Bead Mediated Microscopy: from high resolution microscopy to nano-Raman

Solid-state physics, material science, as well as biology, need continuously more and more information from their samples. High spatial resolution information such as optical or electrical properties, chemical species identification as well as topography are important information that optical microscopy or Scanning Probe Microscopy (SPM) can provide. Although electron microscopy (SEM and TEM) certainly assumes a position of absolute importance in the field, its cost and its need to be used by highly specialised personnel still make it an instrument of limited everyday use. On the contrary, probe microscopy has now become of very high diffusion in research labs. To develop my thesis I focused myself on three main and somehow related microscopy techniques: high resolution Raman microscopy, Scanning Near-field Optical Microscopy (SNOM), and Tip Enhanced Raman Spectroscopy (TERS). All of them are state-of-the-art on surface optical analysis techniques but still present relevant limits; among others, respectively: spatial resolution, local power density, complexity and field of applicability. My approach wants to combine some aspects of these techniques to go beyond their limits. Raman spectroscopy is a powerful optical technique, which measures the inelastic scattering of an incoming EM radiation due to the vibrational modes of the molecules present on the surface of a sample. Thanks to its high specificity, it is very powerful in identifying the chemical components of a sample. Several organic and inorganic molecules have their typical Raman spectral peaks, hence, by the Raman spectra, it\u2019s possible to provide a qualitative and quantitative analysis of the elements of a sample. High spatial resolution Raman setups uses the combination of a confocal microscope with a spectrometer assisted by a series of long pass and band pass filters. Despite its extreme versatility, basing Raman spectroscopy on a confocal system also constrains it to acquire its limit in spatial resolution determined by the limit of diffraction. To overcome this limit the most used techniques in SPM are Scanning Near-field Optical Microscopy (SNOM) and Tip Enhanced Raman Spectroscopy (TERS). Both of them exploits evanescent field, which is an electric field that is created by oscillating charges and/or currents and does not propagate in the far field as a classical electromagnetic wave, but is spatially concentrated very near to its source. This confinement allows to obtain field sources definitely smaller than in confocal systems. In SNOM technique, the excitation light is focused through an aperture smaller than the wavelength, creating an evanescent field strongly localized near the aperture itself. Scanning the sample in this near range brings the spatial resolution down to the aperture dimension. The main disadvantage of aperture SNOM is that the overall optical efficiency of probes is very low. The excitation power cannot be too high in order to prevent any damage of the probe, hence the energy that reaches the sample is usually not enough for Raman analysis. TERS instead is more suitable for this purpose. It basically exploits Surface Enhanced Raman Spectroscopy (SERS) principles, using a laser irradiated gold sharp tip to obtain a local enhancement at its apex. Its good efficiency permits to analyze Raman effects with a spatial super-resolution, but, on the other hand, TERS probes usually lack of reprodubility and require very skilled and specialised users. My PhD project has been focused to investigate and optimize an original approach to perform high resolution optical microscopy and Raman spectroscopy, well below the diffraction limit. The concept is to exploit the optical proprieties of a dielectric micro bead lens to achieve a powerful nanoscale near field confinement of light and the Scanning Probe Microscopy (SPM) technique to scan a sample to acquire optical maps. When a dielectric micro bead is hit by an Electromagnetic (EM) wave its effect is to transmit and concentrate the incident EM radiation in a specific area called nanojet, at first glance similar to that created with a standard lens. Some optical proprieties of the nanojets have been already introduced in the literature, but their application in the world of SPM, their employment in Raman microscopy and their combination with nanostructures to improve the spatial resolution are novel features whose investigation is promising. I gave to this technique the name of Beam Mediated Microscopy (BeMM). The combination of super resolution bead mediated SPM with Raman spectroscopy opens interesting perspectives about powerful surface analysis for samples that need a versatile optical probe with a high spatial resolution and soft interaction with the sample, like soft matter substrates or biosamples

Restrictive cardiomyopathy and hypertrophic cardiomyopathy overlap: the importance of the phenotype

Restrictive cardiomyopathy (RCM) is defined on the basis of the haemodynamic finding of restrictive ventricular physiology. However, restrictive ventricular pathophysiology is also a feature of other subtypes of cardiomyopathy, including hypertrophic cardiomyopathy (HCM). Clinically and aetiologically, there is an overlap between RCM and HCM with restrictive physiology. However, the clinical distinction between these two entities can be an important pointer towards the underlying aetiology. This review highlights the importance of the recognition of the clinical phenotype as the first step in the classification of cardiomyopathies

