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Background and Objectives : Drug delivery systems (DDSs) offer efficient treatment solutions to challenging diseases such as central nervous system (CNS) diseases by bypassing biological barriers such as the blood-brain barrier (BBB). Among DDSs, polymeric nanoparticles (NPs), particularly poly(lactic-co-glycolic acid) (PLGA) NPs, hold an outstanding position due to their biocompatible and biodegradable qualities. Despite their potential, the translation of PLGA NPs from laboratory-scale production to clinical applications remains a significant challenge. This study aims to address these limitations by developing scalable PLGA NPs and evaluating their potential biological applications. Methods : We prepared blank and model-protein-loaded (albumin-FITC and wheat germ agglutinin-488 (WGA-488)) fluorescent PLGA NPs using the traditional double-emulsion method combined with the micro-spray-reactor system, a novel approach that enables fine particle production enabling scale-up applications. We tested the biocompatibility of the NPs in living RPMI 2650 and neuroblastoma cell lines, as well as their trafficking and uptake. Release kinetics of the encapsulated proteins were investigated through confocal microscopy and in vitro release studies, providing insights into the stability and functionality of the released proteins. Results : The formulation demonstrated sustained and prolonged protein release profiles. Importantly, cellular uptake studies revealed that the NPs were not internalized. Furthermore, encapsulated WGA-488 protein retained its functional activity after release, validating the integrity of the encapsulation and release processes. Conclusions : The proof-of-concept study on NP manufacturing and an innovative drug trafficking and release approach can bring new perspectives on scalable preparations of PLGA NPs and their biological applications.

Advanced 3D imaging techniques and image segmentation and classification methods can profoundly transform biomedical research by offering deep insights into the cytoarchitecture of the human brain in relation to pathological conditions. Here, we propose a comprehensive pipeline for performing 3D imaging and automated quantitative cellular phenotyping on Formalin-Fixed Paraffin-Embedded (FFPE) human brain specimens, a valuable yet underutilized resource. We exploited the versatility of our method by applying it to different human specimens from both adult and pediatric, normal and abnormal brain regions. Quantitative data on neuronal volume, ellipticity, local density, and spatial clustering level were obtained from a machine learning-based analysis of the 3D cytoarchitectural organization of cells identified by different molecular markers in two subjects with malformations of cortical development (MCD). This approach will grant access to a wide range of physiological and pathological paraffin-embedded clinical specimens, allowing for volumetric imaging and quantitative analysis of human brain samples at cellular resolution. Possible genotype-phenotype correlations can be unveiled, providing new insights into the pathogenesis of various brain diseases and enlarging treatment opportunities.

Although visible light-based stereolithography (SLA) represents an affordable technology for the rapid prototyping of 3D scaffolds for in vitro support of cells, its potential could be limited by the lack of functional photocurable biomaterials that can be SLA-structured at micrometric resolution. Even if innovative photocomposites showing biomimetic, bioactive, or biosensing properties have been engineered by loading inorganic particles into photopolymer matrices, main examples rely on UV-assisted extrusion-based low-resolution processes. Here, SLA-printable composites were obtained by mixing a polyethylene glycol diacrylate (PEGDA) hydrogel with multibranched gold nanoparticles (NPs). NPs were engineered to copolymerize with the PEGDA matrix by implementing a functionalization protocol involving covalent grafting of allylamine molecules that have C═C pendant moieties. The formulations of gold nanocomposites were tailored to achieve high-resolution fast prototyping of composite scaffolds via visible light-based SLA. Furthermore, it was demonstrated that, after mixing with a polymer and after laser structuring, gold NPs still retained their unique plasmonic properties and could be exploited for optical detection of analytes through surface-enhanced Raman spectroscopy (SERS). As a proof of concept, SERS-sensing performances of 3D printed plasmonic scaffolds were successfully demonstrated with a Raman probe molecule (e.g., 4-mercaptobenzoic acid) from the perspective of future extensions to real-time sensing of cell-specific markers released within cultures. Finally, biocompatibility tests preliminarily demonstrated that embedded NPs also played a key role by inducing physiological cell-cytoskeleton rearrangements, further confirming the potentialities of such hybrid nanocomposites as groundbreaking materials in laser-based bioprinting.

3D reconstruction of human brain volumes at high resolution is now possible thanks to advancements in tissue clearing methods and fluorescence microscopy techniques. Analyzing the massive data produced with these approaches requires automatic methods able to perform fast and accurate cell counting and localization. Recent advances in deep learning have enabled the development of various tools for cell segmentation. However, accurate quantification of neurons in the human brain presents specific challenges, such as high pixel intensity variability, autofluorescence, non-specific fluorescence and very large size of data. In this paper, we provide a thorough empirical evaluation of three techniques based on deep learning (StarDist, CellPose and BCFind-v2, an updated version of BCFind) using a recently introduced three-dimensional stereological design as a reference for large-scale insights. As a representative problem in human brain analysis, we focus on a 4 -cm 3 portion of the Broca's area. We aim at helping users in selecting appropriate techniques depending on their research objectives. To this end, we compare methods along various dimensions of analysis, including correctness of the predicted density and localization, computational efficiency, and human annotation effort. Our results suggest that deep learning approaches are very effective, have a high throughput providing each cell 3D location, and obtain results comparable to the estimates of the adopted stereological design., (© 2024. The Author(s).)

Large-scale cortical dynamics play a crucial role in many cognitive functions such as goal-directed behaviors, motor learning and sensory processing. It is well established that brain states including wakefulness, sleep, and anesthesia modulate neuronal firing and synchronization both within and across different brain regions. However, how the brain state affects cortical activity at the mesoscale level is less understood. This work aimed to identify the cortical regions engaged in different brain states. To this end, we employed group ICA (Independent Component Analysis) to wide-field imaging recordings of cortical activity in mice during different anesthesia levels and the awake state. Thanks to this approach we identified independent components (ICs) representing elements of the cortical networks that are common across subjects under decreasing levels of anesthesia toward the awake state. We found that ICs related to the retrosplenial cortices exhibited a pronounced dependence on brain state, being most prevalent in deeper anesthesia levels and diminishing during the transition to the awake state. Analyzing the occurrence of the ICs we found that activity in deeper anesthesia states was characterized by a strong correlation between the retrosplenial components and this correlation decreases when transitioning toward wakefulness. Overall these results indicate that during deeper anesthesia states coactivation of the posterior-medial cortices is predominant over other connectivity patterns, whereas a richer repertoire of dynamics is expressed in lighter anesthesia levels and the awake state., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Scaglione, Resta, Goretti and Pavone.)

