Dynamic Ca2+ imaging with a simplified lattice light-sheet microscope: A sideways view of subcellular Ca2+ puffs |
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| Institution: | 1. Department of Neurobiology and Behavior, University of California, Irvine, CA 92697, United States;2. Department of Neurobiology and Behavior, Department of Physiology and Biophysics, University of California, Irvine, CA 92697, United States;1. Department of Neurobiology and Behavior, University of California, Irvine, CA, 92697, United States;2. Department of Physiology and Biophysics, University of California, Irvine, CA, 92697, United States;1. Department of Molecular Genetics and Microbiology, Duke University, Durham, NC 27710, USA;2. University Program in Genetics and Genomics, Duke University, Durham, NC 27710, USA;3. Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147, USA;4. Department of Biostatistics and Bioinformatics, Duke University, Durham, NC 27710, USA;5. Department of Immunology, Duke University, Durham, NC 27710, USA;2. The John Radcliffe Hospital, Nuffield Department of Medicine, Oxford University Hospital, Oxford, United Kingdom;3. Department of Cardiac Surgery, The Johns Hopkins Hospital, Baltimore, Maryland |
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| Abstract: | We describe the construction of a simplified, inexpensive lattice light-sheet microscope, and illustrate its use for imaging subcellular Ca2+ puffs evoked by photoreleased i-IP3 in cultured SH-SY5Y neuroblastoma cells loaded with the Ca2+ probe Cal520. The microscope provides sub-micron spatial resolution and enables recording of local Ca2+ transients in single-slice mode with a signal-to-noise ratio and temporal resolution (2 ms) at least as good as confocal or total internal reflection microscopy. Signals arising from openings of individual IP3R 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 IP3-evoked Ca2+ 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. |
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| Keywords: | Lattice light-sheet Microscopy |
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