Optical recording of membrane potential permits spatially resolved measurement of electrical activity in subcellular regions of single cells, which would be inaccessible to electrodes, and imaging of spatiotemporal patterns of action potential propagation in excitable tissues, such as the brain or heart. However, the available voltage-sensitive dyes (VSDs) are not always spectrally compatible with newly available optical technologies for sensing or manipulating the physiological state of a system. Here, we describe a series of 19 fluorinated VSDs based on the hemicyanine class of chromophores. Strategic placement of the fluorine atoms on the chromophores can result in either blue or red shifts in the absorbance and emission spectra. The range of one-photon excitation wavelengths afforded by these new VSDs spans 440-670 nm; the twophoton excitation range is 900-1,340 nm. The emission of each VSD is shifted by at least 100 nm to the red of its one-photon excitation spectrum. The set of VSDs, thus, affords an extended toolkit for optical recording to match a broad range of experimental requirements. We show the sensitivity to voltage and the photostability of the new VSDs in a series of experimental preparations ranging in scale from single dendritic spines to whole heart. Among the advances shown in these applications are simultaneous recording of voltage and calcium in single dendritic spines and optical electrophysiology recordings using two-photon excitation above 1,100 nm.fluorescence | microscopy O ptical recording techniques provide powerful tools for neurobiologists (1) and cardiac physiologists (2) to study detailed patterns of electrical activity over time and space in cells, tissues, and organs. Rational design methods, based on molecular orbital calculations of the dye chromophores and characterization of their binding and orientations in membranes (3-5), were used to engineer dye structures. The general class of dye chromophores called hemicyanine (also referred to as styryl dyes) has emerged from this effort as a good foundation for voltage-sensitive dyes (VSDs), because they exhibit electrochromism. This mechanism, also referred to as the molecular Stark effect, involves the differential interaction of the electric field in the membrane with the ground and excited states of the dye chromophore. Several important hemicyanine dyes were produced over the years, including di-4-ANEPPS (6, 7), di-8-ANEPPS (8), di-2-ANEPEQ (also known as JPW-1114) (9, 10), RH-421 and RH-795 (11), ANNINE-6 and ANNINE-6+ (12, 13), di-3-ANEPPDHQ (14, 15), di-4-ANBDQBS, and di-4-ANBDQPQ (16,17). Because the electrochromic mechanism is a direct interaction of the electric field with the chromophore and does not require any movement of the dye molecule, all of these dyes provide rapid absorbance and fluorescence responses to membrane potential (V m ); they are, therefore, capable of recording action potentials (APs). Other mechanisms can give more sensitive voltage responses in specialized applications (18)(19)(20)(21)(22). Addit...

The plasma membrane of cardiac myocytes presents complex invaginations known as the transverse-axial tubular system (TATS). Despite TATS's crucial role in excitation-contraction coupling and morphological alterations found in pathological settings, TATS's electrical activity has never been directly investigated in remodeled tubular networks. Here we develop an ultrafast random access multiphoton microscope that, in combination with a customly synthesized voltage-sensitive dye, is used to simultaneously measure action potentials (APs) at multiple sites within the sarcolemma with submillisecond temporal and submicrometer spatial resolution in real time. We find that the tight electrical coupling between different sarcolemmal domains is guaranteed only within an intact tubular system. In fact, acute detachment by osmotic shock of most tubules from the surface sarcolemma prevents AP propagation not only in the disconnected tubules, but also in some of the tubules that remain connected with the surface. This indicates that a structural disorganization of the tubular system worsens the electrical coupling between the TATS and the surface. The pathological implications of this finding are investigated in failing hearts. We find that AP propagation into the pathologically remodeled TATS frequently fails and may be followed by local spontaneous electrical activity. Our findings provide insight on the relationship between abnormal TATS and asynchronous calcium release, a major determinant of cardiac contractile dysfunction and arrhythmias.cardiac disease | nonlinear microscopy | voltage imaging | t tubules T he transverse-axial tubular system (TATS) is a complex network characterized by transverse (t tubules, TT) and longitudinal (axial tubules, AT) components running from one transverse tubule to the next (1-3). The TATS of a myocyte rapidly conducts depolarization of the surface sarcolemma (SS) to the core of the cardiomyocyte (4). The coupling between sarcolemmal Ca 2+ entry during an action potential (AP) and Ca 2+ release from sarcoplasmic reticulum (SR) promotes synchronous myofibril activation throughout the myocyte (5). Recent studies highlight that AP-relevant ion channels and transporters are expressed in TATS membrane with different densities and isoforms from those in SS (6, 7). These findings, in combination with diffusional limitations in TATS's lumen (3,8), raise the possibility that AP may differ among membrane domains. Further interest in the TATS AP stems from the recent finding of loss and disorganization of tubules in several pathological conditions, including heart failure (9-14). Because correlation between morphological TATS alterations and Ca 2+ -release asynchronicity has been observed (14, 15), recording AP propagation in TATS can elucidate the fundamental electrophysiological mechanism linking structural and functional anomalies.Although the uniformity of the AP across the whole sarcolemma has been mathematically (16) and experimentally (17) proved, a potential alteration of the AP propagation i...