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network., (© 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

Systemic administration of Nogo-A-neutralizing antibody ameliorates experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, the blood-brain barrier (BBB) is a major obstacle limiting the passage of systemically applied antibody to the CNS. To bypass the BBB, in the present study we tested the intranasal route of administration by targeting the olfactory mucosa with the Nogo-A-blocking antibody 11C7 mAb in myelin oligodendrocyte glycoprotein-induced EAE. Antibodies were specifically administered onto the olfactory mucosa using a microcatheter. Antibody distribution was examined in the CNS by ELISA and light-sheet microscopy. The effects of 11C7 mAb on Nogo-A signaling were assessed by Western blotting. EAE-induced deficits were monitored daily. Demyelination was observed on spinal cord histological sections. Gene expression changes were followed by trancriptomic analyses. A sensitive capture ELISA revealed a rapid and widespread distribution of 11C7 mAb in the CNS, including the olfactory bulb, the cerebellum and the lumbar spinal cord, but not in the CSF. Light-sheet microscopy allowed to observe antibody accumulation in the parenchyma, thus demonstrating nose-to-brain transfer of IgG. At the functional level, the widespread penetration of 11C7 mAb in the CNS, including the thoracolumbar spinal cord, resulted in the improvement of motor symptoms and in the preservation of myelin in the spinal cord of EAE mice. This was accompanied by Nogo-A signaling downregulation, as reflected by the decreased level of phosphorylated cofilin observed by Western blotting in the cerebellum. In the brain of EAE score-matched animals, 11C7 modified the expression of genes that can influence neurotransmission and cognitive functions, independently of the demyelination phenotype in the spinal cord. In conclusion, our data show the feasibility of olfactory mucosa-directed administration for the delivery of therapeutic antibodies targeting CNS antigens in EAE mice., (© 2023. Cell Death Differentiation Association (ADMC).)

Downsizing surface-enhanced Raman spectroscopy (SERS) within microfluidic devices has opened interesting perspectives for the development of low-cost and portable (bio)sensors for the optical analysis of liquid samples. Despite the research efforts, SERS-fluidic devices still rely either on the use of expensive bulky set-ups or on polymeric devices giving spurious background signals fabricated via expensive manufacturing processes. Here, polymeric platforms integrating fluidics and optics were fabricated with versatile designs allowing easy coupling with fiber-based Raman systems. For the first time, anti-fouling photocurable perfluoropolyether (PFPE) was explored for high-throughput SERS-integrating chip fabrication via replica molding of negative stamps obtained through standard and advanced fabrication processes. The PFPE devices comprised networks of channels for fluid handling and for optical fiber housing with multiple orientations. Embedded microfeatures were used to control the relative positioning of the fibers, thus guaranteeing the highest signal delivering and collection. The feasibility of PFPE devices as fiber-based SERS fluidic platforms was demonstrated through the straightforward acquisition of Raman-SERS spectra of a mixture of gold nanoparticles as SERS substrates with rhodamine 6G (Rh6G) at decreasing concentrations. In the presence of high-performing gold nanostars, the Rh6G signal was detectable at dilutions down to the nanomolar level even without tight focusing and working at low laser power-a key aspect for analyte detection in real-world biomedical and environmental applications.

Trodusquemine is an aminosterol with a variety of biological and pharmacological functions, such as acting as an antimicrobial, stimulating body weight loss and interfering with the toxicity of proteins involved in the development of Alzheimer's and Parkinson's diseases. The mechanisms of interaction of aminosterols with cells are, however, still largely uncharacterized. Here, by using fluorescently labeled trodusquemine (TRO-A594 and TRO-ATTO565), we show that trodusquemine binds initially to the plasma membrane of living cells, that the binding affinity is dependent on cholesterol, and that trodusquemine is then internalized and mainly targeted to lysosomes after internalization. We also found that TRO-A594 is able to strongly and selectively bind to myelinated fibers in fixed mouse brain slices, and that it is a marker compatible with tissue clearing and light-sheet fluorescence microscopy or expansion microscopy. In conclusion, this work contributes to further characterize the biology of aminosterols and provides a new tool for nerve labeling suitable for the most advanced microscopy techniques., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Recent studies evidence that blue-LED-light irradiation can modulate cell responses in the wound healing process within 24 h from treatment. This study aims to investigate blue-light (410-430 nm) photobiomodulation used in a murine wound model within six days post-treatment. Methods: A superficial wound was made in 30 CD1 male mice. The injuries were treated with a blue LED light (20.6 J/cm 2 ), and biopsies were collected at 24, 72, and 144 h. Histology, fluorescence analysis, and advanced microscopy techniques were used. Results: We can observe an increase in the cellular infiltrate response, and in mast-cell density and their degranulation index correlated to the expression of the major histocompatibility complex after 24 h. Furthermore, after six days, the vessel density increases with the expression of the platelet-derived growth factor in the mast cells. Finally, collagen deposition and morphology in the treated wounds appear more similar to unwounded skin. Conclusions: Blue-light photobiomodulation stimulates several cellular processes that are finely coordinated by mast cells, leading to more rapid wound healing and a better-recovered skin morphology.

Effective targeting of α v β 3 integrin is of high relevance in cancer research as this protein is overexpressed on several types of tumor cells, making such receptor ideal for the development of therapeutics and of diagnostic imaging agents. In this paper, the synthesis of a novel functionalized triazole-based RGD peptidomimetic and its covalent conjugation on pegylated gold nanostars is reported. These highly stable nanoconstructs showed a multivalent effect in binding α v β 3 integrin receptors and proved to inhibit M21 cell adhesion at 25 pM concentration. Thanks to their peculiar surface plasmon resonance in the "NIR transparent window", targeted gold nanostars may represent a promising agent for anticancer multi-modality treatments. 2009 Elsevier Ltd. All rights reserved., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The combination of optical tissue transparency with immunofluorescence allows the molecular characterization of biological tissues in 3D. However, adult human organs are particularly challenging to become transparent because of the autofluorescence contributions of aged tissues. To meet this challenge, we optimized SHORT (SWITCH-H 2 O 2 -antigen Retrieval-TDE), a procedure based on standard histological treatments in combination with a refined clearing procedure to clear and label portions of the human brain. 3D histological characterization with multiple molecules is performed on cleared samples with a combination of multi-colors and multi-rounds labeling. By performing fast 3D imaging of the samples with a custom-made inverted light-sheet fluorescence microscope (LSFM), we reveal fine details of intact human brain slabs at subcellular resolution. Overall, we proposed a scalable and versatile technology that in combination with LSFM allows mapping the cellular and molecular architecture of the human brain, paving the way to reconstruct the entire organ., (© 2022. The Author(s).)

