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Preferred IUPAC name 3-[10-(Dibutylamino)chryseno[2,1-f]isoquinolin-2-ium-2-yl]-N,N,N-trimethylpropan-1-aminium dibromide | |
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CompTox Dashboard (EPA) | |
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Properties | |
C39H47Br2N3 | |
Molar mass | 717.634 g·mol−1 |
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa). |
ANNINE-6plus is a water soluble voltage sensitive dye (also called potentiometric dyes). This compound was developed at the Max Planck Institute for Biochemistry in Germany. [1] It is used to optically measure the changes in transmembrane voltage of excitable cells, including neurons, skeletal and cardiac myocytes.
ANNINE-6plus has a fractional fluorescent intensity change (ΔF/F per 100 mV change) of about 30% with single-photon excitation (~488 nm) and >50% with two-photon excitation (~1060 nm).
ANNINE-6plus has been applied in the microscopic imaging of action potentials of cardiomyocyte in perfused mice heart. [2] Using confocal microscopy in conjunction with ANNINE-6plus, single sweep action potentials with high peak signal-to-noise ratio (SNR) have been recorded from single transverse tubule (t-tubule) of a few micrometers in the ventricular cardiomyocyte.
In physiology, an action potential (AP) occurs when the membrane potential of a specific cell location rapidly rises and falls. This depolarization then causes adjacent locations to similarly depolarize. Action potentials occur in several types of animal cells, called excitable cells, which include neurons, muscle cells, endocrine cells and in some plant cells.
Cardiac muscle is one of three types of vertebrate muscle tissue, with the other two being skeletal muscle and smooth muscle. It is involuntary, striated muscle that constitutes the main tissue of the wall of the heart. The cardiac muscle (myocardium) forms a thick middle layer between the outer layer of the heart wall and the inner layer, with blood supplied via the coronary circulation. It is composed of individual cardiac muscle cells joined by intercalated discs, and encased by collagen fibers and other substances that form the extracellular matrix.
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.
A muscle cell is also known as a myocyte when referring to either a cardiac muscle cell (cardiomyocyte), or a smooth muscle cell as these are both small cells. A skeletal muscle cell is long and threadlike with many nuclei and is called a muscle fiber. Muscle cells develop from embryonic precursor cells called myoblasts.
The cardiac action potential is a brief change in voltage across the cell membrane of heart cells. This is caused by the movement of charged atoms between the inside and outside of the cell, through proteins called ion channels. The cardiac action potential differs from action potentials found in other types of electrically excitable cells, such as nerves. Action potentials also vary within the heart; this is due to the presence of different ion channels in different cells.
Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to about one millimeter in thickness, with 0.64 μm lateral and 3.35 μm axial spatial resolution. Unlike traditional fluorescence microscopy, in which the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. Two-photon excitation microscopy typically uses near-infrared (NIR) excitation light which can also excite fluorescent dyes. However, for each excitation, two photons of NIR light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased penetration depth for this technique. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced photobleaching.
T-tubules are extensions of the cell membrane that penetrate into the centre of skeletal and cardiac muscle cells. With membranes that contain large concentrations of ion channels, transporters, and pumps, T-tubules permit rapid transmission of the action potential into the cell, and also play an important role in regulating cellular calcium concentration.
Cyanines, also referred to as tetramethylindo(di)-carbocyanines are a synthetic dye family belonging to the polymethine group. Although the name derives etymologically from terms for shades of blue, the cyanine family covers the electromagnetic spectrum from near IR to UV.
A calcium spark is the microscopic release of calcium (Ca2+) from a store known as the sarcoplasmic reticulum (SR), located within muscle cells. This release occurs through an ion channel within the membrane of the SR, known as a ryanodine receptor (RyR), which opens upon activation. This process is important as it helps to maintain Ca2+ concentration within the cell. It also initiates muscle contraction in skeletal and cardiac muscles and muscle relaxation in smooth muscles. Ca2+ sparks are important in physiology as they show how Ca2+ can be used at a subcellular level, to signal both local changes, known as local control, as well as whole cell changes.
Ryanodine receptor 2 (RYR2) is one of a class of ryanodine receptors and a protein found primarily in cardiac muscle. In humans, it is encoded by the RYR2 gene. In the process of cardiac calcium-induced calcium release, RYR2 is the major mediator for sarcoplasmic release of stored calcium ions.
