Fluorescent bicolour sensor for low-background neutrinoless double ß decay experiments.

Observation of the neutrinoless double ß decay is the only practical way to establish that neutrinos are their own antiparticles1. Because of the small masses of neutrinos, the lifetime of neutrinoless double ß decay is expected to be at least ten orders of magnitude greater than the typical lifetimes of natural radioactive chains, which can mimic the experimental signature of neutrinoless double ß decay2. The most robust identification of neutrinoless double ß decay requires the definition of a signature signal-such as the observation of the daughter atom in the decay-that cannot be generated by radioactive backgrounds, as well as excellent energy resolution. In particular, the neutrinoless double ß decay of 136Xe could be established by detecting the daughter atom, 136Ba2+, in its doubly ionized state3-8. Here we demonstrate an important step towards a 'barium-tagging' experiment, which identifies double ß decay through the detection of a single Ba2+ ion. We propose a fluorescent bicolour indicator as the core of a sensor that can detect single Ba2+ ions in a high-pressure xenon gas detector. In a sensor made of a monolayer of such indicators, the Ba2+ dication would be captured by one of the molecules and generate a Ba2+-coordinated species with distinct photophysical properties. The presence of such a single Ba2+-coordinated indicator would be revealed by its response to repeated interrogation with a laser system, enabling the development of a sensor able to detect single Ba2+ ions in high-pressure xenon gas detectors for barium-tagging experiments.

Objective analysis of collagen organization in thyroid nodule capsules using second harmonic generation microscopy images and the Hough transform.

Papillary carcinoma is the most prevalent type of thyroid cancer. Its diagnosis requires accurate and subjective analyses from expert pathologists. Here we propose a method based on the Hough transform (HT) to detect and objectively quantify local structural differences in collagen thyroid nodule capsules. Second harmonic generation (SHG) microscopy images were acquired on non-stained histological sections of capsule fragments surrounding the healthy thyroid gland and benign and tumoral/malignant nodules. The HT was applied to each SHG image to extract numerical information on the organization of the collagen architecture in the tissues under analysis. Results show that control thyroid capsule samples present a non-organized structure composed of wavy collagen distribution with local orientations. On the opposite, in capsules surrounding malignant nodules, a remodeling of the collagen network takes place and local undulations disappear, resulting in an aligned pattern with a global preferential orientation. The HT procedure was able to quantitatively differentiate thyroid capsules from capsules surrounding papillary thyroid carcinoma (PTC) nodules. Moreover, the algorithm also reveals that the collagen arrangement of the capsules surrounding benign nodules significantly differs from both the thyroid control and PTC nodule capsules. Combining SHG imaging with the HT results thus in an automatic and objective tool to discriminate between the pathological modifications that affect the capsules of thyroid nodules across the progressions of PTC, with potential to be used in clinical settings to complement current state-of-the-art diagnostic methods.

Tear-film dynamics by combining double-pass images, pupil retro-illumination, and contrast sensitivity.

Tear-film dynamics were analyzed by a synchronizing recording of double-pass (DP) and pupil retro-illumination (RI) images with contrast sensitivity (CS) measurements. Simultaneous DP and RI images were acquired in three subjects wearing contact lenses while keeping the eye open. Changes in contrast sensitivity for an 18 c/deg green grating were also estimated. From the DP retinal images, the effect of the tear film is described through the objective scattering index (OSI). This presented a negative correlation with the increase in CS during tear-film deterioration (as observed by RI imaging). These results show a relationship between visual outcome degradation due to tear-film breakup and the increase in intraocular scattering. This work shows a combined methodology for the evaluation of tear-film dynamics.

Comparing the performance of a femto fiber-based laser and a Ti:sapphire used for multiphoton microscopy applications.

Ti:sapphire laser systems are the more extended excitation sources in multiphoton (MP) microscopy. Although tunable, the cost, size, and lack of portability often limit their use in some research fields. Femtosecond fiber-based lasers represent an attractive alternative since they are portable, compact, and affordable. Most MP applications using these devices employ wavelengths beyond 1000 nm. This work evaluates the performance of a mode-locked fiber-based laser emitting at 780 nm (within the spectral region often used with Ti:sapphire devices) for use in MP imaging microscopy. MP images acquired with this laser system have been compared with those obtained with a "regular" solid-state source. Results herein show that the images recorded with both laser sources were similar, independently of the depth location of the imaged plane. The structural information contained in the images hardly differed. Moreover, the images of deeper layers improved by means of adaptive optics were also similar. These ultrafast laser sources are expected to enhance the impact of MP microscopy in basic research, as well as in biomedical environments.

Wavefront correction in two-photon microscopy with a multi-actuator adaptive lens.