Colorectal cancer presents one of the most elevated incidences of cancer worldwide. Colonoscopy relies on histopathology analysis of hematoxylin-eosin (H&E) images of the removed tissue. Novel techniques such as multi-photon microscopy (MPM) show promising results for performing real-time optical biopsies. However, clinicians are not used to this imaging modality and correlation between MPM and H&E information is not clear. The objective of this paper is to describe and make publicly available an extensive dataset of fully co-registered H&E and MPM images that allows the research community to analyze the relationship between MPM and H&E histopathological images and the effect of the semantic gap that prevents clinicians from correctly diagnosing MPM images. The dataset provides a fully scanned tissue images at 10x optical resolution (0.5 µm/px) from 50 samples of lesions obtained by colonoscopies and colectomies. Diagnostics capabilities of TPF and H&E images were compared. Additionally, TPF tiles were virtually stained into H&E images by means of a deep-learning model. A panel of 5 expert pathologists evaluated the different modalities into three classes (healthy, adenoma/hyperplastic, and adenocarcinoma). Results showed that the performance of the pathologists over MPM images was 65% of the H&E performance while the virtual staining method achieved 90%. MPM imaging can provide appropriate information for diagnosing colorectal cancer without the need for H&E staining. However, the existing semantic gap among modalities needs to be corrected., (© 2022 The Authors.)

Fast volumetric imaging is essential for understanding the function of excitable tissues such as those found in the brain and heart. Measuring cardiac voltage transients in tissue volumes is challenging, especially at the high spatial and temporal resolutions needed to give insight to cardiac function. We introduce a new imaging modality based on simultaneous illumination of multiple planes in the tissue and parallel detection with multiple cameras, avoiding compromises inherent in any scanning approach. The system enables imaging of voltage transients in situ , allowing us, for the first time to our knowledge, to map voltage activity in the whole heart volume at KHz rates. The high spatiotemporal resolution of our method enabled the observation of novel dynamics of electrical propagation through the zebrafish atrioventricular canal., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Background: MDR in bacteria is threatening to public health. Overexpression of efflux pumps is an important cause of MDR. The co-administration of antimicrobial drugs and efflux pump inhibitors (EPIs) is a promising approach to address the problem of MDR., Objectives: To identify new putative EPIs and to characterize their mechanisms of action., Methods: The effects of four selected piperazine derivatives on resistance-nodulation-cell division (RND) pumps was evaluated in Escherichia coli strains overexpressing or not expressing RND pumps by assays aimed at evaluating antibiotic potentiation, membrane functionality, ethidium bromide accumulation and AcrB expression. The cytotoxicity of selected piperazines towards primary cultures of human dermal fibroblasts was also investigated., Results: Four molecules enhanced levofloxacin activity against strains overexpressing RND efflux pumps (AcrAB-TolC and AcrEF-TolC), but not against RND pump-deficient strains. They had little effects on membrane potential. Molecule 4 decreased, whereas the other three increased, membrane permeability compared with untreated control cells. The four molecules showed differences in the specificity of interaction with RND efflux pumps, by inactivating the transport of one or more antibiotics, and in the levels of ethidium bromide accumulation and of acrB expression inhibition., Conclusions: Piperazine derivatives are good candidates as inhibitors of RND efflux pumps. They decreased the activity of RND pumps by mixed mechanisms of action. Small structural differences among the molecules can be critical in defining their behaviour., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Tissue cross-linking represents an important and often used technique to enhance the mechanical properties of biomaterials. For the first time, we investigated biochemical and structural properties of genipin (GE) cross-linked equine pericardium (EP) using optical imaging techniques in tandem with quantitative atomic force microscopy (AFM). EP was cross-linked with GE at 37 °C, and its biochemical and biomechanical properties were observed at various time points up to 24 h. GE cross-linked EP was monitored by the normalized ratio between its second-harmonic generation (SHG) and two-photon autofluorescence emissions and remained unchanged for untreated EP; however, a decreasing ratio due to depleted SHG and elevated autofluorescence and a fluorescence band at 625 nm were found for GE cross-linked EP. The mean autofluorescence lifetime of GE cross-linked EP also decreased. The biochemical signature of GE cross-linker and shift in collagen bands were detected and quantified using shifted excitation Raman difference spectroscopy as an innovative approach for tackling artifacts with high fluorescence backgrounds. AFM images indicated a higher and increasing Young's modulus correlated with cross-linking, as well as collagen structural changes in GE cross-linked EP, qualitatively explaining the observed decrease in the second-harmonic signal. In conclusion, we obtained detailed information about the biochemical, structural, and biomechanical effects of GE cross-linked EP using a unique combination of optical and force microscopy techniques in a nondestructive and label-free manner.

The optical clearing of the cardiac tissue has always been a challenging goal to obtain successful three-dimensional reconstructions of entire hearts. Typically, the developed protocols are targeted at the clearing of the brain; cardiac tissue requires proper arrangements to the original protocols, which are usually tough and time-consuming to figure out. Here, we present the application of three different clearing methodologies on mouse hearts: uDISCO, CLARITY, and SHIELD. For each approach, we describe the required optimizations that we have developed to improve the outcome; in particular, we focus on comparing the features of the tissue after the application of each methodology, especially in terms of tissue preservation, transparency, and staining. We found that the uDISCO protocol induces strong fiber delamination of the cardiac tissue, thus reducing the reliability of structural analyses. The CLARITY protocol confers a high level of transparency to the heart and allows deep penetration of the fluorescent dyes; however, it requires long times for the clearing and the tissue loses its robustness. The SHIELD methodology, indeed, is very promising for tissue maintenance since it preserves its consistency and provides ideal transparency, but further approaches are needed to obtain homogeneous staining of the whole heart. Since the CLARITY procedure, despite the disadvantages in terms of tissue preservation and timings, is actually the most suitable approach to image labeled samples in depth, we optimized and performed the methodology also on human cardiac tissue from control hearts and hearts with hypertrophic cardiomyopathy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics ' agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, this status report reviews an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion report, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed, and provide an outlook for the future directions., (© 2022 The Authors.)