Sodium channel blockers are drugs which impair the conduction of sodium ions (Na+) through sodium channels.
Ankyrin-3 (ANK-3), also known as ankyrin-G, is a protein from ankyrin family that in humans is encoded by the ANK3 gene.
Voltage-sensitive dyes, also known as potentiometric dyes, are dyes which change their spectral properties in response to voltage changes. They are able to provide linear measurements of firing activity of single neurons, large neuronal populations or activity of myocytes. Many physiological processes are accompanied by changes in cell membrane potential which can be detected with voltage sensitive dyes. Measurements may indicate the site of action potential origin, and measurements of action potential velocity and direction may be obtained.
Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field or on the far-field. Among techniques that rely on the latter are those that improve the resolution only modestly beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment, the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.
GCaMP is a genetically encoded calcium indicator (GECI) initially developed in 2001 by Junichi Nakai. It is a synthetic fusion of green fluorescent protein (GFP), calmodulin (CaM), and M13, a peptide sequence from myosin light-chain kinase. When bound to Ca2+, GCaMP fluoresces green with a peak excitation wavelength of 480 nm and a peak emission wavelength of 510 nm. It is used in biological research to measure intracellular Ca2+ levels both in vitro and in vivo using virally transfected or transgenic cell and animal lines. The genetic sequence encoding GCaMP can be inserted under the control of promoters exclusive to certain cell types, allowing for cell-type specific expression of GCaMP. Since Ca2+ is a second messenger that contributes to many cellular mechanisms and signaling pathways, GCaMP allows researchers to quantify the activity of Ca2+-based mechanisms and study the role of Ca2+ ions in biological processes of interest.
Photo-activated localization microscopy and stochastic optical reconstruction microscopy (STORM) are widefield fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. The methods were proposed in 2006 in the wake of a general emergence of optical super-resolution microscopy methods, and were featured as Methods of the Year for 2008 by the Nature Methods journal. The development of PALM as a targeted biophysical imaging method was largely prompted by the discovery of new species and the engineering of mutants of fluorescent proteins displaying a controllable photochromism, such as photo-activatible GFP. However, the concomitant development of STORM, sharing the same fundamental principle, originally made use of paired cyanine dyes. One molecule of the pair, when excited near its absorption maximum, serves to reactivate the other molecule to the fluorescent state.
Calcium imaging is a microscopy technique to optically measure the calcium (Ca2+) status of an isolated cell, tissue or medium. Calcium imaging takes advantage of calcium indicators, fluorescent molecules that respond to the binding of Ca2+ ions by fluorescence properties. Two main classes of calcium indicators exist: chemical indicators and genetically encoded calcium indicators (GECI). This technique has allowed studies of calcium signalling in a wide variety of cell types. In neurons, electrical activity is always accompanied by an influx of Ca2+ ions. Thus, calcium imaging can be used to monitor the electrical activity in hundreds of neurons in cell culture or in living animals, which has made it possible to dissect the function of neuronal circuits.
Genetically encoded voltage indicator is a protein that can sense membrane potential in a cell and relate the change in voltage to a form of output, often fluorescent level. It is a promising optogenetic recording tool that enables exporting electrophysiological signals from cultured cells, live animals, and ultimately human brain. Examples of notable GEVIs include ArcLight, ASAP1, ASAP3, Archons, SomArchon, and Ace2N-mNeon.
Three-photon microscopy (3PEF) is a high-resolution fluorescence microscopy based on nonlinear excitation effect. Different from two photon excitation microscopy, it uses three exciting photons. It typically uses 1300 nm or longer wavelength lasers to excite the fluorescent dyes with three simultaneously absorbed photons. The fluorescent dyes then emit one photon whose energy is three times the energy of each incident photon. Compared to two-photon microscopy, three-photon microscopy reduces the fluorescence away from the focal plane by , which is much faster than that of two-photon microscopy by . In addition, three-photon microscopy employs near-infrared light with less tissue scattering effect. This causes three photon microscopy to have higher resolution than conventional microscopy.
Brian M. Salzberg is an American neuroscientist, biophysicist and professor. He is Professor of Neuroscience and of Physiology at the Perelman School of Medicine, University of Pennsylvania.