements are expensive and require a high degree of technical expertise, which limits their broad use. Wide-field imaging of genetically encoded indicators is a high-throughput, cost-effective and flexible approach to measure activity of specific cell populations with high temporal resolution and a cortex-wide field of view. Here we outline our protocol for assembling a wide-field macroscope setup, performing surgery to prepare the intact skull and imaging neural activity chronically in behaving, transgenic mice. Further, we highlight a processing pipeline that leverages novel, cloud-based methods to analyze large-scale imaging datasets. The protocol targets laboratories that are seeking to build macroscopes, optimize surgical procedures for long-term chronic imaging and/or analyze cortex-wide neuronal recordings. The entire protocol, including steps for assembly and calibration of the macroscope, surgical preparation, imaging and data analysis, requires a total of 8 h. It is designed to be accessible to laboratories with limited expertise in imaging methods or interest in high-throughput imaging during behavior.

Novel multifocal flat metasurface (MS) lenses are developed using two techniques: (i) polarization diversity, and (ii) annular segmentation of the lens aperture. The polarization-diversity technique enables overall lens aperture reuse, thus doubling the number of foci through the simultaneous focusing of two orthogonal linearly polarized incident beams at two distinct foci using the lens aperture. The annular-segmentation technique, on the other hand, is independent of incident beam polarization and is only based on dividing the lens aperture into concentric annular segments that converge different portions of the illuminating beam at different foci. The total number of foci can be further increased by combining the polarization-diversity and the annular-segmentation techniques. Subsequently, the concept of multifocality is further extended to design a novel flat lens with an overall single needle-like focal region with elongated depth of focus (DOF) without loss of lateral resolution. To this goal, we design a multifocal lens with overlapping profiles of foci superposed into a single elongated needle-shaped focal region. Using the combination of polarization-diversity and annular-segmentation techniques, we develop a novel MS flat lens made of Y-shaped nanoantennas, whose polarization-dependent reflection phase and amplitude can be controlled independently via their geometrical parameters. Via numerical calculations, we demonstrate that the proposed MS lens has an overall single focal region with an extremely long DOF of about 74.1 λ, a lateral full width at half maximum varying in the range of 1.37 λ to 2.8 λ, and a numerical aperture of about 0.26 (considering the center of the focal region as the effective focal point). Here, the MS lens's capability to synthesize extremely long DOF is conceptually demonstrated without resorting to time-consuming and complicated wavefront synthesis methods. The engineering of focal intensity profiles with flat MSs may introduce significant advancement in nanoimaging, many areas of microscopy, and ophthalmic applications.

Genetically encoded biosensors function by linking structural change in a protein construct, typically tagged with one or more fluorescent proteins, to changes in a biological parameter of interest (such as calcium concentration, pH, phosphorylation-state, etc.). Typically, the structural change triggered by alterations in the bio-parameter is monitored as a change in either fluorescent intensity, or lifetime. Potentially, other photo-physical properties of fluorophores, such as fluorescence anisotropy, molecular brightness, concentration, and lateral and/or rotational diffusion could also be used. Furthermore, while it is likely that multiple photo-physical attributes of a biosensor might be altered as a function of the bio-parameter, standard measurements monitor only a single photo-physical trait. This limits how biosensors are designed, as well as the accuracy and interpretation of biosensor measurements. Here we describe the design and construction of an automated multimodal-mi...

Collectively known as congenital heart defects (CHDs), cardiac abnormalities at birth are the most common forms of neonatal defects. Being principally responsible for the heart‘s pumping power, ventricles are particularly affected by developmental abnormalities, such as flow disturbances or genomic defects. Hypoplastic Right Heart Syndrome (HRHS) is a rare disease where the right ventricle is underdeveloped. In this study, we introduce a surgical procedure performed on chick embryo, termed right atrial ligation (RAL) for disturbing hemodynamics within the right heart aiming in order to generate an animal model of HRHS. RAL is a new surgical manipulation, similar to the well-studied left atrial ligation (LAL) surgery but it induces the hemodynamic change into the right side of the heart. After inducing RAL, We utilized techniques such as Doppler ultrasound, x-ray micro-CT, histology, and computational fluid dynamics (CFD) analysis, for a comprehensive functional and structural analys...