Proper three-dimensional (3D)-cardiomyocyte orientation is important for an effective tension production in cardiac muscle. Cardiac diseases can cause severe remodeling processes in the heart, such as cellular misalignment, that can affect both the electrical and mechanical functions of the organ. To date, a proven methodology to map and quantify myocytes disarray in massive samples is missing. In this study, we present an experimental pipeline to reconstruct and analyze the 3D cardiomyocyte architecture in massive samples. We employed tissue clearing, staining, and advanced microscopy techniques to detect sarcomeres in relatively large human myocardial strips with micrometric resolution. Z-bands periodicity was exploited in a frequency analysis approach to extract the 3D myofilament orientation, providing an orientation map used to characterize the tissue organization at different spatial scales. As a proof-of-principle, we applied the proposed method to healthy and pathologically remodeled human cardiac tissue strips. Preliminary results suggest the reliability of the method: strips from a healthy donor are characterized by a well-organized tissue, where the local disarray is log-normally distributed and slightly depends on the spatial scale of analysis; on the contrary, pathological strips show pronounced tissue disorganization, characterized by local disarray significantly dependent on the spatial scale of analysis. A virtual sample generator is developed to link this multi-scale disarray analysis with the underlying cellular architecture. This approach allowed us to quantitatively assess tissue organization in terms of 3D myocyte angular dispersion and may pave the way for developing novel predictive models based on structural data at cellular resolution., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Giardini, Lazzeri, Vitale, Ferrantini, Costantini, Pavone, Poggesi, Bocchi and Sacconi.)

The combination of tissue clearing techniques with advanced optical microscopy facilitates the achievement of three-dimensional (3D) reconstruction of macroscopic specimens at high resolution. Whole mouse organs or even bodies have been analyzed, while the reconstruction of the human nervous system remains a challenge. Although several tissue protocols have been proposed, the high autofluorescence and variable post-mortem conditions of human specimens negatively affect the quality of the images in terms of achievable transparency and staining contrast. Moreover, homogeneous staining of high-density epitopes, such as neuronal nuclear antigen (NeuN), creates an additional challenge. Here, we evaluated different tissue transformation approaches to find the best solution to uniformly clear and label all neurons in the human cerebral cortex using anti-NeuN antibodies in combination with confocal and light-sheet fluorescence microscopy (LSFM). Finally, we performed mesoscopic high-resolution 3D reconstruction of the successfully clarified and stained samples with LSFM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Scardigli, Pesce, Brady, Mazzamuto, Gavryusev, Silvestri, Hof, Destrieux, Costantini and Pavone.)

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

Current techniques for fast characterization of cardiac electrophysiology employ optical technologies to control and monitor action potential features of single cells or cellular monolayers placed in multiwell plates. High-speed investigation capacities are commonly achieved by serially analyzing well after well employing fully automated fluorescence microscopes. Here, we describe an alternative cost-effective optical approach (MULTIPLE) that exploits high-power LED arrays to globally illuminate a culture plate and an sCMOS sensor for parallel detection of the fluorescence coming from multiple wells. MULTIPLE combines optical detection of action potentials using a red-shifted voltage-sensitive fluorescent dye (di-4-ANBDQPQ) with optical stimulation, employing optogenetic actuators, to ensure excitation of cardiomyocytes at constant rates. MULTIPLE was first characterized in terms of interwell uniformity of the illumination intensity and optical detection performance. Then, it was applied for probing action potential features in HL-1 cells (i.e., mouse atrial myocyte-like cells) stably expressing the blue light-activatable cation channel CheRiff. Under proper stimulation conditions, we were able to accurately measure action potential dynamics across a 24-well plate with variability across the whole plate of the order of 10%. The capability of MULTIPLE to detect action potential changes across a 24-well plate was demonstrated employing the selective K v 11.1 channel blocker (E-4031), in a dose titration experiment. Finally, action potential recordings were performed in spontaneous beating human induced pluripotent stem cell derived cardiomyocytes following pharmacological manipulation of their beating frequency. We believe that the simplicity of the presented optical scheme represents a valid complement to sophisticated and expensive state-of-the-art optical systems for high-throughput cardiac electrophysiological investigations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Credi, Balducci, Munagala, Cianca, Bigiarini, de Vries, Loew, Pavone, Cerbai, Sartiani and Sacconi.)

Background: Colorectal cancer has a high incidence rate worldwide, with over 1.8 million new cases and 880,792 deaths in 2018. Fortunately, its early detection significantly increases the survival rate, reaching a cure rate of 90% when diagnosed at a localized stage. Colonoscopy is the gold standard technique for detection and removal of colorectal lesions with potential to evolve into cancer. When polyps are found in a patient, the current procedure is their complete removal. However, in this process, gastroenterologists cannot assure complete resection and clean margins which are given by the histopathology analysis of the removed tissue, which is performed at laboratory., Aims: In this paper, we demonstrate the capabilities of multiphoton microscopy (MPM) technology to provide imaging biomarkers that can be extracted by deep learning techniques to identify malignant neoplastic colon lesions and distinguish them from healthy, hyperplastic, or benign neoplastic tissue, without the need for histopathological staining., Materials and Methods: To this end, we present a novel MPM public dataset containing 14,712 images obtained from 42 patients and grouped into 2 classes. A convolutional neural network is trained on this dataset and a spatially coherent predictions scheme is applied for performance improvement., Results: We obtained a sensitivity of 0.8228 ± 0.1575 and a specificity of 0.9114 ± 0.0814 on detecting malignant neoplastic lesions. We also validated this approach to estimate the self-confidence of the network on its own predictions, obtaining a mean sensitivity of 0.8697 and a mean specificity of 0.9524 with the 18.67% of the images classified as uncertain., Conclusions: This work lays the foundations for performing in vivo optical colon biopsies by combining this novel imaging technology together with deep learning algorithms, hence avoiding unnecessary polyp resection and allowing in situ diagnosis assessment., Competing Interests: There are no conflicts of interest., (Copyright: © 2021 Journal of Pathology Informatics.)

