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.

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.

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.

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.

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.

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.

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.

Assessment of the corneal collagen organization after chemical burn using second harmonic generation microscopy.

The organization of the corneal stoma is modified due to different factors, including pathology, surgery or external damage. Here the changes in the organization of the corneal collagen fibers during natural healing after chemical burn are investigated using second harmonic generation (SHG) imaging. Moreover, the structure tensor (ST) was used as an objective tool for morphological analyses at different time points after burn (up to 6 months). Unlike control corneas that showed a regular distribution, the collagen pattern at 1 month of burn presented a non-organized arrangement. SHG signal levels noticeably decreased and individual fibers were hardly visible. Over time, the healing process led to a progressive re-organization of the fibers that could be quantified through the ST. At 6 months, the stroma distribution reached values similar to those of control eyes and a dominant direction of the fibers re-appeared. The present results show that SHG microscopy imaging combined with the ST method is able to objectively monitor the temporal regeneration of the corneal organization after chemical burn. Future implementations of this approach into clinically adapted devices would help to diagnose and quantify corneal changes, not only due to chemical damages, but also as a result of disease or surgical procedures.

Arrangement of the photoreceptor mosaic in a diabetic rat model imaged with multiphoton microscopy.

Diabetic retinopathy (DR) is defined as a microvascular pathology. However, some data have suggested that the retinal photoreceptors (PRs) might be important in the pathogenesis of this ocular disease. In this study the organization of the PRs in control and diabetic-induced rats was compared using multiphoton microscopy. The PR mosaic was imaged at different locations in non-stained retinas. The density of PRs was directly quantified from cell counting. The spatially resolved density presents a double-slope pattern (from the central retina towards the periphery) in both healthy and pathological samples, although the values for the latter were significantly lower all across the retina. Moreover, Voronoi analysis was performed to explore changes in PR topography. In control specimens a hexagonally packed structure was dominant. However, despite the non-controlled effects of the disease in retinal structures, this PR regularity was fairly maintained in diabetic retinas.

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.

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.

Quantitative Discrimination of Healthy and Diseased Corneas With Second Harmonic Generation Microscopy.

PURPOSE: To analyze the spatial organization of pathological corneas with second harmonic generation (SHG) imaging and to provide a proof of concept to objectively distinguish these from the healthy corneas. METHODS: A custom-built SHG microscope was used to image the anterior stroma of ex vivo corneas, both control and affected by some representative pathologies. The structure tensor (ST) was employed as a metric to explore and quantify the alterations in the spatial distribution of the collagen lamellae. RESULTS: The collagen arrangement differed between healthy and pathological samples. The former showed a regular distribution and a low structural dispersion (SD < 40°) within the stroma with a well-defined dominant orientation. This regular arrangement drastically turns into a disorganized pattern in pathological corneas (SD > 40°). CONCLUSIONS: The combination of SHG imaging and the ST allows obtaining quantitative information to differentiate the stromal collagen organization in healthy and diseased corneas. This approach represents a feasible and powerful technique with potential applications in clinical corneal diagnoses. TRANSLATIONAL RELEVANCE: The ST applied to SHG microscopy images of the corneal stroma provides an experimental objective score to differentiate control from pathological or damaged corneas. Future implementations of this technique in clinical environments might might be a promising tool in Ophthalmology, not only to diagnose and monitor corneal diseases, but also to follow-up surgical outcome.

In vivo two-photon microscopy of the human eye.

Two-photon (2P) microscopy is a powerful tool for imaging and exploring label-free biological tissues at high resolution. Although this type of microscopy has been demonstrated in ex vivo ocular tissues of both humans and animal models, imaging the human eye in vivo has always been challenging. This work presents a novel compact 2P microscope for non-contact imaging of the anterior part of the living human eye. The performance of the instrument was tested and the maximum permissible exposure to protect ocular tissues established. To the best of our knowledge, 2P images of the in vivo human cornea, the sclera and the trabecular meshwork are shown for the very first time. Acquired images are of enough quality to visualize collagen arrangement and morphological features of clinical interest. Future implementations of this technique may constitute a potential tool for early diagnosis of ocular diseases at submicron scale.

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.

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.

Quantitative Analysis of the Corneal Collagen Distribution after In Vivo Cross-Linking with Second Harmonic Microscopy.

Corneal cross-linking (CXL) is a surgical procedure able to modify corneal biomechanics and stabilize keratoconus progression. Although it is known that CXL produces changes in corneal collagen distribution, these are still a topic of discussion. Here we quantitatively compare the corneal stroma architecture between two animal models four weeks after in vivo conventional CXL treatment, with second harmonic generation (SHG) imaging microscopy and the structure tensor (ST). The healing stage and the stroma recovery were also analyzed by means of histological sections. Results show that the CXL effects depend on the initial arrangement of the corneal collagen. While the treatment increases the order in corneas with a low level of initial organization, corneas presenting a fairly regular pattern are hardly affected. Histological samples showed active keratocytes in anterior and middle stroma, what means that the recovery is still in progress. The combination of SHG imaging and the ST is able to objectively discriminate the changes suffered by the collagen arrangement after the CXL treatment, whose effectiveness depends on the initial organization of the collagen fibers within the corneal stroma.

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.

Author Correction: A Study on Image Quality in Polarization-Resolved Second Harmonic Generation Microscopy.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Comparison of second harmonic microscopy images of collagen-based ocular tissues with 800 and 1045 nm.

Second harmonic generation (SHG) imaging is a well-suited multiphoton technique allowing visualization of biological tissues mainly composed of collagen with submicron resolution. Despite its inherent confocal properties, imaging of deeper layers within thick samples has still some limitations. Although the use of longer wavelengths might help to overcome this, the dependence between SHG signals and wavelength is still under discussion. We report here on the dependence with wavelength of SHG signals from collagen-based ocular tissues. The quality of SHG images for two commonly used excitation wavelengths (800 and 1045 nm) is studied. The analysis of the collagen structural information reveals that the information provided by both wavelengths is similar. It was also found that, independently of the depth location, 1045-nm SHG images presented always lower signal levels than those acquired with 800 nm. However, the contrast of the former images was higher, what may improve the visualization of certain features of interest.