Biomaterial Database

Indicators and optical configuration for simultaneous high-resolution recording of membrane potential and intracellular calcium using laser scanning microscopy. 1998

A Bullen, and P Saggau

Division of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, TX-77030, USA.

The instrumental design and experimental conditions for high-speed, simultaneous optical recording of membrane potential and intracellular Ca2+ with subcellular resolution are presented. This method employs an extended version of a high-speed, random-access, laser-scanning fluorescence microscope designed to record fast physiological signals from small neuronal structures with high spatiotemporal resolution (Bullen, Patel, Saggau, Biophys J 73:477-491, 1997). With this instrument, imaging and optical recording functions are conducted separately allowing frame rates up to 3 kHz. Individual scanning points are selected interactively from a reference image collected with differential interference contrast (DIC) optics. At each recording site, fluorescence from two indicators is measured simultaneously by independent photodetectors. To optimize signal strength, spectral separation and the achievable signal-to-noise ratio, several combinations of voltage-sensitive dye, Ca2+ indicator and optical elements (dichroic mirrors, filters, etc.) were considered. The best results were achieved from the combination of the intracellular voltage-sensitive dye Di-2-ANEPEQ and the Ca2+ indicator Calcium Green-1. These indicators have overlapping absorption spectra allowing simultaneous excitation with a single laser line (488 nm). Spectral separation of the fluorescence from these two indicators was accomplished using a secondary dichroic mirror (DCLP580) and emission filters (535/45 and OG590). Representative records obtained with this instrument and this combination of indicators demonstrate the feasibility of simultaneous high fidelity measurements of membrane potential and intracellular Ca2+ from the same point at high spatial (2 micrometer) and temporal (<ms) resolution without requiring signal averaging.

UI	MeSH Term	Description	Entries
D007202	Indicators and Reagents	Substances used for the detection, identification, analysis, etc. of chemical, biological, or pathologic processes or conditions. Indicators are substances that change in physical appearance, e.g., color, at or approaching the endpoint of a chemical titration, e.g., on the passage between acidity and alkalinity. Reagents are substances used for the detection or determination of another substance by chemical or microscopical means, especially analysis. Types of reagents are precipitants, solvents, oxidizers, reducers, fluxes, and colorimetric reagents. (From Grant & Hackh's Chemical Dictionary, 5th ed, p301, p499)	Indicator,Reagent,Reagents,Indicators,Reagents and Indicators
D007424	Intracellular Fluid	The fluid inside CELLS.	Fluid, Intracellular,Fluids, Intracellular,Intracellular Fluids
D008564	Membrane Potentials	The voltage differences across a membrane. For cellular membranes they are computed by subtracting the voltage measured outside the membrane from the voltage measured inside the membrane. They result from differences of inside versus outside concentration of potassium, sodium, chloride, and other ions across cells' or ORGANELLES membranes. For excitable cells, the resting membrane potentials range between -30 and -100 millivolts. Physical, chemical, or electrical stimuli can make a membrane potential more negative (hyperpolarization), or less negative (depolarization).	Resting Potentials,Transmembrane Potentials,Delta Psi,Resting Membrane Potential,Transmembrane Electrical Potential Difference,Transmembrane Potential Difference,Difference, Transmembrane Potential,Differences, Transmembrane Potential,Membrane Potential,Membrane Potential, Resting,Membrane Potentials, Resting,Potential Difference, Transmembrane,Potential Differences, Transmembrane,Potential, Membrane,Potential, Resting,Potential, Transmembrane,Potentials, Membrane,Potentials, Resting,Potentials, Transmembrane,Resting Membrane Potentials,Resting Potential,Transmembrane Potential,Transmembrane Potential Differences
D009474	Neurons	The basic cellular units of nervous tissue. Each neuron consists of a body, an axon, and dendrites. Their purpose is to receive, conduct, and transmit impulses in the NERVOUS SYSTEM.	Nerve Cells,Cell, Nerve,Cells, Nerve,Nerve Cell,Neuron
D009930	Organic Chemicals	A broad class of substances containing carbon and its derivatives. Many of these chemicals will frequently contain hydrogen with or without oxygen, nitrogen, sulfur, phosphorus, and other elements. They exist in either carbon chain or carbon ring form.	Organic Chemical,Chemical, Organic,Chemicals, Organic
D002118	Calcium	A basic element found in nearly all tissues. It is a member of the alkaline earth family of metals with the atomic symbol Ca, atomic number 20, and atomic weight 40. Calcium is the most abundant mineral in the body and combines with phosphorus to form calcium phosphate in the bones and teeth. It is essential for the normal functioning of nerves and muscles and plays a role in blood coagulation (as factor IV) and in many enzymatic processes.	Coagulation Factor IV,Factor IV,Blood Coagulation Factor IV,Calcium-40,Calcium 40,Factor IV, Coagulation
D005456	Fluorescent Dyes	Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags.	Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D006624	Hippocampus	A curved elevation of GRAY MATTER extending the entire length of the floor of the TEMPORAL HORN of the LATERAL VENTRICLE (see also TEMPORAL LOBE). The hippocampus proper, subiculum, and DENTATE GYRUS constitute the hippocampal formation. Sometimes authors include the ENTORHINAL CORTEX in the hippocampal formation.	Ammon Horn,Cornu Ammonis,Hippocampal Formation,Subiculum,Ammon's Horn,Hippocampus Proper,Ammons Horn,Formation, Hippocampal,Formations, Hippocampal,Hippocampal Formations,Hippocampus Propers,Horn, Ammon,Horn, Ammon's,Proper, Hippocampus,Propers, Hippocampus,Subiculums
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D013343	Styrenes	Derivatives and polymers of styrene. They are used in the manufacturing of synthetic rubber, plastics, and resins. Some of the polymers form the skeletal structures for ion exchange resin beads.