1,567 research outputs found
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Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning
We present a new method for high-resolution, three-dimensional fluorescence imaging. In contrast to beam-scanning confocal microscopy, where the laser focus must be scanned both laterally and axially to collect a volume, we obtain depth information without the necessity of depth scanning. In this method, the emitted fluorescence is collected in the backward direction and is sent through a phase plate that encodes the depth information into the phase of a spectrally resolved interference pattern. We demonstrate that decoding this phase information allows for depth localization accuracy better than 4 µm over a 500 µm depth-of-field. In a high numerical aperture configuration with a much smaller depth of field, a localization accuracy of tens of nanometers can be achieved. This approach is ideally suited for miniature endoscopes, where space limitations at the endoscope tip render depth scanning difficult. We illustrate the potential for 3D visualization of complex biological samples by constructing a three-dimensional volume of the microvasculature of ex vivo murine heart tissue from a single 2D scan
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Comprehensive Evaluation of Peripheral Nerve Regeneration in the Acute Healing Phase Using Tissue Clearing and Optical Microscopy in a Rodent Model
Peripheral nerve injury (PNI), a common injury in both the civilian and military arenas, is usually associated with high healthcare costs and with patients enduring slow recovery times, diminished quality of life, and potential long-term disability. Patients with PNI typically undergo complex interventions but the factors that govern optimal response are not fully characterized. A fundamental understanding of the cellular and tissue-level events in the immediate postoperative period is essential for improving treatment and optimizing repair. Here, we demonstrate a comprehensive imaging approach to evaluate peripheral nerve axonal regeneration in a rodent PNI model using a tissue clearing method to improve depth penetration while preserving neural architecture. Sciatic nerve transaction and end-to-end repair were performed in both wild type and thy-1 GFP rats. The nerves were harvested at time points after repair before undergoing whole mount immunofluorescence staining and tissue clearing. By increasing the optic depth penetration, tissue clearing allowed the visualization and evaluation of Wallerian degeneration and nerve regrowth throughout entire sciatic nerves with subcellular resolution. The tissue clearing protocol did not affect immunofluorescence labeling and no observable decrease in the fluorescence signal was observed. Large-area, high-resolution tissue volumes could be quantified to provide structural and connectivity information not available from current gold-standard approaches for evaluating axonal regeneration following PNI. The results are suggestive of observed behavioral recovery in vivo after neurorrhaphy, providing a method of evaluating axonal regeneration following repair that can serve as an adjunct to current standard outcomes measurements. This study demonstrates that tissue clearing following whole mount immunofluorescence staining enables the complete visualization and quantitative evaluation of axons throughout nerves in a PNI model. The methods developed in this study could advance PNI research allowing both researchers and clinicians to further understand the individual events of axonal degeneration and regeneration on a multifaceted level
Prospects for detecting gravitational waves at 5 Hz with ground-based detectors
We propose an upgrade to Advanced LIGO (aLIGO), named LIGO-LF, that focuses
on improving the sensitivity in the 5-30 Hz low-frequency band, and we explore
the upgrade's astrophysical applications. We present a comprehensive study of
the detector's technical noises and show that with technologies currently under
development, such as interferometrically sensed seismometers and
balanced-homodyne readout, LIGO-LF can reach the fundamental limits set by
quantum and thermal noises down to 5 Hz. These technologies are also directly
applicable to the future generation of detectors. We go on to consider this
upgrade's implications for the astrophysical output of an aLIGO-like detector.
