voltage imaging Dotaz Zobrazit nápovědu
Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.
- MeSH
- chování zvířat MeSH
- dánio pruhované MeSH
- fluorescence MeSH
- genetické inženýrství MeSH
- larva MeSH
- luminescentní proteiny chemie genetika MeSH
- mezencefalon cytologie fyziologie MeSH
- monitorování fyziologických funkcí metody MeSH
- myši MeSH
- neurony fyziologie MeSH
- neurozobrazování metody MeSH
- optogenetika MeSH
- plavání MeSH
- proteinové domény MeSH
- rezonanční přenos fluorescenční energie MeSH
- rhodopsiny mikrobiální chemie genetika MeSH
- zobrazování pomocí barviva citlivého na potenciál metody MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- luminescentní proteiny MeSH
- rhodopsiny mikrobiální MeSH
Measuring the transduction of electrical signals within neurons is a key capability in neuroscience. Fluorescent voltage sensitive dyes (VSDs) were early tools that complemented classical electrophysiology by enabling the optical recording of membrane potential changes from many cells simultaneously. Recent advances in the VSD field have led to bright and highly sensitive sensors that can be targeted to the desired cell populations in live brain tissue. Despite this progress, recently, protein-based genetically encoded voltage indicators (GEVIs) have become the go-to tools for targeted voltage imaging in complex environments. In this Perspective, we summarize progress in developing targetable VSDs, discuss areas where these synthetic sensors are or could become relevant, and outline hurdles that need to be overcome to promote the routine use of targetable VSDs in neuroscience research.
- Klíčová slova
- cell-selective targeting, fluorescent sensor, imaging probe, membrane potential, voltage-sensitive dye,
- MeSH
- akční potenciály fyziologie účinky léků MeSH
- fluorescenční barviva MeSH
- lidé MeSH
- membránové potenciály fyziologie MeSH
- neurony * fyziologie MeSH
- zobrazování pomocí barviva citlivého na potenciál metody trendy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- fluorescenční barviva MeSH
The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.
- Klíčová slova
- biosensors, fluorescence imaging, fluorescent proteins, genetically encoded indicators, voltage imaging,
- MeSH
- akční potenciály fyziologie MeSH
- angiotensin-konvertující enzym 2 * MeSH
- mutace genetika MeSH
- myši MeSH
- neurony fyziologie MeSH
- rodopsin * genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- angiotensin-konvertující enzym 2 * MeSH
- rodopsin * MeSH
BACKGROUND: Electroanatomical voltage mapping (EAVM) has been compared with late gadolinium enhancement cardiovascular magnetic resonance (LGE-CMR), which cannot delineate diffuse fibrosis. T1-mapping CMR overcomes the limitations of LGE-CMR, but it has not been directly compared against EAVM. OBJECTIVES: This study aims to assess the relationship between left ventricular (LV) endocardial voltage obtained by EAVM and extracellular volume (ECV) obtained by T1 mapping. METHODS: The study investigated patients who underwent endocardial EAVM for ventricular arrhythmias (CARTO 3, Biosense Webster) together with preprocedural contrast-enhanced T1 mapping (Ingenia 3T, Philips Healthcare). After image integration, EAVM datapoints were projected onto LGE-CMR and ECV-encoded images. Average values of unipolar voltage (UV), bipolar voltage (BV), LGE transmurality, and ECV were merged from corresponding cardiac segments (6 per slice) and pooled for analysis. RESULTS: The analysis included data from 628 segments from 18 patients (57 ± 13 years of age, 17% females, LV ejection fraction 48% ± 14%, nonischemic/ischemic cardiomyopathy/controls: 8/6/4 patients). Based on the 95th and 5th percentile values obtained from the controls, ECV >33%, BV <2.9 mV, and UV <6.7 mV were considered abnormal. There was a significant inverse association between voltage and ECV, but only in segments with abnormal ECV. Increased ECV could predict abnormal BV and UV with acceptable accuracy (area under the curve of 0.78 [95% CI: 0.74-0.83] and 0.84 [95% CI: 0.79-0.88]). CONCLUSIONS: This study found a significant inverse relationship between LV endocardial voltage and ECV. Real-time integration of T1 mapping may guide catheter mapping and may allow identification of areas of diffuse fibrosis potentially related to ventricular arrhythmias.
