Flux in Artificial Ca Sparks Generated by 2-Photon Release from a Novel Cage Confocally Imaged at Microsecond Resolution

Control of calcium signaling in striated muscle relies on concurrent actions of calcium ions to promote and inhibit release channel opening. To understand these actions we developed artificial Ca sparks generated by 2-photon (2P) release from NDBF-EGTA (Momotake, Nature Methods 2006) as quantifiable local stimuli. A “Dual Scanner” (Zeiss) delivers IR laser flashes through a LSM 510 scanner, while rapidly imaging fluorescence of a [Ca2+] monitor via a slit scanner (5-LIVE; ca 100 μs/line). Ca sparks of 0.1 to 10 μM (A, B) are elicited in a droplet after microseconds of 2P irradiation at 720 nm and imaged with the low affinity dye fluo 4FF. Reaction-diffusion analysis (Rios, JGP 1999) yields the flux of Ca photorelease (C). This flux, which initially reaches several hundred mM/s, decays with τ of 2-3 ms. The technique is used to measure physico-chemical properties of calcium ligands, including bio-sensors. Applied inside muscle fibers (Figueroa, this meeting) it serves to quantitatively characterize calcium control in cells.Instrument purchased with a S10 NCRR award and Hasterlik Family matching funds. Supported by NIAMS, NHLBI/NIH and MDA.View Large Image | View Hi-Res Image | Download PowerPoint Slide