A single LIGO-LF can detect mergers of stellar-mass black holes (BHs) out to a
redshift of z~6 and would be sensitive to intermediate-mass black holes up to
2000 M_\odot. The detection rate of merging BHs will increase by a factor of 18
compared to aLIGO. Additionally, for a given source the chirp mass and total
mass can be constrained 2 times better than aLIGO and the effective spin 3-5
times better than aLIGO. Furthermore, LIGO-LF enables the localization of
coalescing binary neutron stars with an uncertainty solid angle 10 times
smaller than that of aLIGO at 30 Hz, and 4 times smaller when the entire signal
is used. LIGO-LF also significantly enhances the probability of detecting other
astrophysical phenomena including the tidal excitation of neutron star r-modes
and the gravitational memory effects.Comment: 5 pages, 6 figures, published in PR
Optimization of Transcutaneous Oxygenation Wearable Sensors for Clinical Applications
In this manuscript, the development of an experimental and mathematical toolset is reported that allows for improved in vivo measurements of optical transcutaneous oxygen tension measurements (TCOM) wearable technology in humans. In addition to optimizing O2-sensing films for higher sensitivity oxygen detection, calibration algorithms are additionally developed to account for excitation source leakage, as well as algorithms to combine readings of partial pressure of oxygen (pO2), derived from phosphorescence intensity and lifetime, into a single metric. This new iteration of the TCOM wearable device is then tested in a pilot human study. By implementing characterization and calibration algorithms, the data from the pilot study demonstrates the ability to obtain reliable transcutaneous pO2 readings with a TCOM sensor regardless of size and without the need for strict conditions of constant temperature, humidity, or motion that have limited the range of applications of this technology in the past
Longitudinal, 3D in vivo imaging of sebaceous glands by coherent anti-Stokes Raman scattering microscopy –normal function and response to cryotherapy
Sebaceous glands perform complex functions, and are centrally involved in the pathogenesis of acne vulgaris. Current techniques for studying sebaceous glands are mostly static in nature, whereas the gland’s main function – excretion of sebum via the holocrine mechanism – can only be evaluated over time. We present a longitudinal, real-time alternative – the in vivo, label-free imaging of sebaceous glands using Coherent Anti-Stokes Raman Scattering (CARS) microscopy, which is used to selectively visualize lipids. In mouse ears, CARS microscopy revealed dynamic changes in sebaceous glands during the holocrine secretion process, as well as in response to damage to the glands caused by cooling. Detailed gland structure, plus the active migration of individual sebocytes and cohorts of sebocytes were measured. Cooling produced characteristic changes in sebocyte structure and migration. This study demonstrates that CARS microscopy is a promising tool for studying the sebaceous gland and its associated disorders in three-dimensions in vivo
Yambo: an \textit{ab initio} tool for excited state calculations
{\tt yambo} is an {\it ab initio} code for calculating quasiparticle energies
and optical properties of electronic systems within the framework of many-body
perturbation theory and time-dependent density functional theory. Quasiparticle
energies are calculated within the approximation for the self-energy.
Optical properties are evaluated either by solving the Bethe--Salpeter equation
or by using the adiabatic local density approximation. {\tt yambo} is a
plane-wave code that, although particularly suited for calculations of periodic
bulk systems, has been applied to a large variety of physical systems. {\tt
yambo} relies on efficient numerical techniques devised to treat systems with
reduced dimensionality, or with a large number of degrees of freedom. The code
has a user-friendly command-line based interface, flexible I/O procedures and
is interfaced to several publicly available density functional ground-state
codes.Comment: This paper describes the features of the Yambo code, whose source is
available under the GPL license at www.yambo-code.or
Distinct, dosage-sensitive requirements for the autism-associated factor CHD8 during cortical development