A multi-actuator adaptive lens (AL) was incorporated into a multi-photon (MP) microscope to improve the quality of images of thick samples. Through a hill-climbing procedure the AL corrected for the specimen-induced aberrations enhancing MP images. The final images hardly differed when two different metrics were used, although the sets of Zernike coefficients were not identical. The optimized MP images acquired with the AL were also compared with those obtained with a liquid-crystal-on-silicon spatial light modulator. Results have shown that both devices lead to similar images, which corroborates the usefulness of this AL for MP imaging.

Complete genome sequence of bean leaf crumple virus, a novel begomovirus infecting common bean in Colombia.

A copy of the complete genome of a novel bipartite begomovirus infecting common bean (Phaseolus vulgaris L.) in Colombia was obtained by rolling-circle amplification (RCA), cloned, and sequenced. The virus is associated with leaf crumple symptoms and significant yield losses in Andean and Mesoamerican beans. Such symptoms have been reported increasingly in Colombia since at least 2002, and we detected the virus in leaf material collected since 2008. Sequence analysis showed that the virus is a member of a distinct species, sharing 81% and 76% nucleotide (nt) sequence identity (in DNA-A and DNA-B, respectively) to other begomoviruses infecting common bean in the Americas. The data obtained support the taxonomic status of this virus (putatively named 'bean leaf crumple virus', BLCrV) as a member of a novel species in the genus Begomovirus.

Performance evaluation of a sensorless adaptive optics multiphoton microscope.

A wavefront sensorless adaptive optics technique was combined with a custom-made multiphoton microscope to correct for specimen-induced aberrations. A liquid-crystal-on-silicon (LCoS) modulator was used to systematically generate Zernike modes during image recording. The performance of the instrument was evaluated in samples providing different nonlinear signals and the benefit of correcting higher order aberrations was always noticeable (in both contrast and resolution). The optimum aberration pattern was stable in time for the samples here involved. For a particular depth location within the sample, the wavefront to be precompensated was independent on the size of the imaged area (up to ∼ 360 × 360 µm(2)). The mode combination optimizing the recorded image depended on the Zernike correction control sequence; however, the final images hardly differed. At deeper locations, a noticeable dominance of spherical aberration was found. The influence of other aberration terms was also compared to the effect of the spherical aberration.

Analysis and quantification of collagen organization with the structure tensor in second harmonic microscopy images of ocular tissues.

The important biological role of collagen-based tissues and the changes produced in the fiber distribution under particular situations (surgery, pathology, external damage, etc.) require tools for the analysis of the collagen organization that might potentially help in early diagnoses. Since collagen structures provide efficient second harmonic generation (SHG) signals, SHG microscopy has emerged as a powerful technique to visualize collagen fibers and qualitatively discriminate normal from abnormal tissues. Here we propose a quantitative method based on the structure tensor to quantify the different organization of collagen patterns in SHG images of ocular tissues. Results show that well-organized collagen distributions present a high degree of isotropy (DoI), a dominant orientation (PO), and a low structural dispersion (SD). On the other hand, the PO vanishes when the collagen tissue is not organized as a consequence of an increase in the SD and a decrease in the DoI. The proposed method is also able to discriminate partially organized samples. The combination of SHG microscopy and the structure tensor is a useful method to objectively classify collagen distributions. Clinical applications of this technique could help in the diagnosis and tracking of pathologies related to collagen disorders in connective tissue.

Spherical Aberration and Scattering Compensation in Microscopy Images through a Blind Deconvolution Method.

The optical quality of an image depends on both the optical properties of the imaging system and the physical properties of the medium the light passes while travelling from the object to the image plane. The computation of the point spread function (PSF) associated to the optical system is often used to assess the image quality. In a non-ideal optical system, the PSF is affected by aberrations that distort the final image. Moreover, in the presence of turbid media, the scattering phenomena spread the light at wide angular distributions that contribute to reduce contrast and sharpness. If the mathematical degradation operator affecting the recorded image is known, the image can be restored through deconvolution methods. In some scenarios, no (or partial) information on the PSF is available. In those cases, blind deconvolution approaches arise as useful solutions for image restoration. In this work, a new blind deconvolution method is proposed to restore images using spherical aberration (SA) and scatter-based kernel filters. The procedure was evaluated in different microscopy images. The results show the capability of the algorithm to detect both degradation coefficients (i.e., SA and scattering) and to restore images without information on the real PSF.

Structural changes in the crystalline lens as a function of the postmortem interval assessed with two-photon imaging microscopy.

