Temporally optimized and spectrally shaped hyperspectral coherent anti-Stokes Raman scattering microscopy.

Coherent anti-Stokes Raman scattering (CARS) microscopy offers label-free chemical contrasts based on molecular vibrations. Hyperspectral CARS (HS-CARS) microscopy enables comprehensive microscale chemical characterization of biological samples. Various HS-CARS methods have been developed with individual advantages and disadvantages. We present what we believe to be a new temporally optimized and spectrally shaped (TOSS) HS-CARS method to overcome the limitations of existing techniques by providing precise control of the spatial and temporal profiles of the excitation beams for efficient and accurate measurements. This method uniquely uses Fourier transform pulse shaping based on a two-dimensional spatial light modulator to control the phase and amplitude of the excitation beams. TOSS-HS-CARS achieves fast, stable, and flexible acquisition, minimizes photodamage, and is highly adaptable to a multimodal multiphoton imaging system.

Programmable hyperspectral coherent anti-Stokes Raman scattering microscopy.

Hyperspectral coherent Raman scattering microscopy provides a significant improvement in acquisition time compared to spontaneous Raman scattering yet still suffers from the time required to sweep through individual wavenumbers. To address this, we present the use of a pulse shaper with a 2D spatial light modulator for phase- and amplitude-based shaping of the Stokes beam to create programmable spectrally tailored excitation envelopes. This enables collection of useful spectral information in a more rapid and efficient manner.

Depixelation and enhancement of fiber bundle images by bundle rotation.

Fiber bundles have become widely adopted for use in endoscopy, live-organism imaging, and other imaging applications. An inherent consequence of imaging with these bundles is the introduction of a honeycomb-like artifact that arises from the inter-fiber spacing, which obscures features of objects in the image. This artifact subsequently limits applicability and can make interpretation of the image-based data difficult. This work presents a method to reduce this artifact by on-axis rotation of the fiber bundle. Fiber bundle images were first low-pass and median filtered to improve image quality. Consecutive filtered images with rotated samples were then co-registered and averaged to generate a final, reconstructed image. The results demonstrate removal of the artifacts, in addition to increased signal contrast and signal-to-noise ratio. This approach combines digital filtering and spatial resampling to reconstruct higher-quality images, enhancing the utility of images acquired using fiber bundles.

Large field-of-view metabolic profiling of murine brain tissue following morphine incubation using label-free multiphoton microscopy.

BACKGROUND: Although the effects on neural activation and glucose consumption caused by opiates such as morphine are known, the metabolic machinery underlying opioid use and misuse is not fully explored. Multiphoton microscopy (MPM) techniques have been developed for optical imaging at high spatial resolution. Despite the increased use of MPM for neural imaging, the use of intrinsic optical contrast has seen minimal use in neuroscience. NEW METHOD: We present a label-free, multimodal microscopy technique for metabolic profiling of murine brain tissue following incubation with morphine sulfate (MSO4). We evaluate two- and three-photon excited autofluorescence, and second and third harmonic generation to determine meaningful intrinsic contrast mechanisms in brain tissue using simultaneous label-free, autofluorescence multi-harmonic (SLAM) microscopy. RESULTS: Regional differences quantified in the cortex, caudate, and thalamus of the brain demonstrate region-specific changes to metabolic profiles measured from FAD intensity, along with brain-wide quantification. While the overall intensity of FAD signal significantly decreased after morphine incubation, this metabolic molecule accumulated near the nucleus accumbens. COMPARISON WITH EXISTING METHODS: Histopathology requires tissue fixation and staining to determine cell type and morphology, lacking information about cellular metabolism. Tools such as fMRI or PET imaging have been widely used, but lack cellular resolution. SLAM microscopy obviates the need for tissue preparation, permitting immediate use and imaging of tissue with subcellular resolution in its native environment. CONCLUSIONS: This study demonstrates the utility of SLAM microscopy for label-free investigations of neural metabolism, especially the intensity changes in FAD autofluorescence and structural morphology from third-harmonic generation.

Simultaneous label-free autofluorescence multi-harmonic microscopy driven by the supercontinuum generated from a bulk nonlinear crystal.