Antithrombotic Management during Percutaneous Mitral Valve Repair with the Mitraclip System in a Patient with Heparin-Induced Thrombocytopenia

Interventional cardiology procedures require full anticoagulation to prevent thrombus formation on catheters and devices with potential development of embolic complications. Bivalirudin, a short half-life direct thrombin inhibitor, has been largely used during percutaneous coronary interventions and represents the preferred alternative to heparin in patients with heparin-induced thrombocytopenia (HIT). However, few data are available about intraprocedural use of bivalirudin during transcatheter structural heart disease interventions. Activated clotting time (ACT) monitoring during bivalirudin infusion pre- sents some limitations and it is not mandatory. We report a case of bivalirudin use in a patient with type-2 HIT during percutaneous mitral valve repair with the Mitraclip system (Abbott, Abbott Park, Illinois, United States). Despite use of standard bivalirudin dose (0.75 mg/kg bolus and 1.4 mg/kg/min infusion—reduced infusion rate was motivated by a glomerular filtration rate of 37 mL/min), the patient developed a large thrombus on the second clip during its orientation toward the mitral orifice. ACT was measured at that time and was suboptimal (240 seconds). The case was successfully managed with clip and thrombus retrieval, adjunctive 0.3 mg/kg bivalirudin bolus and increased infusion rate, and clip repositioning with ACT monitoring. This report makes the case for mandatory ACT checking and drug titration during high-risk catheter–based structural heart disease interventions, even when thromboprophylaxis is performed with bivalirudin. Additional coagulation tests may be useful to monitor bivalirudin response in similar cases

Novel α-actin gene mutation p.(ala21val) causing familial hypertrophic cardiomyopathy, myocardial noncompaction, and transmural crypts. clinical-pathologic correlation

.Background: Mutations of α-actin gene (ACTC1) have been phenotypically related to various cardiac anomalies, including hypertrophic cardiomyopathy and dilated cardiomyopathy and left ventricular (LV) myocardial noncompaction. A novel ACTC mutation is reported as cosegregating for familial hypertrophic cardiomyopathy and LV myocardial noncompaction with transmural crypts. Methods and results: In an Italian family of 7 subjects, 4 aged 10 (II-1), 14 (II-2), 43 (I-4) and 46 years (I-5), presenting abnormal ECG changes, dyspnea and palpitation (II-2, I-4, and I-5), and recurrent cerebral ischemic attack (I-5), underwent 2-dimensional echo, cardiac magnetic resonance, Holter monitoring, and next-generation sequencing gene analysis. Patients II-2 and I-5 with ventricular tachycardia underwent a cardiac invasive study, including coronary with LV angiography and endomyocardial biopsy. In all the affected members, ECG showed right bundle branch block and left anterior hemiblock with age-related prolongation of QRS duration. Two-dimensional echo and cardiac magnetic resonance documented LV myocardial noncompaction in all and in I-4, I-5, and II-2 a progressive LV hypertrophy up to 22-mm maximal wall thickness. Coronary arteries were normal. LV angiography showed transmural crypts progressing to spongeous myocardial transformation with LV dilatation and dysfunction in the oldest subject. At histology and electron microscopy detachment of myocardiocytes were associated with cell and myofibrillar disarray and degradation of intercalated discs causing disanchorage of myofilaments to cell membrane. Next-generation sequencing showed in affected members an unreported p.(Ala21Val) mutation of ACTC. Conclusions: Novel p.(Ala21Val) mutation of ACTC1 causes myofibrillar and intercalated disc alteration leading to familial hypertrophic cardiomyopathy and LV myocardial noncompaction with transmural crypt

Dilated-Hypokinetic Evolution of Hypertrophic Cardiomyopathy Prevalence, Incidence, Risk Factors, and Prognostic Implications in Pediatric and Adult Patients

