Grating-Coupled Surface Plasmon Resonance (GC-SPR) Optimization for Phase-Interrogation Biosensing in a Microfluidic Chamber.

Surface Plasmon Resonance (SPR)-based sensors have the advantage of being label-free, enzyme-free and real-time. However, their spreading in multidisciplinary research is still mostly limited to prism-coupled devices. Plasmonic gratings, combined with a simple and cost-effective instrumentation, have been poorly developed compared to prism-coupled system mainly due to their lower sensitivity. Here we describe the optimization and signal enhancement of a sensing platform based on phase-interrogation method, which entails the exploitation of a nanostructured sensor. This technique is particularly suitable for integration of the plasmonic sensor in a lab-on-a-chip platform and can be used in a microfluidic chamber to ease the sensing procedures and limit the injected volume. The careful optimization of most suitable experimental parameters by numerical simulations leads to a 30–50% enhancement of SPR response, opening new possibilities for applications in the biomedical research field while maintaining the ease and versatility of the configuration

Multiphoton Imaging of Ca2+ Instability in Acute Myocardial Slices from a RyR2R2474S Murine Model of Catecholaminergic Polymorphic Ventricular Tachycardia

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a familial stress-induced arrhythmia syndrome, mostly caused by mutations in Ryanodine receptor 2 (RyR2), the sarcoplasmic reticulum (SR) Ca2+ release channel in cardiomyocytes. Pathogenetic mutations lead to gain of function in the channel, causing arrhythmias by promoting diastolic spontaneous Ca2+ release (SCR) from the SR and delayed afterdepolarizations. While the study of Ca2+ dynamics in single cells from murine CPVT models has increased our understanding of the disease pathogenesis, questions remain on the mechanisms triggering the lethal arrhythmias at tissue level. Here, we combined subcellular analysis of Ca2+ signals in isolated cardiomyocytes and in acute thick ventricular slices of RyR2R2474S knock-in mice, electrically paced at different rates (1-5 Hz), to identify arrhythmogenic Ca2+ dynamics, from the sub- to the multicellular perspective. In both models, RyR2R2474S cardiomyocytes had increased propensity to develop SCR upon adrenergic stimulation, which manifested, in the slices, with Ca2+ alternans and synchronous Ca2+ release events in neighboring cardiomyocytes. Analysis of Ca2+ dynamics in multiple cells in the tissue suggests that SCRs beget SCRs in contiguous cells, overcoming the protective electrotonic myocardial coupling, and potentially generating arrhythmia triggering foci. We suggest that intercellular interactions may underscore arrhythmic propensity in CPVT hearts with 'leaky' RyR2

Multiphoton Label-Free ex-vivo imaging using a custom-built dual-wavelength microscope with chromatic aberrations compensation

Label-Free Multiphoton Microscopy is a very powerful optical microscopy that can be applied to study samples with no need for exogenous fluorescent probes, keeping the main benefits of a Multiphoton approach, like longer penetration depths and intrinsic optical sectioning, while opening the possibility of serial examinations with different kinds of techniques. Among the many variations of Label-Free MPM, Higher Harmonic Generation (HHG) is one of the most intriguing due to its generally low photo-toxicity, which enables the examination of specimens particularly susceptible to photo-damages. HHG and common Two-Photon Microscopy (TPM) are well-established techniques, routinely used in several research fields. However, they require a significant amount of fine-tuning in order to be fully exploited and, usually, the optimized conditions greatly differ, making them quite difficult to perform in parallel without any compromise on the extractable information. Here we present our custom-built Multiphoton microscope capable of performing simultaneously TPM and HHG without any kind of compromise on the results thanks to two, separate, individually optimized laser sources with full chromatic aberration compensation. We also apply our setup to the examination of a plethora of ex vivo samples in order to prove the significant advantages of our approach

Optogenetic determination of the myocardial requirements for extrasystoles by cell type-specific targeting of ChannelRhodopsin-2

Extrasystoles lead to several consequences, ranging from uneventful palpitations to lethal ventricular arrhythmias, in the presence of pathologies, such as myocardial ischemia. The role of working versus conducting cardiomyocytes, as well as the tissue requirements (minimal cell number) for the generation of extrasystoles, and the properties leading ectopies to become arrhythmia triggers (topology), in the normal and diseased heart, have not been determined directly in vivo. Here, we used optogenetics in transgenic mice expressing ChannelRhodopsin-2 selectively in either cardiomyocytes or the conduction system to achieve cell type-specific, noninvasive control of heart activity with high spatial and temporal resolution. By combining measurement of optogenetic tissue activation in vivo and epicardial voltage mapping in Langendorff-perfused hearts, we demonstrated that focal ectopies require, in the normal mouse heart, the simultaneous depolarization of at least 1,300–1,800 working cardiomyocytes or 90–160 Purkinje fibers. The optogenetic assay identified specific areas in the heart that were highly susceptible to forming extrasystolic foci, and such properties were correlated to the local organization of the Purkinje fiber network, which was imaged in three dimensions using optical projection tomography. Interestingly, during the acute phase of myocardial ischemia, focal ectopies arising from this location, and including both Purkinje fibers and the surrounding working cardiomyocytes, have the highest propensity to trigger sustained arrhythmias. In conclusion, we used cell-specific optogenetics to determine with high spatial resolution and cell type specificity the requirements for the generation of extrasystoles and the factors causing ectopies to be arrhythmia triggers during myocardial ischemia

Customized bioreactor enables the production of 3D diaphragmatic constructs influencing matrix remodeling and fibroblast overgrowth