We demonstrate the ability of nondestructive optical imaging techniques such as second-harmonic generation (SHG), two-photon fluorescence (TPF), fluorescence lifetime imaging (FLIM), and Raman spectroscopy (RS) to monitor biochemical and mechanical alterations in tissues upon collagen degradation. Decellularized equine pericardium (EP) was treated with 50 μg/mL bacterial collagenase at 37 °C for 8, 16, 24, and 32 h. The SHG ratio (defined as the normalized ratio between SHG and TPF signals) remained unchanged for untreated EP (stored in phosphate-buffered solution (PBS)), whereas treated EP showed a trend of a decreasing SHG ratio with increasing collagen degradation. In the fluorescence domain, treated EP experienced a red-shifted emission and the fluorescence lifetime had a trend of decreasing lifetime with increasing collagen digestion. RS monitors collagen degradation, the spectra had less intense Raman bands at 814, 852, 938, 1242, and 1270 cm -1 . Non-negative least-squares (NNLS) modeling quantifies collagen loss and relative increase of elastin. The Young's modulus, derived from atomic force microscope-based nanoindentation experiments, showed a rapid decrease within the first 8 h of collagen degradation, whereas more gradual changes were observed for optical modalities. We conclude that optical imaging techniques like SHG, RS, and FLIM can monitor collagen degradation in a label-free manner and coarsely access mechanical properties in a nondestructive manner.

Malignant melanoma is an aggressive form of skin cancer, which develops from the genetic mutations of melanocytes - the most frequent involving BRAF and NRAS genes. The choice and the effectiveness of the therapeutic approach depend on tumour mutation; therefore, its assessment is of paramount importance. Current methods for mutation analysis are destructive and take a long time; instead, Raman spectroscopy could provide a fast, label-free and non-destructive alternative. In this study, confocal Raman microscopy has been used for examining three in vitro melanoma cell lines, harbouring different molecular profiles and, in particular, specific BRAF and NRAS driver mutations. The molecular information obtained from Raman spectra has served for developing two alternative classification algorithms based on linear discriminant analysis and artificial neural network. Both methods provide high accuracy (≥90%) in discriminating all cell types, suggesting that Raman spectroscopy may be an effective tool for detecting molecular differences between melanoma mutations., (© 2020 Wiley-VCH GmbH.)

In recent years, photobiomodulation (PBM) has been recognized as a physical therapy in wound management. Despite several published research papers, the mechanism underlying photobiomodulation is still not completely understood. The investigation about application of blue light to improve wound healing is a relatively new research area. Tests in selected patients evidenced a stimulation of the healing process in superficial and chronic wounds treated with a blue LED light emitting at 420 nm; a study in animal model pointed out a faster healing process in superficial wound, with an important role of fibroblasts and myofibroblasts. Here, we present a study aiming at evidencing the effects of blue light on the proliferation and metabolism in fibroblasts from healthy skin and keratinocytes. Different light doses (3.43, 6.87, 13.7, 20.6, 30.9 and 41.2 J/cm2) were used to treat the cells, evidencing inhibitory and stimulatory effects following a biphasic dose behavior. Electrophysiology was used to investigate the effects on membrane currents: healthy fibroblasts and keratinocytes showed no significant differences between treated and not treated cells. Raman spectroscopy revealed the mitochondrial Cytochrome C (Cyt C) oxidase dependence on blue light irradiation: a significant decrease in peak intensity of healthy fibroblast was evidenced, while it is less pronounced in keratinocytes. In conclusion, we observed that the blue LED light can be used to modulate metabolism and proliferation of human fibroblasts, and the effects in wound healing are particularly evident when studying the fibroblasts and keratinocytes co-cultures.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.

Keloids are an exuberant response to wound healing, characterized by an exaggerated synthesis of collagen, probably due to the increase of fibroblasts activity and to the reduction of their apoptosis rate: currently no standard treatments or pharmacological therapies are able to prevent keloid recurrence. To reach this goal, in recent years some physical treatments have been proposed, and among them the PhotoBioModulation therapy (PBM). This work analyses the effects of a blue LED light irradiation (410-430 nm, 0.69 W/cm 2 power density) on human fibroblasts, isolated from both keloids and perilesional tissues. Different light doses (3.43-6.87-13.7-20.6-30.9 and 41.2 J/cm 2 ) were tested. Biochemical assays and specific staining were used to assess cell metabolism, proliferation and viability. Micro-Raman spectroscopy was used to explore direct effects of the blue LED light on the Cytochrome C (Cyt C) oxidase. We also investigated the effects of the irradiation on ionic membrane currents by patch-clamp recordings. Our results showed that the blue LED light can modulate cell metabolism and proliferation, with a dose-dependent behavior and that these effects persist at least till 48 h after treatment. Furthermore, we demonstrated that the highest fluence value can reduce cell viability 24 h after irradiation in keloid-derived fibroblasts, while the same effect is observed 48 h after treatment in perilesional fibroblasts. Electrophysiological recordings showed that the medium dose (20.6 J/cm 2 ) of blue LED light induces an enhancement of voltage-dependent outward currents elicited by a depolarizing ramp protocol. Overall, these data demonstrate the potentials that PBM shows as an innovative and minimally-invasive approach in the management of hypertrophic scars and keloids, in association with current treatments.

Visualizing neuronal activation on a brain-wide scale yet with cellular resolution is a fundamental technical challenge for neuroscience. This would enable analyzing how different neuronal circuits are disrupted in pathology and how they could be rescued by pharmacological treatments. Although this goal would have appeared visionary a decade ago, recent technological advances make it eventually feasible. Here, we review the latest developments in the fields of genetics, sample preparation, imaging, and image analysis that could be combined to afford whole-brain cell-resolution activation mapping. We show how the different biochemical and optical methods have been coupled to study neuronal circuits at different spatial and temporal scales, and with cell-type specificity. The inventory of techniques presented here could be useful to find the tools best suited for a specific experiment. We envision that in the next years, mapping of neuronal activation could become routine in many laboratories, allowing dissecting the neuronal counterpart of behavior., (Copyright © 2020 Franceschini, Costantini, Pavone and Silvestri.)

Significance: Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults. With a worldwide incidence rate of 2 to 3 per 100,000 people, it accounts for more than 60% of all brain cancers; currently, its 5-year survival rate is < 5 % . GBM treatment relies mainly on surgical resection. In this framework, multimodal optical spectroscopy could provide a fast and label-free tool for improving tumor detection and guiding the removal of diseased tissues. Aim: Discriminating healthy brain from GBM tissues in an animal model through the combination of Raman and reflectance spectroscopies. Approach: EGFP-GL261 cells were injected into the brains of eight laboratory mice for inducing murine GBM in these animals. A multimodal optical fiber probe combining fluorescence, Raman, and reflectance spectroscopy was used to localize in vivo healthy and tumor brain areas and to collect their spectral information. Results: Tumor areas were localized through the detection of EGFP fluorescence emission. Then, Raman and reflectance spectra were collected from healthy and tumor tissues, and later analyzed through principal component analysis and linear discriminant analysis in order to develop a classification algorithm. Raman and reflectance spectra resulted in 92% and 93% classification accuracy, respectively. Combining together these techniques allowed improving the discrimination between healthy and tumor tissues up to 97%. Conclusions: These preliminary results demonstrate the potential of multimodal fiber-probe spectroscopy for in vivo label-free detection and delineation of brain tumors, and thus represent an additional, encouraging step toward clinical translation and deployment of fiber-probe spectroscopy., (© 2020 The Authors.)