- Klíčová slova
- T1 mapping, extracellular volume, fibrosis, image integration, voltage,
- MeSH
- fibróza MeSH
- gadolinium MeSH
- kontrastní látky * MeSH
- lidé MeSH
- magnetická rezonance kinematografická * MeSH
- srdeční arytmie diagnostické zobrazování MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- gadolinium MeSH
- kontrastní látky * MeSH
Sample preparation protocols for conventional high voltage transmission electron microscopy (TEM) heavily rely on the usage of staining agents containing various heavy metals, most commonly uranyl acetate and lead citrate. However high toxicity, rising legal regulations, and problematic waste disposal of uranyl acetate have increased calls for the reduction or even complete replacement of this staining agent. One of the strategies for uranyless imaging is the employment of low-voltage transmission electron microscopy. To investigate the influence of different imaging and staining strategies on the final image of cyanobacterial cells, samples stained by uranyl acetate with lead citrate, as well as unstained samples, were observed using TEM and accelerating voltages of 200 kV or 25 kV. Moreover, to examine the possibilities of reducing chromatic aberration, which often causes issues when imaging using electrons of lower energies, samples were also imaged using a scanning transmission electron microscopy at 15 kV accelerating voltages. The results of this study demonstrate that low-voltage electron microscopy offers great potential for uranyless electron microscopy.
- Klíčová slova
- Synechocystis, contrasting agents, low voltage electron microscopy, polyhydroxyalkanoates, transmission electron microscopy, uranyl acetate,
- Publikační typ
- časopisecké články MeSH
Optical mapping is a fluorescence-based physiological method to image spreading of action potential in excitable tissues, such as the heart and central nervous system. Because of the requirements for high speed imaging in low light conditions, highly sensitive high-speed cameras together with an optical system with maximum photon efficiency are required. While the optimization of these two components is relatively straightforward, the choice of the perfect light source is less simple; depending on the other (usually fixed) components, various parameters may acquire different weight in decision-making process. Here we describe the rationale for building an optical mapping setup and consider the relative advantages and disadvantages of three different commonly available light sources: mercury vapor lamp (HBO), xenon lamp (XBO), and light emitting diode (LED). Using the same optical system (fluorescence macroscope) and high-speed camera (Ultima L), we have tested each of the sources for its ability to provide bright and even illumination of the field of view and measured its temporal fluctuations in intensity. Then we used each in the actual optical mapping experiment using isolated, perfused adult mouse heart or chick embryonic heart to determine the actual signal to noise ratio at various acquisition rates. While the LED sources have undergone significant improvements in the recent past, the other alternatives may still surpass them in some parameters, so they may not be the automatic number one choice for every application.
- MeSH
- akční potenciály MeSH
- fluorescenční barviva chemie MeSH
- kuřecí embryo MeSH
- myši MeSH
- srdce fyziologie MeSH
- světlo MeSH
- vápník analýza metabolismus MeSH
- zobrazování pomocí barviva citlivého na potenciál metody normy MeSH
- zvířata MeSH
- Check Tag
- kuřecí embryo MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fluorescenční barviva MeSH
- vápník MeSH
BACKGROUND AND AIMS: A significant source of man-made radiation is now linked to medical devices especially X-ray imaging based ones like CT scans which expose the body to cumulative ionizing radiation and thus attendant cancer risks. The aim of this study was to determine whether using a combination of Automatic Tube Current Modulation (ATCM) and Automatic Tube Voltage Selection (ATVS) during two-phase whole-body CT (2PWBCT) examinations would reduce the radiation dose while preserving the image quality. PATIENTS AND METHODS: This was a prospective, observational, single-centre study of 127 adult patients who had undergone the 2PWBCT polytraumatic protocol. All were examined on a Somatom Drive scanner (Siemens). The patients were divided into two groups: ATCM only (42 patients) and ATCM +ATVS (85 patients). Patients' arm positions during examination and the examination dose length product (DLP) values were recorded, as well the standard deviations (SD) of the density in reference areas on CT scans for the image quality assessment. The DLP values and image quality in the groups were compared using ANOVA. RESULTS: Mean Total DLP (in mGy*cm): ATCM only: 3337 +/-797, ATCM+ATVS: 3402 +/-830; P=0.674. No effect of arm position (P=0.586). Mean density SD values in reference areas (in HU) in ATCM only: 49 +/-45, 15 +/-6, 9 +/-2, 12 +/-4, 10 +/-3, in ATCM+ATVS: 48 +/-45, 17 +/-6, 11 +/-3, 15 +/-6, 12 +/-4. SD values was higher in ATCM+ATVS group (P<0.001). CONCLUSION: Combination of ATVS and ATCM in polytraumatic 2PWBCT leads to no significant radiation load reduction compared with ATCM only but does lead to a slight degradation of image quality. The radiation load is significantly reduced if the patient has their arms behind the head when scanning, regardless of the activation of ATVS.
