Abstract
SummaryWe report the first demonstration of a fast wavelength‐switchable 340/380 nm light‐emitting diode (LED) illuminator for Fura‐2 ratiometric Ca2+ imaging of live cells. The LEDs closely match the excitation peaks of bound and free Fura‐2 and enables the precise detection of cytosolic Ca2+ concentrations, which is only limited by the Ca2+ response of Fura‐2. Using this illuminator, we have shown that Fura‐2 acetoxymethyl ester (AM) concentrations as low as 250 nM can be used to detect induced Ca2+ events in tsA‐201 cells and while utilising the 150 μs switching speeds available, it was possible to image spontaneous Ca2+ transients in hippocampal neurons at a rate of 24.39 Hz that were blunted or absent at typical 0.5 Hz acquisition rates. Overall, the sensitivity and acquisition speeds available using this LED illuminator significantly improves the temporal resolution that can be obtained in comparison to current systems and supports optical imaging of fast Ca2+ events using Fura‐2.
Highlights
Calcium (Ca2+) plays a varied and integral role in mediating and controlling many biological processes including the regulation of muscle contractions (Szent-Gyorgyi, 1975), triggering insulin release from pancreatic cells (Dyachok & Gylfe, 2001), and the release of neurotransmitters in neurons (Frenguellil & Malinow, 1996)
Increases in cytosolic Ca2+ levels can originate from a number of sources including release from internal stores triggered by activation of G-proteincoupled receptors by both endogenous and exogenous stimuli (Bootman, 2012) or from external sources via influx through
We demonstrate its application in microscopy by performing Fura-2 ratiometric Ca2+ imaging in both an immortalised cell line and in primary cultured neurons exhibiting pharmacologically induced and synaptically driven Ca2+ responses
Summary
Calcium (Ca2+) plays a varied and integral role in mediating and controlling many biological processes including the regulation of muscle contractions (Szent-Gyorgyi, 1975), triggering insulin release from pancreatic cells (Dyachok & Gylfe, 2001), and the release of neurotransmitters in neurons (Frenguellil & Malinow, 1996). Electrophysiological studies are still the gold standard for measuring electrical activity within and between excitable cells due to their high temporal resolution (Scanziani & Hausser, 2009), the spatial resolution is low and the nature of the technique leads to low throughput data production. The development of Ca2+ specific fluorescent indicators allows for high throughput data acquisition with good spatial resolution (Tsien, 1999; Hell, 2007; Scanziani & Hausser, 2009), which has allowed intracellular Ca2+ dynamics to be investigated noninvasively in multiple cells simultaneously using widefield epifluorescence microscopy. The Ca2+ concentration changes are identifiable through an emission intensity change (Paredes et al, 2008) These indicators are unable to provide quantitative Ca2+ data because the emission intensities may be influenced by dye concentration or photobleaching during imaging (Maravall et al, 2000)
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