Being able to replicate real experiments with computational simulations is a unique opportunity to refine and validate models with experimental data and redesign the experiments based on simulations. However, since it is technically demanding to model all components of an experiment, traditional approaches to modeling reduce the experimental setups as much as possible. In this study, our goal is to replicate all the relevant features of an experiment on motor control and motor rehabilitation after stroke. To this aim, we propose an approach that allows continuous integration of new experimental data into a computational modeling framework. First, results show that we could reproduce experimental object displacement with high accuracy via the simulated embodiment in the virtual world by feeding a spinal cord model with experimental registration of the cortical activity. Second, by using computational models of multiple granularities, our preliminary results show the possibility of simulating several features of the brain after stroke, from the local alteration in neuronal activity to long-range connectivity remodeling. Finally, strategies are proposed to merge the two pipelines. We further suggest that additional models could be integrated into the framework thanks to the versatility of the proposed approach, thus allowing many researchers to achieve continuously improved experimental design., (Copyright © 2020 Allegra Mascaro, Falotico, Petkoski, Pasquini, Vannucci, Tort-Colet, Conti, Resta, Spalletti, Ramalingasetty, von Arnim, Formento, Angelidis, Blixhavn, Leergaard, Caleo, Destexhe, Ijspeert, Micera, Laschi, Jirsa, Gewaltig and Pavone.)

Aim: To investigate the action mechanism of 1-benzyl-1,4-diazepane (1-BD) as efflux pump inhibitor (EPI) in Escherichia coli mutants: Δ acrAB or overexpressing AcrAB and AcrEF efflux pumps. Materials & methods: Effect of 1-BD on: antibiotic potentiation, by microdilution method; membrane functionality, by fluorimetric assays; ethidium bromide accumulation, by fluorometric real-time efflux assay; AcrB expression, by quantitative photoactivated localization microscopy. Results: 1-BD decreases the minimal inhibitory concentration of levofloxacin and other antibiotics and increase ethidium bromide accumulation in E. coli overexpressing efflux pumps but not in the Δ acrAB strain. 1-BD increases membranes permeability, without sensibly affecting inner membrane polarity and decreases acrAB transcription. Conclusion: 1-BD acts as an EPI in E. coli with a mixed mechanism, different from that of major reference EPIs.

Autofluorescence spectroscopy has emerged in recent years as a powerful tool to report label-free contrast between normal and diseased tissues, both in vivo and ex vivo. We report the development of an instrument employing Single Photon Avalanche Diode (SPAD) arrays to realize real-time multispectral autofluorescence lifetime imaging at a macroscopic scale using handheld single-point fibre optic probes, under bright background conditions. At the detection end, the fluorescence signal is passed through a transmission grating and both spectral and temporal information are encoded in the SPAD array. This configuration allows interrogation in the spectral range of interest in real time. Spatial information is provided by an external camera together with a guiding beam that provides a visual reference that is tracked in real-time. Through fast image processing and data analysis, fluorescence lifetime maps are augmented on white light images to provide feedback of the measurements in real-time. We validate and demonstrate the practicality of this technique in the reference fluorophores and in articular cartilage samples mimicking the degradation that occurs in osteoarthritis. Our results demonstrate that SPADs together with fibre probes can offer means to report autofluorescence spectral and lifetime contrast in real-time and thus are suitable candidates for in situ tissue diagnostics.

Time-correlated single photon counting is the "gold-standard" method for fluorescence lifetime measurements and has demonstrated potential for clinical deployment. However, the translation of the technology into clinic is hindered by the use of ultrasensitive detectors, which make the fluorescence acquisition impractical with bright lighting conditions such as in clinical settings. We address this limitation by interleaving periodic fluorescence detection with synchronous out-of-phase externally modulated light source, thus guaranteeing specimen illumination and a fluorescence signal free from bright background light upon temporal separation. Fluorescence lifetime maps are generated in real-time from single-point measurements by tracking a reference beam and using the phasor approach. We demonstrate the feasibility and practicality of this technique in a number of biological specimens, including real-time mapping of degraded articular cartilage. This method is compatible and can be integrated with existing clinical microscopic, endoscopic and robotic modalities, thus offering a new pathway towards label-free diagnostics and surgical guidance in a number of clinical applications., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Urothelial carcinoma (UC) is the most common bladder tumour. Proper treatment requires tumour resection for diagnosing its grade (aggressiveness) and stage (invasiveness). White-light cystoscopy and histopathological examination are the gold standard procedures for clinical and histopathological diagnostics, respectively. However, cystoscopy is limited in terms of specificity, histology requires long tissue processing, both procedures rely on operator's experience. Multimodal optical spectroscopy can provide a powerful tool for detecting, staging and grading bladder tumours in a fast, reliable and label-free modality. In this study, we collected fluorescence, Raman and reflectance spectra from 50 biopsies obtained from 32 patients undergoing transurethral resection of bladder tumour using a multimodal fibre-probe. Principal component analysis allowed distinguishing normal from pathological tissues, as well as discriminating tumour stages and grades. Each individual spectroscopic technique provided high specificity and sensitivity in classifying all tissues; however, a multimodal approach resulted in a considerable increase in diagnostic accuracy (≥95%), which is of paramount importance for tumour grading and staging. The presented method offers the potential for being applied in cystoscopy and for providing an automated diagnosis of UC at the clinical level, with an improvement with respect to current state-of-the-art procedures., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) has been established as a gold standard method to improve image quality. The selective line detection of a complementary metal–oxide–semiconductor camera (CMOS) working in rolling shutter mode allows the rejection of out-of-focus and scattered light, thus reducing background signal during image formation. Most modern CMOS have two rolling shutters, but usually only a single illuminating beam is used, halving the maximum obtainable frame rate. We report on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acousto-optic deflector. Such a simple solution enables us to independently generate, control and synchronize two beams with the two rolling slits on the camera. We show that the doubling of the imaging speed does not affect the confocal detection high contrast.

