title: Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets.
abstract: The observation of ionic signaling dynamics in intact pancreatic islets has contributed greatly to our understanding of both α- and β-cell function. Insulin secretion from β-cells depends on the firing of action potentials and consequent rises of intracellular calcium activity ([Ca(2+)]i). Zinc (Zn(2+)) is cosecreted with insulin, and has been postulated to play a role in cell-to-cell cross talk within an islet, in particular inhibiting glucagon secretion from α-cells. Thus, measuring [Ca(2+)]i and Zn(2+) dynamics from both α- and β-cells will elucidate mechanisms underlying islet hormone secretion. [Ca(2+)]i and intracellular Zn(2+) can be measured using fluorescent biosensors, but the most efficient sensors have overlapping spectra that complicate their discrimination. Hyperspectral imaging can be used to distinguish signals from multiple fluorophores, but available hyperspectral implementations are either too slow to measure the dynamics of ionic signals or not suitable for thick samples. We have developed a five-dimensional (x,y,z,t,λ) imaging system that leverages a snapshot hyperspectral imaging method, image mapping spectrometry, and light-sheet microscopy. This system provides subsecond temporal resolution from deep within multicellular structures. Using a single excitation wavelength (488 nm) we acquired images from triply labeled samples with two biosensors and a genetically expressing fluorescent protein (spectrally overlapping with one of the biosensors) with high temporal resolution. Measurements of [Ca(2+)]i and Zn(2+) within both α- and β-cells as a function of glucose concentration show heterogeneous uptake of Zn(2+) into α-cells that correlates to the known heterogeneities in [Ca(2+)]i. These differences in intracellular Zn(2+) among α-cells may contribute to the inhibition in glucagon secretion observed at elevated glucose levels.
authors.affiliation: Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee; Cell Biology and Physiology, Washington University in St. Louis, St. Louis, Missouri.
authors.forename: Zeno
authors.initials: Z
authors.lastname: Lavagnino
authors.affiliation: Department of Bioengineering, Rice University, Houston, Texas.
authors.forename: Jason
authors.initials: J
authors.lastname: Dwight
authors.affiliation: Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee; Cell Biology and Physiology, Washington University in St. Louis, St. Louis, Missouri.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Ustione
authors.affiliation: Department of Bioengineering, Rice University, Houston, Texas.
authors.forename: Thuc-Uyen
authors.initials: TU
authors.lastname: Nguyen
authors.affiliation: Department of Bioengineering, Rice University, Houston, Texas.
authors.forename: Tomasz S
authors.initials: TS
authors.lastname: Tkaczyk
authors.affiliation: Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee; Cell Biology and Physiology, Washington University in St. Louis, St. Louis, Missouri. Electronic address: piston@wustl.edu.
authors.forename: David W
authors.initials: DW
authors.lastname: Piston
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival; D042541:Intracellular Space / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D057054:Molecular Imaging; D015398:Signal Transduction; D015032:Zinc / Q000378:metabolism
chemical_list: D015032:Zinc; D002118:Calcium