For more than a century, radiotherapy has been an effective treatment for oncologic patients. The Authors report a brief history of the radiation therapy and its actual indication for the treatments of cutaneous malignant diseases.

OBJECTIVE:Neauvia Stimulate is biocompatible, injectable hyaluronic acid (HA) filler (26 mg/ml) PEG cross-linked with 1% of calcium hydroxyapatite (CaHA) for facial soft-tissue augmentation that provides volume to tissues, followed by process of neocollagenesis for improving skin quality.AIM:The aim of the present study is to evaluate the biosafety of the product (Lot. 160517-26-1/2 PEG) on human keratinocytes cultured in vitro.MATERIAL AND METHODS:The experimental model proposed, despite being an in vitro system, allows the derivation of useful information to predict the possible activity of the product in further in vivo application. Human keratinocytes (HaCaT cells) were treated with the product for 24h at increasing concentrations of product respect to control (untreated cells).RESULTS:The biosafety of the product to be tested has been evaluated performing different methods: MTT test, NRU test, Kenacid Blue assay. Moreover, any possible effect on the structure, morphology, and viability of cells has been evaluated.CONCLUSION:In conclusion, the results obtained by the different methods show that the product Neauvia Stimulate® does not cause any cytotoxic effect and does not affect the correct structure and morphology of cells cultures.

BACKGROUND:Vitiligo is an acquired pigmentary cutaneous disease, characterised by the progressive loss of melanocytes, resulting in hypopigmented skin areas which progressively become amelanotic. Classically, vitiligo treatments are unsatisfactory and challenging. Despite the continuous introduction of new therapies, phototherapy is still the mainstay for vitiligo repigmentation.AIM:The aim of this multicenter observational retrospective study was to evaluate the efficacy and safety of the nb - UVB micro - phototherapy (BIOSKIN EVOLUTION®), used alone or in associations with an oral Janus kinase inhibitor (Tofacitinib citrate), in the treatment of stable or active forms of localised vitiligo.MATERIAL AND METHODS:Fifty eight patients had been treated with n-UVB micro-photootherapy (Group A); 9 patients had been treated with phototherapy plus Tofacitinb citrate (Group B).RESULTS:Among Group A, 42 patients (72%) obtained a re-pigmentation rate higher than 75%, with a medium value of 77%. 11 patients (19%) achieved a marked improvement of the clinical findings with a repigmentation rate between 50-75%; 4 patients (8%) showed a moderate response with a lesional repigmentation of 25-50%. Only one patient (1%) had a poor response to the phototherapeutic treatmentCONCLUSION:Nb - UVB micro-focused phototherapy is one of the most effective therapeutic options for vitiligo treatment. The association of micro-focused phototherapy to Tofacitinib citrate seems to provide better clinical results in term of repigmentation rate.