ObjectivesThis study sought to investigate the incidence, risk factors, and prognosis of dilated-hypokinetic evolution in a large cohort of patients with hypertrophic cardiomyopathy (HCM) followed up at a cardiology center serving both the pediatric and the adult population.BackgroundThe available data on this evolution of HCM mainly regards prevalence (rather than incidence) in adults, with very little being known about the pediatric population.MethodsA total of 222 consecutive HCM patients (65% men, 19% ≤18 years old) were prospectively evaluated for a mean follow-up of 11 ± 9 years.ResultsA diagnosis of dilated-hypokinetic HCM was made in 12 patients at first evaluation (11 without previous septal myectomy surgery; prevalence, 4.9%). Twelve of the 210 patients with classic HCM at first evaluation underwent dilated-hypokinetic evolution (incidence, 5.3/1,000 patient-years). Patients with prevalent/incident dilated-hypokinetic evolution were younger at first evaluation (32 ± 14 years vs. 41 ± 21 years, p = 0.04) and more often had a family history of HCM (61% vs. 26%, p = 0.002) or sudden death (43% vs. 19%, p = 0.01) with respect to patients who maintained classic HCM. Moreover, they showed greater interventricular septum (23 ± 3 mm vs. 19 ± 6 mm, p = 0.004) and posterior wall (15 ± 3 mm vs. 13 ± 4 mm, p = 0.006) thickness. Cardiovascular death-free survival was lower among patients with dilated-hypokinetic HCM (p < 0.04). Cox proportional hazards regression analysis identified left ventricular wall thickness (hazard ratio [HR] = 1.07; 95% confidence interval [CI], 1.01 to 1.14; p = 0.03) and end-diastolic diameter (HR = 1.08; 95% CI 1.04 to 1.11; p = 0.0001) as independent predictors of cardiovascular death.ConclusionsDilated-hypokinetic evolution is rare but not exceptional in HCM. Young age at diagnosis, family history of HCM, and greater wall thickness are incremental risk factors for dilated-hypokinetic HCM, which carries an ominous prognosis

Prediction of thrombo-embolic risk in patients with hypertrophic cardiomyopathy (HCM Risk-CVA)

Aims Atrial fibrillation (AF) and thrombo-embolism (TE) are associated with reduced survival in hypertrophic cardiomyopathy (HCM), but the absolute risk of TE in patients with and without AF is unclear. The primary aim of this study was to derive and validate a model for estimating the risk of TE in HCM. Exploratory analyses were performed to determine predictors of TE, the performance of the CHA2DS2-VASc score, and outcome with vitamin K antagonists (VKAs). Methods and results A retrospective, longitudinal cohort of seven institutions was used to develop multivariable Cox regression models fitted with pre-selected predictors. Bootstrapping was used for validation. Of 4821 HCM patients recruited between 1986 and 2008, 172 (3.6%) reached the primary endpoint of cerebrovascular accident (CVA), transient ischaemic attack (TIA), or systemic peripheral embolus within 10 years. A total of 27.5% of patients had a CHA2DS2-VASc score of 0, of whom 9.8% developed TE during follow-up. Cox regression revealed an association between TE and age, AF, the interaction between age and AF, TE prior to first evaluation, NYHA class, left atrial (LA) diameter, vascular disease, and maximal LV wall thickness. There was a curvilinear relationship between LA size and TE risk. The model predicted TE with a C-index of 0.75 [95% confidence interval (CI) 0.70-0.80] and the D-statistic was 1.30 (95% CI 1.05-1.56). VKA treatment was associated with a 54.8% (95% CI 31-97%, P = 0.037) relative risk reduction in HCM patients with AF. Conclusions The study shows that the risk of TE in HCM patients can be identified using a small number of simple clinical features. LA size, in particular, should be monitored closely, and the assessment and treatment of conventional vascular risk factors should be routine practice in older patients. Exploratory analyses show for the first time evidence for a reduction of TE with VKA treatment. The CHA2DS2-VASc score does not appear to correlate well with the clinical outcome in patients with HCM and should not be used to assess TE risk in this population

Histopathological comparison of intramural coronary artery remodeling and myocardial fibrosis in obstructive versus end-stage hypertrophic cardiomyopathy.