Nonlinear microscopy encompasses several imaging techniques that leverage laser technology to probe intrinsic molecules of biological specimens. These native molecules produce optical fingerprints that allow nonlinear microscopes to reveal the chemical composition and structure of cells and tissues in a label-free and non-destructive fashion, information that enables a plethora of applications, e.g., real-time digital histopathology or image-guided surgery. Because state-of-the-art lasers exhibit either a limited bandwidth or reduced wavelength tunability, nonlinear microscopes lack the spectral support to probe different biomolecules simultaneously, thus losing analytical potential. Therefore, a conventional nonlinear microscope requires multiple or tunable lasers to individually excite endogenous molecules, increasing both the cost and complexity of the system. A solution to this problem is supercontinuum generation, a nonlinear optical phenomenon that supplies broadband femtosecond radiation, granting a wide spectrum for concurrent molecular excitation. This study introduces a source for nonlinear multiphoton microscopy based on the supercontinuum generation from a yttrium aluminum garnet (YAG) crystal, an approach that allows simultaneous label-free autofluorescence multi-harmonic imaging of biological samples and offers a practical and compact alternative for the clinical translation of nonlinear microscopy. While this supercontinuum covered the visible spectrum (550-900 nm) and the near-infrared region (950-1200 nm), the pulses within 1030-1150 nm produced label-free volumetric chemical images of ex vivo chinchilla kidney, thus validating the supercontinuum from bulk crystals as a powerful source for multimodal nonlinear microscopy.

Supercontinuum intrinsic fluorescence imaging heralds free view of living systems.

Optimal imaging strategies remain underdeveloped to maximize information for fluorescence microscopy while minimizing the harm to fragile living systems. Taking hint from the supercontinuum generation in ultrafast laser physics, we generated supercontinuum fluorescence from untreated unlabeled live samples before nonlinear photodamage onset. Our imaging achieved high-content cell phenotyping and tissue histology, identified bovine embryo polarization, quantified aging-related stress across cell types and species, demystified embryogenesis before and after implantation, sensed drug cytotoxicity in real-time, scanned brain area for targeted patching, optimized machine learning to track small moving organisms, induced two-photon phototropism of leaf chloroplasts under two-photon photosynthesis, unraveled microscopic origin of autumn colors, and interrogated intestinal microbiome. The results enable a facility-type microscope to freely explore vital molecular biology across life sciences.

Inspiring a convergent engineering approach to measure and model the tissue microenvironment.

Understanding the molecular and physical complexity of the tissue microenvironment (TiME) in the context of its spatiotemporal organization has remained an enduring challenge. Recent advances in engineering and data science are now promising the ability to study the structure, functions, and dynamics of the TiME in unprecedented detail; however, many advances still occur in silos that rarely integrate information to study the TiME in its full detail. This review provides an integrative overview of the engineering principles underlying chemical, optical, electrical, mechanical, and computational science to probe, sense, model, and fabricate the TiME. In individual sections, we first summarize the underlying principles, capabilities, and scope of emerging technologies, the breakthrough discoveries enabled by each technology and recent, promising innovations. We provide perspectives on the potential of these advances in answering critical questions about the TiME and its role in various disease and developmental processes. Finally, we present an integrative view that appreciates the major scientific and educational aspects in the study of the TiME.

Phase-sensitive detection of anomalous diffusion dynamics in the neuronal membrane induced by ion channel gating.

Non-ergodicity of neuronal dynamics from rapid ion channel gating through the membrane induces membrane displacement statistics that deviate from Brownian motion. The membrane dynamics from ion channel gating were imaged by phase-sensitive optical coherence microscopy. The distribution of optical displacements of the neuronal membrane showed a Lévy-like distribution and the memory effect of the membrane dynamics by the ionic gating was estimated. The alternation of the correlation time was observed when neurons were exposed to channel-blocking molecules. Non-invasive optophysiology by detecting the anomalous diffusion characteristics of dynamic images is demonstrated.

Ultra-parallel label-free optophysiology of neural activity.