- Klíčová slova
- automatic tube current modulation, automatic tube voltage selection, dose length product, polytrauma, whole body CT,
- MeSH
- celotělové zobrazování * metody MeSH
- dávka záření * MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- počítačová rentgenová tomografie * metody MeSH
- polytrauma * diagnostické zobrazování MeSH
- prospektivní studie MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- pozorovací studie MeSH
- srovnávací studie MeSH
Motion artefact (MA) in voltage-sensitive fluorescent signals causes significant debasement of action potential. During ischemia and reperfusion in isolated rabbit heart, this artefact develops in a manner which may be described by the time of its onset, level, and shape. The MA during ischemia: (a) may become substantial with approximately two minutes delay after establishing global ischemia; (b) may be almost twice as high as the physiological action potential and decreases both with time and repetition of ischemia; (c) the MA shape is unpredictable and depends on individual rabbit.
- MeSH
- akční potenciály MeSH
- artefakty * MeSH
- časové faktory MeSH
- fluorescenční barviva * MeSH
- králíci MeSH
- perfuze MeSH
- prediktivní hodnota testů MeSH
- pyridinové sloučeniny * MeSH
- reperfuzní poškození myokardu diagnóza patofyziologie MeSH
- techniky in vitro MeSH
- zobrazování pomocí barviva citlivého na potenciál metody MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 1-(3-sulfonatopropyl)-4-(beta)(2-(di-n-butylamino)-6-naphthylvinyl)pyridinium betaine MeSH Prohlížeč
- fluorescenční barviva * MeSH
- pyridinové sloučeniny * MeSH
Visualization of electrical activity in living cells represents an important challenge in context of basic neurophysiological studies. Here we report a new voltage sensitive fluorescent indicator which response could be detected by fluorescence monitoring in a single red channel. To the best of our knowledge, this is the first fluorescent protein-based voltage sensor which uses insertion-into-circular permutant topology to provide an efficient interaction between sensitive and reporter domains. Its fluorescent core originates from red fluorescent protein (FP) FusionRed, which has optimal spectral characteristics to be used in whole body imaging techniques. Indicators using the same domain topology could become a new perspective for the FP-based voltage sensors that are traditionally based on Förster resonance energy transfer (FRET).
- MeSH
- biosenzitivní techniky metody MeSH
- červený fluorescenční protein MeSH
- elektrofyziologické jevy MeSH
- fluorescenční barviva metabolismus MeSH
- HEK293 buňky MeSH
- krysa rodu Rattus MeSH
- lidé MeSH
- luminescentní proteiny chemie MeSH
- nádorové buněčné linie MeSH
- proteinové domény MeSH
- proteinové inženýrství metody MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fluorescenční barviva MeSH
- luminescentní proteiny MeSH
The Pithoviridae giant virus family exhibits the largest viral particle known so far, a prolate spheroid up to 2.5 μm in length and 0.9 μm in diameter. These particles show significant variations in size. Little is known about the structure of the intact virion due to technical limitations with conventional electron cryo-microscopy (cryo-EM) when imaging thick specimens. Here we present the intact structure of the giant Pithovirus sibericum particle at near native conditions using high-voltage electron cryo-tomography (cryo-ET) and energy-filtered cryo-EM. We detected a previously undescribed low-density outer layer covering the tegument and a periodical structuring of the fibres in the striated apical cork. Energy-filtered Zernike phase-contrast cryo-EM images show distinct substructures inside the particles, implicating an internal compartmentalisation. The density of the interior volume of Pithovirus particles is three quarters lower than that of the Mimivirus. However, it is remarkably high given that the 600 kbp Pithovirus genome is only half the size of the Mimivirus genome and is packaged in a volume up to 100 times larger. These observations suggest that the interior is densely packed with macromolecules in addition to the genomic nucleic acid.