Key Points: The heart is innervated by a dense sympathetic neuron network which, in the short term, controls chronotropy and inotropy and, in the long term, regulates cardiomyocyte size. Acute neurogenic control of heart rate is achieved locally through direct neuro-cardiac coupling at specific junctional sites (neuro-cardiac junctions). The ventricular sympathetic network topology is well-defined and characteristic for each mammalian species. In the present study, we used cell size regulation to determine whether long-term modulation of cardiac structure is achieved via direct sympatho-cardiac coupling. Local density of cardiac innervation correlated with cell size throughout the myocardial walls in all mammalian species analysed, including humans. The data obtained suggest that constitutive neurogenic control of cardiomyocyte trophism occurs through direct intercellular signalling at neuro-cardiac junctions., Abstract: It is widely appreciated that sympathetic stimulation of the heart involves a sharp increase in beating rate and significant enhancement of contractility. We have previously shown that, in addition to these evident functions, sympathetic neurons (SNs) also provide trophic input to cardiomyocytes (CMs), regulating cell and organ size. More recently, we have demonstrated that cardiac neurons establish direct interactions with CMs, allowing neuro-cardiac communication to occur locally, with a 'quasi-synaptic' mechanism. Based on the evidence that cardiac SNs are unevenly distributed throughout the myocardial walls, we investigated the hypothesis that CM size distribution reflects the topology of neuronal density. In vitro analyses of SN/CM co-cultures, ex vivo confocal and multiphoton imaging in clarified hearts, and biochemical and molecular approaches were employed, in both rodent and human heart biopsies. In line with the trophic effect of SNs, and with local neuro-cardiac communication, CMs, directly contacted by SNs in co-cultures, were larger than the non-targeted ones. This property reflects the distribution of CM size throughout the ventricles of intact mouse heart, in which cells in the outer myocardial layers, which were contacted by more neuronal processes, were larger than those in the less innervated subendocardial region. Such differences disappeared upon genetic or pharmacological interference with the trophic SN/CM signalling axis. Remarkably, CM size followed the SN distribution pattern in other mammals, including humans. Our data suggest that both the acute and chronic influence of SNs on cardiac function and structure is enacted as a result of the establishment of specific intercellular neuro-cardiac junctions., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Rationale: Despite major advances in cardiovascular medicine, heart disease remains a leading cause of death worldwide. However, the field of tissue engineering has been growing exponentially in the last decade and restoring heart functionality is now an affordable target; yet, new materials are still needed for effectively provide rapid and long-lasting interventions. Liquid crystalline elastomers (LCEs) are biocompatible polymers able to reversibly change shape in response to a given stimulus and generate movement. Once stimulated, LCEs can produce tension or movement like a muscle. However, so far their application in biology was limited by slow response times and a modest possibility to modulate tension levels during activation., Objective: To develop suitable LCE-based materials to assist cardiac contraction., Methods and Results: Thanks to a quick, simple, and versatile synthetic approach, a palette of biocompatible acrylate-based light-responsive LCEs with different molecular composition was prepared and mechanically characterized. Out of this, the more compliant one was selected. This material was able to contract for some weeks when activated with very low light intensity within a physiological environment. Its contraction was modulated in terms of light intensity, stimulation frequency, and t on /t off ratio to fit different contraction amplitude/time courses, including those of the human heart. Finally, LCE strips were mounted in parallel with cardiac trabeculae, and we demonstrated their ability to improve muscular systolic function, with no impact on diastolic properties., Conclusions: Our results indicated LCEs are promising in assisting cardiac mechanical function and developing a new generation of contraction assist devices.

[This corrects the article DOI: 10.3389/fncel.2018.00315.].

Optogenetics is an emerging method that uses light to manipulate electrical activity in excitable cells exploiting the interaction between light and light-sensitive depolarizing ion channels, such as channelrhodopsin-2 (ChR2). Initially used in the neuroscience, it has been adopted in cardiac research where the expression of ChR2 in cardiac preparations allows optical pacing, resynchronization and defibrillation. Recently, optogenetics has been leveraged to manipulate cardiac electrical activity in the intact heart in real-time. This new approach was applied to simulate a re-entrant circuit across the ventricle. In this technical note, we describe the development and the implementation of a new software package for real-time optogenetic intervention. The package consists of a single LabVIEW program that simultaneously captures images at very high frame rates and delivers precisely timed optogenetic stimuli based on the content of the images. The software implementation guarantees closed-loop optical manipulation at high temporal resolution by processing the raw data in workstation memory. We demonstrate that this strategy allows the simulation of a ventricular tachycardia with high stability and with a negligible loss of data with a temporal resolution of up to 1 ms., Competing Interests: The authors declare no conflict of interest.

Light-sheet microscopy (LSM) has proven a useful tool in neuroscience to image whole brains with high frame rates at cellular resolution and, in combination with tissue clearing methods, is often employed to reconstruct the cyto-architecture over the intact mouse brain. Inherently to LSM, however, residual opaque objects, always present to some extent even in extremely well optically cleared samples, cause stripe artifacts, which, in the best case, severely affect image homogeneity and, in the worst case, completely obscure features of interest. Here, demonstrating two example applications in intact optically cleared mouse brains, we report how Bessel beams reduce streaking artifacts and produce high-fidelity structural data for the brain-wide morphology of neuronal and vascular networks. We found that a third of the imaged volume of the brain was affected by strong striated image intensity inhomogeneity and, furthermore, a significant amount of information content lost with Gaussian illumination was accessible when interrogated with Bessel beams. In conclusion, Bessel beams produce high-fidelity structural data of improved image homogeneity and might significantly relax demands placed on the automated tools to count, trace, or segment fluorescent features of interest.