Abstract Background Although imaging techniques have demonstrated the existence of microvascular abnormalities in hypertrophic cardiomyopathy (HCM), a detailed histopathological assessment is lacking as well as a comparison between different phases of the disease. We aimed to compare microvasculopathy and myocardial fibrosis in hypertrophic obstructive cardiomyopathy (HOCM) versus end-stage (ES) HCM. Methods 27 myectomy specimens of HOCM patients and 30 ES-HCM explanted hearts were analyzed. Myocardial fibrosis was quantitatively determined with dedicated software and qualitatively classified as scar-like or interstitial. Intramural coronary arteries were evaluated separately according to lumen diameter: 100–500 μ versus Results Median value of fibrosis in the anterobasal septum of explanted hearts was 34.6% as opposed to 10.3% of myectomy specimens (p  Conclusions Microvasculopathy is an intrinsic feature of HCM with similar characteristics across the natural phases of the disease. Conversely, myocardial fibrosis changes over time with ES hearts showing a three-fold greater amount, mainly scar-like. ES showed a closer association between microvasculopathy and replacement fibrosis

Targeted next-generation sequencing helps to decipher the genetic and phenotypic heterogeneity of hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is mainly associated with myosin, heavy chain 7 (MYH7) and myosin binding protein C, cardiac (MYBPC3) mutations. In order to better explain the clinical and genetic heterogeneity in HCM patients, in this study, we implemented a target-next generation sequencing (NGS) assay. An Ion AmpliSeq Custom Panel for the enrichment of 19 genes, of which 9 of these did not encode thick/intermediate and thin myofilament (TTm) proteins and, among them, 3 responsible of HCM phenocopy, was created. Ninety-two DNA samples were analyzed by the Ion Personal Genome Machine: 73 DNA samples (training set), previously genotyped in some of the genes by Sanger sequencing, were used to optimize the NGS strategy, whereas 19 DNA samples (discovery set) allowed the evaluation of NGS performance. In the training set, we identified 72 out of 73 expected mutations and 15 additional mutations: the molecular diagnosis was achieved in one patient with a previously wild-type status and the pre-excitation syndrome was explained in another. In the discovery set, we identified 20 mutations, 5 of which were in genes encoding non-TTm proteins, increasing the diagnostic yield by approximately 20%: a single mutation in genes encoding non-TTm proteins was identified in 2 out of 3 borderline HCM patients, whereas co-occuring mutations in genes encoding TTm and galactosidase alpha (GLA) altered proteins were characterized in a male with HCM and multiorgan dysfunction. Our combined targeted NGS-Sanger sequencing-based strategy allowed the molecular diagnosis of HCM with greater efficiency than using the conventional (Sanger) sequencing alone. Mutant alleles encoding non-TTm proteins may aid in the complete understanding of the genetic and phenotypic heterogeneity of HCM: co-occuring mutations of genes encoding TTm and non-TTm proteins could explain the wide variability of the HCM phenotype, whereas mutations in genes encoding only the non-TTm proteins are identifiable in patients with a milder HCM status

Differences in cardiac phenotype and natural history of laminopathies with and without neuromuscular onset

Objective: To investigate differences in cardiac manifestations of patients affected by laminopathy, according to the presence or absence of neuromuscular involvement at presentation.Methods: We prospectively analyzed 40 consecutive patients with a diagnosis of laminopathy followed at a single centre between 1998 and 2017. Additionally, reports of clinical evaluations and tests prior to referral at our centre were retrospectively evaluated.Results: Clinical onset was cardiac in 26 cases and neuromuscular in 14. Patients with neuromuscular presentation experienced first symptoms earlier in life (11 vs 39 years; p &lt; 0.0001) and developed atrial fibrillation/flutter (AF) and required pacemaker implantation at a younger age (28 vs 41 years [p = 0.013] and 30 vs 44 years [p = 0.086] respectively), despite a similar overall prevalence of AF (57% vs 65%; p = 0.735) and atrio-ventricular (A-V) block (50% vs 65%; p = 0.500). Those with a neuromuscular presentation developed a cardiomyopathy less frequently (43% vs 73%; p = 0.089) and had a lower rate of sustained ventricular tachyarrhythmias (7% vs 23%; p = 0.387). In patients with neuromuscular onset rhythm disturbances occurred usually before evidence of cardiomyopathy. Despite these differences, the need for heart transplantation and median age at intervention were similar in the two groups (29% vs 23% [p = 0.717] and 43 vs 46 years [p = 0.593] respectively).Conclusions: In patients with laminopathy, the type of disease onset was a marker for a different natural history. Specifically, patients with neuromuscular presentation had an earlier cardiac involvement, characterized by a linear and progressive evolution from rhythm disorders (AF and/or A-V block) to cardiomyopathy