Engineering of a light-sheet microscope to image intact islets in 3D.

(A) Schematic of the light-sheet microscope showing the optical configuration. (B) Representative light-sheet (upper panel) and spinning disk confocal images (lower panel) of a mouse pancreatic islet expressing β-cell specific H2B-mCherry fluorophore at different 2D focal planes, emphasizing the superior depth penetration of the light-sheet microscope. (C) 3D imaging of β-cells expressing GCaMP6s Ca2+ biosensors and nuclei mCherry biosensors. (D) Using Ins1-Cre:Rosa26GCaMP6s/H2B-mCherry islets, the software-identified center of β-cell nuclei (yellow dots) is used to generate GCaMP6s regions of interest (gray spheres). A representative Ca2+ timecourse is displayed in the right panel for an islet stimulated with glucose and amino acids.

Characterization of single β-cells using 3D network and phase analysis.

(A) Flow diagram illustrating the calculation of cell degree from pairwise comparisons between single β-cells. (B) An example 3D network for a single β-cell within a representative islet is shown with synchronized cell pairs in blue, cells that have other synchronized pairs in black, and cells that are asynchronous in red. This analysis is repeated for all cells in the islet. (C) Frequency distribution of cell degree for all β-cells analyzed. Top 10% (blue box) and bottom 10% (red box) are high and low degree cells. (D) Representative 3D illustration and Ca2+ traces showing the location of high degree cells (blue) and low degree cells (red). (E) Quantification of the normalized distance from the islet center for average degree cells (gray), high degree cells (blue), and low degree cells (red). (F) Flow diagram illustrating the calculation of cell phase, calculated from the correlation coefficient and phase shift. (G) Wave propagation from early phase cells (blue) to late phase cells (red) in 3D space (H) Frequency distribution of cell phase for all β-cells analyzed. Top 10% (blue box) and bottom 10% (red box) are early and late phase cells. (I) Representative 3D illustration and Ca2+ traces showing the location of high phase cells (blue) and low degree cells (red). (J) Quantification of the normalized distance from the islet center for average phase cells (gray), early phase cells (blue), and late phase cells (red). Data represents n = 28855 cells, 33 islets, 7 mice. Data are displayed as mean ± SEM. ****P < 0.0001 by 1-way ANOVA.

The network of highly synchronized β-cells is consistent between oscillations, while the Ca2+ wave axis rotates.

(A) 3D representation of the islet showing the location of high degree cells (blue) and low degree cells (red) over three consecutive oscillations (top panel) and their corresponding Ca2+ traces (bottom panel). (B) 3D representation of the islet showing the location of early phase cells (blue) and late phase cells (red) over three consecutive oscillations (top panel) and their corresponding Ca2+ traces (bottom panel). (C) Quantification of the retention rate of high degree and early phase cells. (D) Relative spatial change in the center of gravity of β-cell network vs. the β-cell Ca2+ wave. (E) Frequency distribution showing the normalized change in Ca2+ wave axis for islets. Data represent. Data are displayed as mean ± SEM. ****P < 0.0001 by unpaired Student’s t-test.

Cellular and regional consistency of the β-cell network and Ca2+ wave quantified by KL divergence.

(A) Schematic showing cellular consistency analysis and regional consistency analysis. (B) Schematic depicting the use of KL divergence to determine consistency between consecutive oscillations. Every β-cell in the islet is ranked, with near-zero KL divergence values indicating high consistency between oscillations and near-unity KL divergence indicating randomness. (C-D) Comparison of cellular vs. regional consistency of the network (C) and wave (D) by KL divergence. Data are displayed as mean ± SEM. *P < 0.05, ****P < 0.0001 by Student’s t-test.

3D analysis is more robust than 2D analysis.

(A) Example islet showing the locations of the ¼-depth (red) and ½-depth (blue) 2D planes used for analysis. (B) Comparison of wave axis change from 2D and 3D analysis (C) Comparison of distance from center for average and high degree cells based on either 3D (left panel) or 2D planes (middle and right panels). (D) Comparison of distance from center for average and early phase cells based on either 3D (left panel) or 2D planes (middle and right panels). (E-F) Comparison of cellular and regional consistency of the network (E) and Ca2+ wave (F) based on either 3D (left panel) or 2D planes (middle and right panels). Data are displayed as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001 by 1-way ANOVA (B) or Student’s t-test (C-F).

Effect of glycolytic activators on β-cell oscillations.

(A) Schematic of glycolysis showing the targets of glucokinase activator (GKa) and pyruvate kinase activator (PKa). (B) Illustration indicating the oscillation period, active phase duration, silent phase duration, and duty cycle (active phase/period) calculated at half-maximal Ca2+. (C-E) Sample traces and comparison of period, active phase duration, silent phase duration and duty cycle before and after vehicle (0.1% DMSO) (n=11284 cells, 13 islets, 7 mice) (C), GKa (50 nM RO-28-1675) (n=6871 cells, 8 islets, 7 mice) (D) and PKa (n=10700 cells, 13 islets, 7 mice) (10 µM TEPP-46) (E). Data are displayed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s t-test.

Glucokinase activity determines the origin of Ca2+ waves in 3D space.

(A) Illustrations showing the location change of early phase cells (blue) and late phase cells (red) before and after vehicle (left panel), GKa (middle panel) and PKa (right panel). (B-H) Effect of vehicle, GKa, and PKa on regional consistency of the Ca2+ wave (B), wave axis change (C), early phase cell retention (D), late phase cell retention (E). Data are displayed as mean ± SEM. *P < 0.05 by Student’s t-test.

Hardware wiring diagram of the light-sheet microscope.

Hardware integration (top panel) for camera-triggered activation of the excitation lasers and piezo z-stage that limits communication to a single instruction from the computer every 3 minutes. Wiring diagram (bottom panel): two Nikon ‘standard cables’ connect to the NiDAQ card installed in the computer. These two cables link to the laser control box, stage controller and camera. The images captured are received by the computer through a camera link PCIe card. and

NIS-Elements software configuration.

(A) Schematic for comparing continuous acquisition and looped acquisition. The red box indicates the 3-minute window where storage speed is higher than imaging speed. (B) Devices linked to NIS-Elements. (C) NIS-Elements JOBS module configured for looped acquisition. (D) Optical configuration for simultaneous GCaMP6s/H2B-mCherry excitation.

Location of early and late phase cells in an islet with stable wave axis.

3D representation of the islet showing the location of early phase cells (blue) and late phase cells (red) over three consecutive oscillations (top panel) and their corresponding Ca2+ traces (bottom panel).

Effect of glycolytic activators on the β-cell network.

(A-C) Effect of vehicle, glucokinase activator (GKa), and pyruvate kinase activator (PKa) on regional consistency of the β-cell network (A), high degree cell retention (B), and low degree cells retention (C). Data are displayed as mean ± SEM.