The electrical activity of neurons has a spatiotemporal footprint that spans three orders of magnitude. Traditional electrophysiology lacks the spatial throughput to image the activity of an entire neural network; besides, labeled optical imaging using voltage-sensitive dyes and tracking Ca2+ ion dynamics lack the versatility and speed to capture fast-spiking activity, respectively. We present a label-free optical imaging technique to image the changes to the optical path length and the local birefringence caused by neural activity, at 4,000 Hz, across a 200 × 200 µm2 region, and with micron-scale spatial resolution and 300-pm displacement sensitivity using Superfast Polarization-sensitive Off-axis Full-field Optical Coherence Microscopy (SPoOF OCM). The undulations in the optical responses from mammalian neuronal activity were matched with field-potential electrophysiology measurements and validated with channel blockers. By directly tracking the widefield neural activity at millisecond timescales and micrometer resolution, SPoOF OCM provides a framework to progress from low-throughput electrophysiology to high-throughput ultra-parallel label-free optophysiology.

Tracking the binding of multi-functional fluorescent tags for Alzheimer's disease using quantitative multiphoton microscopy.

A recent theranostic approach to address Alzheimer's disease (AD) utilizes multifunctional targets that both tag and negate the toxicity of AD biomarkers. These compounds, which emit fluorescence with both an activation and a spectral shift in the presence of Aß, were previously characterized with traditional fluorescence imaging for binary characterization. However, these multifunctional compounds have broad and dynamic emission spectra that are dependent on factors such as the local environment, presence of Aß deposits, etc. Since quantitative multiphoton microscopy is sensitive to the binding dynamics of molecules, we characterized the performance of two such compounds, LS-4 and ZY-12-OMe, using Simultaneous Label-free Autofluorescence Multi-harmonic (SLAM) microscopy and Fast Optical Coherence, Autofluorescence Lifetime imaging and Second harmonic generation (FOCALS) microscopy. This study shows that the combination of quantitative multiphoton imaging with multifunctional tags for AD offers new insights into the interaction of these tags with AD biomarkers and the theranostic mechanisms.

Label-free metabolic and structural profiling of dynamic biological samples using multimodal optical microscopy with sensorless adaptive optics.

Label-free optical microscopy has matured as a noninvasive tool for biological imaging; yet, it is criticized for its lack of specificity, slow acquisition and processing times, and weak and noisy optical signals that lead to inaccuracies in quantification. We introduce FOCALS (Fast Optical Coherence, Autofluorescence Lifetime imaging, and Second harmonic generation) microscopy capable of generating NAD(P)H fluorescence lifetime, second harmonic generation (SHG), and polarization-sensitive optical coherence microscopy (OCM) images simultaneously. Multimodal imaging generates quantitative metabolic and morphological profiles of biological samples in vitro, ex vivo, and in vivo. Fast analog detection of fluorescence lifetime and real-time processing on a graphical processing unit enables longitudinal imaging of biological dynamics. We detail the effect of optical aberrations on the accuracy of FLIM beyond the context of undistorting image features. To compensate for the sample-induced aberrations, we implemented a closed-loop single-shot sensorless adaptive optics solution, which uses computational adaptive optics of OCM for wavefront estimation within 2 s and improves the quality of quantitative fluorescence imaging in thick tissues. Multimodal imaging with complementary contrasts improves the specificity and enables multidimensional quantification of the optical signatures in vitro, ex vivo, and in vivo, fast acquisition and real-time processing improve imaging speed by 4-40 × while maintaining enough signal for quantitative nonlinear microscopy, and adaptive optics improves the overall versatility, which enable FOCALS microscopy to overcome the limits of traditional label-free imaging techniques.

High-speed label-free two-photon fluorescence microscopy of metabolic transients during neuronal activity.

The brain is an especially active metabolic system, requiring a large supply of energy following neuronal activation. However, direct observation of cellular metabolic dynamics associated with neuronal activation is challenging with currently available imaging tools. In this study, an optical imaging approach combining imaging of calcium transients and the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) is utilized to track the metabolic dynamics in hippocampal neuron cultures. Results show distinct cellular components for the NAD(P)H response following neuronal activity, where notable differences in the NAD(P)H dynamics between neurons and astrocytes can be directly observed. Additionally, tracking of these responses across a large field of view is demonstrated for metabolic profiling of neuronal activation. Observation of neuronal dynamics using these methods allows for closer examination of the complex metabolic machinery of the brain, and may lead to a better understanding of the cellular metabolism of neuronal activation.

Dynamic Tracking Algorithm for Time-Varying Neuronal Network Connectivity using Wide-Field Optical Image Video Sequences.