Background: CHD8 haploinsufficiency causes autism and macrocephaly with high penetrance in the human population. Chd8 heterozygous mice exhibit relatively subtle brain overgrowth and little gene expression changes in the embryonic neocortex. The purpose of this study was to generate new, sub-haploinsufficient Chd8 mouse models to allow us to identify and study the functions of CHD8 during embryonic cortical development. Methods: To examine the possibility that certain phenotypes may only appear at sub-heterozygous Chd8 levels in the mouse, we created an allelic series of Chd8-deficient mice to reduce CHD8 protein levels to approximately 35% (mild hypomorph), 10% (severe hypomorph) and 0% (neural-specific conditional knockout) of wildtype levels. We used RNA sequencing to compare transcriptional dysregulation, structural MRI and brain weight to investigate effects on brain size, and cell proliferation, differentiation and apoptosis markers in immunostaining assays to quantify changes in neural progenitor fate. Results: Mild Chd8 hypomorphs displayed significant postnatal lethality, with surviving animals exhibiting more pronounced brain hyperplasia than heterozygotes. Over 2000 genes were dysregulated in mild hypomorphs, including autism-associated neurodevelopmental and cell cycle genes. We identify increased proliferation of non-ventricular zone TBR2+ intermediate progenitors as one potential cause of brain hyperplasia in these mutants. Severe Chd8 hypomorphs displayed even greater transcriptional dysregulation, including evidence for p53 pathway upregulation. In contrast to mild hypomorphs, these mice displayed reduced brain size and increased apoptosis in the embryonic neocortex. Homozygous, conditional deletion of Chd8 in early neuronal progenitors resulted in pronounced brain hypoplasia, partly caused by p53 target gene derepression and apoptosis in the embryonic neocortex. Limitations Our findings identify an important role for the autism-associated factor CHD8 in controlling the proliferation of intermediate progenitors in the mouse neocortex. We propose that CHD8 has a similar function in human brain development, but studies on human cells are required to confirm this. Because many of our mouse mutants with reduced CHD8 function die shortly after birth, it is not possible to fully determine to what extent reduced CHD8 function results in autism-associated behaviours in mice. Conclusions: Together, these findings identify important, dosage-sensitive functions for CHD8 in p53 pathway repression, neurodevelopmental gene expression and neural progenitor fate in the embryonic neocortex. We conclude that brain development is acutely sensitive to reduced CHD8 expression and that the varying sensitivities of different progenitor populations and cellular processes to CHD8 dosage result in non-linear effects on gene transcription and brain growth. Shaun Hurley, Conor Mohan and Philipp Suetterlin have contributed equally to this work
In vitro ovarian tumor growth and treatment response dynamics visualized with time-lapse OCT imaging
In vitro three-dimensional models for metastatic ovarian cancer have been useful for recapitulating the human disease. These spheroidal tumor cultures, however, can grow in excess of 1 mm in diameter, which are difficult to visualize without suitable imaging technology. Optical coherence tomography (OCT) is an ideal live imaging method for non-perturbatively visualizing these complex systems. OCT enabled detailed observations of the model at both nodular and cellular levels, revealing growth dynamics not previously observed. The development ofa time-lapse OCT system, capable of automated, multidimensional acquisition, further provided insights into the growth and chemotherapeutic response of ovarian cancer.© 2009 Optical Society of America
In vivo coherent Raman imaging of the melanomagenesis-associated pigment pheomelanin
Melanoma is the most deadly form of skin cancer with a yearly global incidence over 232,000 patients. Individuals with fair skin and red hair exhibit the highest risk for developing melanoma, with evidence suggesting the red/blond pigment known as pheomelanin may elevate melanoma risk through both UV radiation-dependent and -independent mechanisms. Although the ability to identify, characterize, and monitor pheomelanin within skin is vital for improving our understanding of the underlying biology of these lesions, no tools exist for real-time, in vivo detection of the pigment. Here we show that the distribution of pheomelanin in cells and tissues can be visually characterized non-destructively and noninvasively in vivo with coherent anti-Stokes Raman scattering (CARS) microscopy, a label-free vibrational imaging technique. We validated our CARS imaging strategy in vitro to in vivo with synthetic pheomelanin, isolated melanocytes, and the Mc1re/e, red-haired mouse model. Nests of pheomelanotic melanocytes were observed in the red-haired animals, but not in the genetically matched Mc1re/e; Tyrc/c (“albino-red-haired”) mice. Importantly, samples from human amelanotic melanomas subjected to CARS imaging exhibited strong pheomelanotic signals. This is the first time, to our knowledge, that pheomelanin has been visualized and spatially localized in melanocytes, skin, and human amelanotic melanomas
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