TY - JOUR
T1 - Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets
AU - Lavagnino, Zeno
AU - Dwight, Jason
AU - Ustione, Alessandro
AU - Nguyen, Thuc Uyen
AU - Tkaczyk, Tomasz S.
AU - Piston, David W.
N1 - Publisher Copyright:
© 2016 Biophysical Society
PY - 2016/7/26
Y1 - 2016/7/26
N2 - 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 ([Ca2+]i). Zinc (Zn2+) 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 [Ca2+]i and Zn2+ dynamics from both α- and β-cells will elucidate mechanisms underlying islet hormone secretion. [Ca2+]i and intracellular Zn2+ 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 [Ca2+]i and Zn2+ within both α- and β-cells as a function of glucose concentration show heterogeneous uptake of Zn2+ into α-cells that correlates to the known heterogeneities in [Ca2+]i. These differences in intracellular Zn2+ among α-cells may contribute to the inhibition in glucagon secretion observed at elevated glucose levels.
AB - 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 ([Ca2+]i). Zinc (Zn2+) 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 [Ca2+]i and Zn2+ dynamics from both α- and β-cells will elucidate mechanisms underlying islet hormone secretion. [Ca2+]i and intracellular Zn2+ 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 [Ca2+]i and Zn2+ within both α- and β-cells as a function of glucose concentration show heterogeneous uptake of Zn2+ into α-cells that correlates to the known heterogeneities in [Ca2+]i. These differences in intracellular Zn2+ among α-cells may contribute to the inhibition in glucagon secretion observed at elevated glucose levels.
UR - http://www.scopus.com/inward/record.url?scp=84979517434&partnerID=8YFLogxK
U2 - 10.1016/j.bpj.2016.06.014
DO - 10.1016/j.bpj.2016.06.014
M3 - Article
C2 - 27463142
AN - SCOPUS:84979517434
SN - 0006-3495
VL - 111
SP - 409
EP - 417
JO - Biophysical Journal
JF - Biophysical Journal
IS - 2
ER -