Coronary heart disease is the most common type of heart disease caused by atherosclerosis. In fact, an arterial wall lesion centered on the accumulation of cholesterol-rich lipids and the accompanying inflammatory response generates a plaque, whose rupture may result in a thrombus with fatal consequences. Plaque characterization for assessing the severity of atherosclerosis is generally performed through standard histopathological examination based on hematoxylin/eosin staining, which is operator-dependent and requires relatively long procedures. In this framework, nonlinear optical microscopy is a valid, label-free alternative to standard diagnostic methods. We combined second-harmonic generation (SHG), two-photon excited fluorescence (TPEF) and fluorescence lifetime imaging microscopy in a multimodal scheme for obtaining morphological and molecular information on human carotid ex vivo specimens affected by atherosclerosis. In this study, discrimination between different tissues within the atherosclerotic plaque was achieved based on both lifetime, TPEF-to-SHG ratio, and image pattern analysis. The presented methodology aims to be a starting point for future fully automated and fast characterization of atherosclerotic biopsies; moreover, it could be extended to the study of other tissues and pathologies. Combined TPEF/SHG mapping of a carotid specimen affected by atherosclerosis., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Asynchronous Ca 2+ release promotes non-homogeneous myofilament activation, leading to mechanical dysfunction, as well as initiation of propagated calcium waves and arrhythmias. Recent advances in microscopy techniques have allowed for optical recordings of local Ca 2+ fluxes and action potentials from multiple sub-cellular domains within cardiac cells with unprecedented spatial and temporal resolution. Since then, sub-cellular local information of the spatio-temporal relationship between Ca 2+ release and action potential propagation have been unlocked, providing novel mechanistic insights in cardiac excitation-contraction coupling (ECC). Here, we review the promising perspectives arouse from repeatedly probing Ca 2+ release at the same sub-cellular location while simultaneously probing multiple locations at the same time within a single cardiac cell. We also compare the results obtained in three different rodent models of cardiac diseases, highlighting disease-specific mechanisms. Slower local Ca 2+ release has been observed in regions with defective action potential conduction in diseased cardiac cells. Moreover, significant increment of Ca 2+ variability (both in time and in space) has been found in diseased cardiac cells but does not directly correlate with local electrical defects nor with the degree of structural aberrations of the cellular membrane system, suggesting a role for other players of the ECC machinery. We finally explore exciting opportunities provided by the technology for studying different cardiomyocyte populations, as well as for dissecting the mechanisms responsible for subcellular spatio-temporal variability of Ca 2+ release.

Rationale: Recently, abundant axial tubule (AT) membrane structures were identified deep inside atrial myocytes (AMs). Upon excitation, ATs rapidly activate intracellular Ca 2+ release and sarcomeric contraction through extensive AT junctions, a cell-specific atrial mechanism. While AT junctions with the sarcoplasmic reticulum contain unusually large clusters of ryanodine receptor 2 (RyR2) Ca 2+ release channels in mouse AMs, it remains unclear if similar protein networks and membrane structures exist across species, particularly those relevant for atrial disease modeling. Objective: To examine and quantitatively analyze the architecture of AT membrane structures and associated Ca 2+ signaling proteins across species from mouse to human. Methods and Results: We developed superresolution microscopy (nanoscopy) strategies for intact live AMs based on a new custom-made photostable cholesterol dye and immunofluorescence imaging of membraneous structures and membrane proteins in fixed tissue sections from human, porcine, and rodent atria. Consistently, in mouse, rat, and rabbit AMs, intact cell-wide tubule networks continuous with the surface membrane were observed, mainly composed of ATs. Moreover, co-immunofluorescence nanoscopy showed L-type Ca 2+ channel clusters adjacent to extensive junctional RyR2 clusters at ATs. However, only junctional RyR2 clusters were highly phosphorylated and may thus prime Ca 2+ release at ATs, locally for rapid signal amplification. While the density of the integrated L-type Ca 2+ current was similar in human and mouse AMs, the intracellular Ca 2+ transient showed quantitative differences. Importantly, local intracellular Ca 2+ release from AT junctions occurred through instantaneous action potential propagation via transverse tubules (TTs) from the surface membrane. Hence, sparse TTs were sufficient as electrical conduits for rapid activation of Ca 2+ release through ATs. Nanoscopy of atrial tissue sections confirmed abundant ATs as the major network component of AMs, particularly in human atrial tissue sections. Conclusion: AT junctions represent a conserved, cell-specific membrane structure for rapid excitation-contraction coupling throughout a representative spectrum of species including human. Since ATs provide the major excitable membrane network component in AMs, a new model of atrial "super-hub" Ca 2+ signaling may apply across biomedically relevant species, opening avenues for future investigations about atrial disease mechanisms and therapeutic targeting.

Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a method of choice to observe brain function with high frame rates at cellular resolution. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20-fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams.

Myosin-5B is one of three members of the myosin-5 family of actin-based molecular motors. Despite its fundamental role in recycling endosome trafficking and in collective actin network dynamics, the molecular mechanisms underlying its motility are inherently unknown. Here we combine single-molecule imaging and high-speed laser tweezers to dissect the mechanoenzymatic properties of myosin-5B. We show that a single myosin-5B moves processively in 36-nm steps, stalls at ~2 pN resistive forces, and reverses its directionality at forces >2 pN. Interestingly, myosin-5B mechanosensitivity differs from that of myosin-5A, while it is strikingly similar to kinesin-1. In particular, myosin-5B run length is markedly and asymmetrically sensitive to force, a property that might be central to motor ensemble coordination. Furthermore, we show that Ca 2+ does not affect the enzymatic activity of the motor unit, but abolishes myosin-5B processivity through calmodulin dissociation, providing important insights into the regulation of postsynaptic cargoes trafficking in neuronal cells.

In any living cell, genome maintenance is carried out by DNA-binding proteins that recognize specific sequences among a vast amount of DNA. This includes fundamental processes such as DNA replication, DNA repair, and gene expression and regulation. Here, we study the mechanism of DNA target search by a single lac repressor protein (LacI) with ultrafast force-clamp spectroscopy, a sub-millisecond and few base-pair resolution technique based on laser tweezers. We measure 1D-diffusion of proteins on DNA at physiological salt concentrations with 20 bp resolution and find that sliding of LacI along DNA is sequence dependent. We show that only allosterically activated LacI slides along non-specific DNA sequences during target search, whereas the inhibited conformation does not support sliding and weakly interacts with DNA. Moreover, we find that LacI undergoes a load-dependent conformational change when it switches between sliding and strong binding to the target sequence. Our data reveal how DNA sequence and molecular switching regulate LacI target search process and provide a comprehensive model of facilitated diffusion for LacI.

Time traces obtained from a variety of biophysical experiments contain valuable information on underlying processes occurring at the molecular level. Accurate quantification of these data can help explain the details of the complex dynamics of biological systems. Here, we describe PLANT (Piecewise Linear Approximation of Noisy Trajectories), a segmentation algorithm that allows the reconstruction of time-trace data with constant noise as consecutive straight lines, from which changes of slopes and their respective durations can be extracted. We present a general description of the algorithm and perform extensive simulations to characterize its strengths and limitations, providing a rationale for the performance of the algorithm in the different conditions tested. We further apply the algorithm to experimental data obtained from tracking the centroid position of lymphocytes migrating under the effect of a laminar flow and from single myosin molecules interacting with actin in a dual-trap force-clamp configuration., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)