The properties and structure of the crystalline lens change as time after death passes. Some experiments have suggested that these might be used to estimate the postmortem interval (PMI). In this study, the organization and texture of the rabbit lens were objectively evaluated as a function of the PMI using two-photon excitation fluorescence (TPEF) imaging microscopy. Between 24 h and 72 h, the lens presented a highly organized structure, although the fiber delineation was progressively vanishing. At 96 h, this turned into a homogeneous pattern where fibers were hardly observed. This behaviour was similar for parameters providing information on tissue texture. On the other hand, the fiber density of the lens is linearly reduced with the PMI. On average, density at 24 h was approximately two-fold when compared to 96 h after death. The present results show that TPEF microscopy combined with different quantitative tools can be used to objectively monitor temporal changes in the lens fiber organization after death. This might help to estimate the PMI, which is one of the most complex problems in forensic science.

Analysis of age-related changes in the left ventricular myocardium with multiphoton microscopy.

Aging induces cardiac remodeling, resulting in an increase in the risk of suffering heart diseases, including heart failure. Collagen deposition increases with age and, together with sarcomeric changes in cardiomyocytes, may lead to ventricular stiffness. Multiphoton (MP) microscopy is a useful technique to visualize and detect variations in cardiac structures in a label free fashion. Here, we propose a method based on MP imaging (both two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) modalities) to explore and objectively quantify age-related structural differences in various components of cardiac tissues. Results in transmural porcine left ventricle (LV) sections reveal significant differences when comparing samples from young and old animals. Collagen and myosin SHG signals in old specimens are respectively 3.8x and >6-fold larger than in young ones. Differences in TPEF signals from cardiomyocyte were ∼3x. Moreover, the increased amount of collagen in old specimens results in a more organized pattern when compared to young LV tissues. Since changes in collagen and myosin are associated with cardiac dysfunction, the technique used herein might be a useful tool to accurately predict and measure changes associated with age-related myocardium fibrosis, tissue remodeling and sarcomeric alterations, with potential implications in preventing heart disease.

Blind deconvolution of second harmonic microscopy images of the living human eye.

Second harmonic generation (SHG) imaging microscopy of thick biological tissues is affected by the presence of aberrations and scattering within the sample. Moreover, additional problems, such as uncontrolled movements, appear when imaging in-vivo. Deconvolution methods can be used to overcome these limitations under some conditions. In particular, we present here a technique based on a marginal blind deconvolution approach for improving SHG images obtained in vivo in the human eye (cornea and sclera). Different image quality metrics are used to quantify the attained improvement. Collagen fibers in both cornea and sclera are better visualized and their spatial distributions accurately assessed. This might be a useful tool to better discriminate between healthy and pathological tissues, especially those where changes in collagen distribution occur.

Three-dimensional spatiotemporal pulse characterization with an acousto-optic pulse shaper and a Hartmann-Shack wavefront sensor.

We demonstrate a simplified arrangement for spatiotemporal ultrashort pulse characterization called Hartmann-Shack assisted, multidimensional, shaper-based technique for electric-field reconstruction. It employs an acousto-optic pulse shaper in combination with a second-order nonlinear crystal and a Hartmann-Shack wavefront sensor. The shaper is used as a tunable bandpass filter, and the wavefronts and intensities of quasimonochromatic spectral slices of the pulse are obtained using the Hartmann-Shack wavefront sensor. The wavefronts and intensities of the spectral slices are related to one another using shaper-assisted frequency-resolved optical gating measurements, performed at particular points in the beam. This enables a three-dimensional reconstruction of the amplitude and phase of the pulse. We present some example pulse measurements and discuss the operating parameters of the device.

The wide-angle point spread function of the human eye reconstructed by a new optical method.

The point spread function (PSF) of the human eye spans over a wide angular distribution where the central part is associated mostly to optical aberrations while the peripheral zones are associated to light scattering. There is a plethora of optical methods for the direct and indirect measurements of the central part of the PSF as a result of monochromatic and polychromatic aberrations. The impact of the spatial characteristics of this central part of the PSF on the retinal image quality and visual function has been extensively analyzed and documented both by optical and psychophysical methods. However, the more peripheral areas of the PSF in the living human eye, ranging from about 1 to 10 degrees of eccentricity, have been investigated only psychophysically. We report here a new optical method for the accurate reconstruction of the wide-angle PSF in the living human eye up to 8 degrees. The methodology consists of projecting disks of uniform radiance on the retina, recording the images after reflection and double pass through the eye's optics and performing a proper analysis of the images. Examples of application of the technique in real eyes with different amount of scatter artificially induced are presented. This procedure allows the direct, accurate, and in vivo measurement of the effect of intraocular scattering and may be a step toward the comprehensive optical evaluation of the optics of the living human eye.

Superpixel-Based Optic Nerve Head Segmentation Method of Fundus Images for Glaucoma Assessment.