The production of skeletal muscle constructs useful for replacing large defects in vivo, such as in congenital diaphragmatic hernia (CDH), is still considered a challenge. The standard application of prosthetic material presents major limitations, such as hernia recurrences in a remarkable number of CDH patients. With this work, we developed a tissue engineering approach based on decellularized diaphragmatic muscle and human cells for the in vitro generation of diaphragmatic-like tissues as a proof-of-concept of a new option for the surgical treatment of large diaphragm defects. A customized bioreactor for diaphragmatic muscle was designed to control mechanical stimulation and promote radial stretching during the construct engineering. In vitro tests demonstrated that both ECM remodeling and fibroblast overgrowth were positively influenced by the bioreactor culture. Mechanically stimulated constructs also increased tissue maturation, with the formation of new oriented and aligned muscle fibers. Moreover, after in vivo orthotopic implantation in a surgical CDH mouse model, mechanically stimulated muscles maintained the presence of human cells within myofibers and hernia recurrence did not occur, suggesting the value of this approach for treating diaphragm defects

A Comprehensive Comparison of Bovine and Porcine Decellularized Pericardia: New Insights for Surgical Applications

Xenogeneic pericardium-based substitutes are employed for several surgical indications after chemical shielding, limiting their biocompatibility and therapeutic durability. Adverse responses to these replacements might be prevented by tissue decellularization, ideally removing cells and preserving the original extracellular matrix (ECM). The aim of this study was to compare the mostly applied pericardia in clinics, i.e. bovine and porcine tissues, after their decellularization, and obtain new insights for their possible surgical use. Bovine and porcine pericardia were submitted to TRICOL decellularization, based on osmotic shock, detergents and nuclease treatment. TRICOL procedure resulted in being effective in cell removal and preservation of ECM architecture of both species' scaffolds. Collagen and elastin were retained but glycosaminoglycans were reduced, significantly for bovine scaffolds. Tissue hydration was varied by decellularization, with a rise for bovine pericardia and a decrease for porcine ones. TRICOL significantly increased porcine pericardial thickness, while a non-significant reduction was observed for the bovine counterpart. The protein secondary structure and thermal denaturation profile of both species' scaffolds were unaltered. Both pericardial tissues showed augmented biomechanical compliance after decellularization. The ECM bioactivity of bovine and porcine pericardia was unaffected by decellularization, sustaining viability and proliferation of human mesenchymal stem cells and endothelial cells. In conclusion, decellularized bovine and porcine pericardia demonstrate possessing the characteristics that are suitable for the creation of novel scaffolds for reconstruction or replacement: differences in water content, thickness and glycosaminoglycans might influence some of their biomechanical properties and, hence, their indication for surgical use

Study of the tissue determinants of cardiac arrhythmias with novel biophysical approaches

Alterations in cardiomyocyte Ca2+ handling initiate and sustain arrhythmias. Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), is an inherited arrhythmogenic disease due to a gain-of-function mutation of RyR2 Ca2+ channels, causing Ca2+ leak from the sarcoplasmic reticulum and abnormal membrane depolarization. How these events are linked to the onset of tissue-wide arrhythmias remains unclear. Ca2+ signaling was investigated in a multicellular model of cardiac tissue, by using a multispot multiphoton microscope that unmasked fast occurring Ca2+ dependent signals. The multicellular model allowed to visualize Ca2+ alternans and synchronized spontaneous Ca2+ releases in CPVT. Such synchronization, would be necessary to overcome the source-sink mismatch in the heart and generate ectopic foci of depolarization that serve as arrhythmia trigger. We thus used optogenetics to calculate the critical mass (<2000 cells) necessary to form such ectopic foci and, with the same method we mimicked the bidirectional ventricular tachycardias characteristic of the clinical manifestation of CPVT.Alterazioni nell’omeostasi del Ca2 + nei cardiomiociti possono avviare e sostenere aritmie. La tachicardia catecolaminergica polimorfica ventricolare (CPVT), è una malattia aritmogenica ereditaria dovuta ad una mutazione dei canali RyR2 del Ca2 +, causando un’incontrollata fuoriuscita di Ca2+ dal reticolo sarcoplasmatico e un’anormale depolarizzazione della membrana. Come questi eventi siano legati alla comparsa di aritmie a livello del tessuto rimane poco chiaro. Il signaling del Ca2 + è stato studiato in un modello multicellulare di tessuto cardiaco, utilizzando un microscopio multi fotone a multipunto che ha smascherato i veloci segnali dipendenti dal Ca2 +. Il modello multicellulare ha permesso di visualizzare alternans di Ca2 + e la sincronizzazione dei rilasci spontanei di Ca2 + in modelli di CPVT. Tale sincronizzazione è necessaria per superare il mismatch source-sink nel cuore e generare foci ectopici di depolarizzazione che servono come innesco aritmico. Abbiamo usato inoltre l’optogenetica per calcolare la massa critica (<2000 cellule) necessaria per formare tali foci ectopici e, con lo stesso metodo abbiamo imitato le tachicardie ventricolari bidirezionali caratteristiche della manifestazione clinica della CPVT

Label-Free Multiphoton Microscopy: Much More Than Fancy Images

Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment. Afterwards, we provide a detailed review analysis of the main quantitative methods to extract important information and parameters from acquired images using second harmonic generation. Lastly, we discuss advantages, limitations, and future perspectives in label-free multiphoton microscopy

Label-Free Multiphoton Microscopy: Much More Than Fancy Images

Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment. Afterwards, we provide a detailed review analysis of the main quantitative methods to extract important information and parameters from acquired images using second harmonic generation. Lastly, we discuss advantages, limitations, and future perspectives in label-free multiphoton microscopy