- Biochemistry, Genetics and Molecular Biology
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We describe the construction of a simplified, inexpensive lattice light-sheet microscope, and illustrate its use for imaging subcellular Ca puffs evoked by photoreleased i-IP in cultured SH-SY5Y neuroblastoma cells loaded with the Ca probe Cal520. The microscope provides sub-micron spatial resolution and enables recording of local Ca transients in single-slice mode with a signal-to-noise ratio and temporal resolution (2ms) at least as good as confocal or total internal reflection microscopy. Signals arising from openings of individual IPR channels are clearly resolved, as are stepwise changes in fluorescence reflecting openings and closings of individual channels during puffs. Moreover, by stepping the specimen through the light-sheet, the entire volume of a cell can be scanned within a few hundred ms. The ability to directly visualize a sideways (axial) section through cells directly reveals that IP-evoked Ca puffs originate at sites in very close (≤a few hundred nm) to the plasma membrane, suggesting they play a specific role in signaling to the membrane.