Glaucoma disease is the second leading cause of blindness in the world. This progressive ocular neuropathy is mainly caused by uncontrolled high intraocular pressure. Although there is still no cure, early detection and appropriate treatment can stop the disease progression to low vision and blindness. In the clinical practice, the gold standard used by ophthalmologists for glaucoma diagnosis is fundus retinal imaging, in particular optic nerve head (ONH) subjective/manual examination. In this work, we propose an unsupervised superpixel-based method for the optic nerve head (ONH) segmentation. An automatic algorithm based on linear iterative clustering is used to compute an ellipse fitting for the automatic detection of the ONH contour. The tool has been tested using a public retinal fundus images dataset with medical expert ground truths of the ONH contour and validated with a classified (control vs. glaucoma eyes) database. Results showed that the automatic segmentation method provides similar results in ellipse fitting of the ONH that those obtained from the ground truth experts within the statistical range of inter-observation variability. Our method is a user-friendly available program that provides fast and reliable results for clinicians working on glaucoma screening using retinal fundus images.

Biomimetic Remineralization of an Extracellular Matrix Collagen Membrane for Bone Regeneration.

Natural extracellular matrix (ECM) collagen membranes are frequently used for bone regeneration procedures. Some disadvantages, such as rapid degradation and questionable mechanical properties, limit their clinical use. These membranes have a heterologous origin and may proceed from different tissues. Biomineralization is a process in which hydroxyapatite deposits mainly in collagen fibrils of the matrices. However, when this deposition occurs on the ECM, its mechanical properties are increased, facilitating bone regeneration. The objective of the present research is to ascertain if different membranes from distinct origins may undergo biomineralization. Nanomechanical properties, scanning electron (SEM) and multiphoton (MP) microscopy imaging were performed in three commercially available ECMs before and after immersion in simulated body fluid solution for 7 and 21 d. The matrices coming from porcine dermis increased their nanomechanical properties and they showed considerable mineralization after 21 d, as observed in structural changes detected through SEM and MP microscopy. It is hypothesized that the more abundant crosslinking and the presence of elastin fibers within this membrane explains the encountered favorable behavior.

Dental Age Estimation Using Multiphoton Microscopy: A Potential Tool for Forensic Science.

Normal aging affects the different structures of teeth, in particular, the dentine. These changes are useful in forensic disciplines as a tool for age estimation. Although multiphoton (MP) microscopy has been used to explore dental pieces, a relationship between age and MP response of the human dentine has not been proposed yet. The relationship between MP signals and natural dentine aging is investigated herein. An index of age (INAG) combining two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) images has been used to quantify these changes. The results show that the INAG significantly decreases with age. Moreover, peritubular dentine size and collagen internal properties are also modified with age. This information confirms the usefulness of this technique in forensic age estimation after disasters (natural or manmade) with a lack of comprehensive fingerprint database. Courts and other government authorities might also benefit from this tool when the official age of individuals under special circumstances is required for legal or medical reasons.

Galvanic current activates the NLRP3 inflammasome to promote Type I collagen production in tendon.

The NLRP3 inflammasome coordinates inflammation in response to different pathogen- and damage-associated molecular patterns, being implicated in different infectious, chronic inflammatory, metabolic and degenerative diseases. In chronic tendinopathic lesions, different non-resolving mechanisms produce a degenerative condition that impairs tissue healing and which therefore complicates their clinical management. Percutaneous needle electrolysis consists of the application of a galvanic current and is an emerging treatment for tendinopathies. In the present study, we found that galvanic current activates the NLRP3 inflammasome and induces an inflammatory response that promotes a collagen-mediated regeneration of the tendon in mice. This study establishes the molecular mechanism of percutaneous electrolysis that can be used to treat chronic lesions and describes the beneficial effects of an induced inflammasome-related response.

Wavefront retrieval of amplified femtosecond beams by second-harmonic generation.

We present a new approach for wavefront characterization of near transform-limited intense femtosecond beams using the angular and spectral dependences of the second-harmonic generation conversion efficiency in uniaxial crystals. The method is applied to different aberrated beams and results are compared with the measurements performed with a commercial sensor, finding very good agreement. The phase retrieval dependence with different parameters (e.g. crystal thickness) is discussed. Successful application to sharpen intensity profiles is also demonstrated.

Enhancement of filamentation postcompression by astigmatic focusing.

The energy scaling up of pulse postcompression is still an open issue. In this work we analyze the use of astigmatic focusing to improve the output pulses in a filamentation based postcompression setup. Unlike spherical conditions, astigmatic focusing enhances the output energy and the spectral broadening of the filament. This is due to the increase of critical power, allowing a considerable improvement of the postcompression energy and stability in a simple way. We demonstrated compression from FWHM 100 fs, 10 nm, 3 mJ input pulses to 13 fs, 142 nm, near 1 mJ pulses.