Propagation of signals between neurons and brain regions provides information about the functional properties of neural networks, and thus information transfer. Advances in optical imaging and statistical analyses of acquired optical signals have yielded various metrics for inferring neural connectivity, and hence for mapping signal intercorrelation. However, a single coefficient is traditionally derived to classify the connection strength between two cells, ignoring the fact that neural systems are inherently time-variant systems. To overcome these limitations, we utilized a time-varying Pearson's correlation coefficient, spike-sorting, wavelet transform, and wavelet coherence of calcium transients from DIV 12-15 hippocampal neurons from GCaMP6s mice after applying various concentrations of glutamate. Results provide a comprehensive overview of resulting firing patterns, network connectivity, signal directionality, and network properties. Together, these metrics provide a more comprehensive and robust method of analyzing transient neural signals, and enable future investigations for tracking the effects of different stimuli on network properties.

Simultaneous two-photon activation and imaging of neural activity based on spectral-temporal modulation of supercontinuum light.

SIGNIFICANCE: Recent advances in nonlinear optics in neuroscience have focused on using two ultrafast lasers for activity imaging and optogenetic stimulation. Broadband femtosecond light sources can obviate the need for multiple lasers by spectral separation for chromatically targeted excitation. AIM: We present a photonic crystal fiber (PCF)-based supercontinuum source for spectrally resolved two-photon (2P) imaging and excitation of GCaMP6s and C1V1-mCherry, respectively. APPROACH: A PCF is pumped using a 20-MHz repetition rate femtosecond laser to generate a supercontinuum of light, which is spectrally separated, compressed, and recombined to image GCaMP6s (930 nm excitation) and stimulate the optogenetic protein, C1V1-mCherry (1060 nm excitation). Galvanometric spiral scanning is employed on a single-cell level for multiphoton excitation and high-speed resonant scanning is employed for imaging of calcium activity. RESULTS: Continuous wave lasers were used to verify functionality of optogenetic activation followed by directed 2P excitation. Results from these experiments demonstrate the utility of a supercontinuum light source for simultaneous, single-cell excitation and calcium imaging. CONCLUSIONS: A PCF-based supercontinuum light source was employed for simultaneous imaging and excitation of calcium dynamics in brain tissue. Pumped PCFs can serve as powerful light sources for imaging and activation of neural activity, and overcome the limited spectra and space associated with multilaser approaches.

Oclusiones de las arterias de los miembros superiores / Occlusion of upper limb arteries

Se realiza una revisión de las entidades que producen oclusiones de las arterias de los miembros superiores, se pone especial énfasis en las ocasionadas por estructuras anatómicas, conocidas como síndrome neurovascular de la cintura escapular o síndrome de compresión de la salida torácica

Supervivencia en los aneurismas aórticos operados electivamente / Survival in aortic aneurysms electively operated

El seguimiento se realizó en 28 pacientes operados electivamente por aneurisma de la aorta abdominal. La técnica quirúrgica realizada fue la aneurismectomía con injerto aortobiilíaco o aortobifemoral. Estos pacientes se operaron electivamente en el período comprendido de 1976 a 1981: 25 hombres y 3 mujeres. El promedio de edad fue de 65 años. Sobrevivió al término de la observación, en 1982, el 55%; del 37% de los fallecidos, 8 decesos ocurrieron en el posoperatorio inmediato por diversas causas, entre las que prevalecieron el tromboembolismo pulmonar y la insuficiencia renal aguda. El promedio general de muerte por año de la serie fue de 0,2121. La esperanza de vida al año fue de un 80% y para el sexto año, de un 28%

Valoración hemodinámica del ergocomb en pacientes ateroscleróticos, diabéticos y arteríticos / Hemodynamic appraisment of ergocomb in atherosclerotic, diabetic and arterious patients

Se estudian 26 pacientes divididos en tres grupos de acuerdo con su enfermedad de base, se utilizó un vasodilatador, el ergocomb, por vía intraarterial (femoral), valorando su efectividad por pruebas hemodinámicas. Ambas pruebas se realizaron antes y 15 minutos después de la administración del medicamento, obteniéndose diferentes resultados para los 3 grupos indicados. Se concluye que el ergocomb puede ser un agente de valor en el tratamiento médico de la insuficiencia arterial crónica de los miembros, por lo que se deben efectuar investigaciones posteriores con una mayor casuística, que permita confirmar esta posibilidad