BBB prevents effective chemotherapy due to low BBB permeability of chemotherapeutic agents 8,9. Furthermore, many experimental therapeutic modalities that work on GBM cells in vitro cannot penetrate the BBB, and thus have little effect in vivo 10. Effective treatment of brain tumors requires the high frequency administration of chemotherapeutic agents. For example, current standard glioma chemotherapeutic protocol requires daily application of temozolomide (TMZ) for 5 consecutive days every 28 days. Three different strategies have been developed to overcome the challenge posed by BBB in drug delivery: intra-arterial infusion of hyperosmolar solutions or vasoactive drugs, direct injection of drugs into target area thus bypassing the BBB, or encapsulating drugs in liposomes or nanoparticles to be delivered across the BBB 11. The first approach induces a transient rise in intracranial pressure, causes nonselective opening of the BBB in the vascular territory, and exposes large volumes of brain tissue to potentially toxic substances 12. The second approach requires surgical device implantation and is limited by parenchymal drug diffusion. The efficacy of the third approach is limited by nanoparticle toxicity and bio-distribution 13,14. In recent years, focused ultrasound (FUS) opening of the BBB has emerged as a promising new technique for enhancing drug delivery into the brain 15. By injecting microbubbles (MB) intravenously and simultaneously applying burst-type ultrasound to targeted brain tissue, we can induce site-specific transient BBB opening and enhance CNS drug delivery without causing significant adverse effects 16. Glioma animal experiments have shown that adding FUS-induced BBB opening to TMZ chemotherapy increases drug tumor tissue concentration and improves patient survival 17,18. Focused ultrasound BBB opening is currently being investigated in clinical trials as an adjunct to brain tumor treatment. Despite the great potential of FUS-BBB opening, several recent studies have highlighted possible safety issues associated with such treatment. Other than BBB opening, some reports have demonstrated that FUS + MB induces transient tissue edema, neuronal function suppression 19 , astroglial scarring 20 , transient ischemia, intracerebral hemorrhage 21 , and sterile inflammation 22,23. Furthermore, all of these studies investigated FUS applied either once only or repeated on a weekly/bi-weekly basis 24. However current standard glioma chemotherapeutic protocol requires daily application of TMZ for 5 consecutive days 25. Based on the fact that BBB opening induced by FUS lasts for only 2-3 hours 26 , and that temozolomide has a mean elimination half-life of approximately 1.9 hours 27 , FUS-BBB opening procedure should be given as frequently as possible during the five-day dosing period to maximize its effect. Repeated FUS-BBB opening has been shown to be safe in various animal models 20,24,28,29. However some recent reports indicate that application of FUS capable of disrupting the BBB may cause cellular apoptosis or sterile inflammation in the target tissue 22,23 , raising concerns about the safety of such a treatment modality. Furthermore, these studies only performed single exposures, or were repeated weekly or biweekly to allow for tissue recovery or remodeling. Previous glioma animal experiments have shown that FUS-BBB opening every other day as an adjunct to TMZ chemotherapy increased drug concentrations in the target area, improved tumor growth inhibition, and increased overall survival 17,18. The safety of such frequent and repeated BBB-openings is still unclear. This study seeks to examine whether such frequent FUS-BBB opening induces behavioral or physiological changes or causes aggravated brain damage, and to validate the safety of such procedures. Materials and Methods FUS instrumentation and calibration. The FUS instruments were described previously 19. Briefly, the device is comprised of a function generator (Agilent, USA), a radiofrequency power amplifier (Amplifier Research, USA) and a focused ultrasound transducer (IMASONIC, France). A FUS frequency of 400 kHz was used. The acoustic pressure field was measured in a free field within an acrylic tank filled with deionized/degassed water by moving a calibrated needle hydrophone (HNA-0400, ONDA,USA) in steps of 0.22 mm, and the field of view in the axial and cross sections were respectively 10 × 90 mm and 10 × 10 mm (Fig. 1A). The diameter and length of the half-maximum pressure amplitude of the ultrasound field were respectively 2 and 15 mm. The measured acoustic pressure of 0, 0.2-0.3, 0.5 and 0.9 MPa corresponded to the mechanical index (MI) to 0.33-0.47, 0.8, and 1.4. The transcranial pressure measurement and distribution is shown in the Supplementary Fig. S1.

Highlights d Cortex-wide task-epoch-specific causal neural activity across sensorimotor learning d Rapid inhibitory response of orofacial cortex contributes to delay licking d Secondary whisker motor cortex is a key node converting whisker input to lick plan d Sensory information converges to a focal frontal area critical for delayed response

Phenol-soluble modulin a3 (PSMa3) is a cytotoxic peptide secreted by Staphylococcus aureus. Increased expression of PSMa3 is associated with the enhanced toxicity of community-associated Methicillin-resistant S. aureus (CA-MRSA), a highly virulent strain of MRSA. Though previous reports suggest membrane disruption, the mechanism of PSMa3mediated cell death is not fully understood. Here, we used a stereochemical strategy to examine the mechanism of cytotoxicity. We found that racemic PSMa3 (L and D-PSMa3 mixed at 1:1 ratio) can form non-toxic and stable fibrils, while chiral PSMa3 remains toxic under the same conditions. We solved the crystal structure of racemic PSMa3 at 1.4Å , which shows a cross-a, homo-chiral packing pattern along the crystal growth axis. We conclude that PSMa3 exerts toxicity primarily through interactions with the lipid bilayer of a cell, and that fibril formation is not necessary for cytotoxicity.

Fluorescence microscopy methods have seen an increase in popularity in recent years for detecting protein crystals in screening trays. The fluorescence-based crystal detection methods have thus far relied on intrinsic UV-inducible tryptophan fluorescence, nonlinear optics or fluorescence in the visible light range dependent on crystals soaked with fluorescent dyes. In this paper data are presented on a novel visible-light-inducible autofluorescence arising from protein crystals as a result of general stabilization of conjugated double-bond systems and increased charge delocalization due to crystal packing. The visiblelight-inducible autofluorescence serves as a complementary method to brightfield microscopy in beamline applications where accurate crystal centering about the rotation axis is essential. Owing to temperature-dependent chromophore stabilization, protein crystals exhibit tenfold higher fluorescence intensity at cryogenic temperatures, making the method ideal for experiments where crystals are cooled to 100 K with a cryostream. In addition to the nondamaging excitation wavelength and low laser power required for imaging, the method can also serve a useful role for differentiating protein crystals from salt crystals in screening trays.