Pancreatic islets are functional micro-organs involved in maintaining normoglycemia through regulated secretion of insulin and other hormones. Extracellular glucose stimulates insulin secretion from islet β-cells through an increase in metabolic state, which can be measured using two-photon NAD(P)H imaging. Extracellular glucose concentrations of >7 mM generate synchronous β-cell calcium (Ca2+) oscillations leading to pulsatile insulin secretion. Our studies have focused on the coupling of cells within the islet using quantitative fluorescence imaging of metabolic (NAD(P)H) and electrical (Ca2+) responses. This imaging requires immobilization of the tissue to achieve subcellular spatial and subsecond temporal resolutions. We have developed microfluidic devices to stimulate islets while holding them stationary against a glass coverslip. One device is a dual-channel microfluidic that allows heterogeneous islet stimulation by introducing a standing gradient in glucose concentration across the islet. The second device is a single channel microfluidic with the general utility to hold islets static. This chapter will describe the fabrication and use of these devices, with specific reference to their demonstrated utility for quantitative and dynamic imaging of living pancreatic islets. We will also highlight the general utility of these devices as a paradigm transferable to the study of other tissues.