What is ratiometric calcium imaging?
Ratiometric methods are based on the use of a ratio between two fluorescence intensities. This allows correction of arctifacts due to bleaching, changes in focus, variations in laser intensity, etc. but makes measurements and data processing more complicated.
How does Fura 2AM work?
Fura-2AM is a ratiometric dye. It can be excited by two wavelengths, 340 nm for calcium bound and 380 nm for unbound. Irrespective of the excitation wavelength, the dye will emit at 510 nm wavelength. The ratio of 340/380 is usually calculated for normalizing unequal loading of the dye into the cells.
How does Fura red work?
Fura Red is a visible light-excitable analog of Fura-2 that can be used to ratiometrically measure Ca2+ in single cells. It’s visible-light excitation (∼488 nm) and long-wavelength emission maxima (∼639 nm) minimizes background interference and enables multicolor analysis with green fluorescent probes.
How do you measure calcium concentration in cells?
Changes in intracellular calcium concentration can be measured using the calcium sensitive fluorescent ratiometric dye fura-2 AM. This method is a high throughput way to measure agonist mediated calcium responses.
What does ratiometric mean?
ratiometric (not comparable) (electronics) Describing any system in which an output is directly proportional to an input. (physics) Relating to the measurement of the ratio between two or more factors.
What is ratiometric imaging?
Calcium Imaging The basis of ratiometric imaging is the sensitivity of certain fluorophores to specific environmental factors. These environmental factors can include pH, the concentration of certain ions like Ca2+ or the proximity of other fluorophore species (as in FRET).
What does Bapta stand for?
BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) is a calcium-specific aminopolycarboxylic acid. The presence of four carboxylic acid functional groups makes possible the binding of two calcium ions.
How do you test for intracellular calcium?
Measuring intracellular calcium using fluorescence The concentration of free calcium in intact cells can be monitored by using polycyclic chelators such as Fura-2 or Indo-1. Fura-2 and Indo-1 provide a ratio metric readout reducing effects caused by leaking or bleached dyes or by varying assay conditions.
How is cytoplasmic calcium measured?
There are various methods to detect cytosolic calcium signals. Classically, signals from a ratiometric calcium indicator, such as Fura-2, are monitored in live cells using a fluorescence imaging setup and absolute calcium concentrations can be derived from determining minimum and maximum fluorescence intensities.
What is ratiometric sensing?
Ratiometric is used to describe an output signal which changes in proportion to a change input or supply voltage. A typical ratiometric device would be a strain gauge output pressure sensor, where the output sensitivity is described as a ratio between output and input supply voltage.
What is a ratiometric indicator for calcium?
Ratiometric calcium indicators. Additionally, ratiometric calcium indicators reduce the problems associated with measuring Ca 2+ in cells of unequal thickness. Fura-2 and Indo-1 are typically used to measure changes in calcium concentration by either monitoring excitation or emission, respectively.
Why use ratiometric analysis for Ca2 + mobilization?
Ratiometric analysis of Ca 2 + mobilization reduces errors due to variations in cell size and shape, indicator loading and instrument variability that can otherwise limit the usefulness of fluorescent Ca 2 + indicators.
What are rhodamine-based calcium indicators?
HeLa cells loaded with 5 µM Fluo-4. Rhodamine-based calcium indicators comprise a range of probes for large or small changes in Ca 2+ concentration. They exhibit a 50-fold increase in fluorescence upon calcium binding and offer a range of wavelengths that can be used in conjunction with GFP or green-fluorescent dyes for multiplexing.
How can calcium be used as a measure of neuron activity?
Functional imaging of calcium as a measure of neuronal activity is a key technique in neuroscience research. We’ve developed a number of Molecular Probes ion indicators to track calcium with intense fluorescent signals and a range of wavelength options.