Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer (IMS)

Abstract

The development of multi-colored fluorescent proteins, nanocrystals, and organic fluorophores, along with the resulting engineered biosensors, has revolutionized the study of protein localization and dynamics in living cells. Hyperspectral imaging has proven to be a useful approach for such studies, but this technique is often limited by low signal and insufficient temporal resolution. Here, we present an implementation of the snapshot hyperspectral imaging device, the Image Mapping Spectrometer (IMS), which acquires full spectral information simultaneously from each pixel in the field without scanning. The IMS is capable of real-time signals capture signals from multiple fluorophores with high collection efficiency (∼65%) and image acquisition rate (up to 7.2 fps). To demonstrate the capabilities of the IMS in cellular applications, we have combined FP-FRET and [Ca2+]i biosensors to measure simultaneously intracellular cAMP and [Ca2+]i signaling in β-cells. Additionally, we have compared quantitatively the IMS detection efficiency with a laser-scanning confocal microscope.