Real-Time Hyperspectral Imaging of Multiple Biosensors in Pancreatic Beta Cells

Abstract

Spectral imaging techniques are important for many biological experiments, particularly live-cell quantitative imaging of multiple fluorescence probes. Existing hyperspectral imaging systems require sequential techniques, limiting the data acquisition rate. A newly-developed snapshot device, the Image Mapping Spectrometer (IMS), acquires full spectral information simultaneously from every pixel in the field with image acquisition rates up to 10 frames/second. The IMS maps adjacent pixels from the object to create space between them in the image, and then uses a grating to spread wavelength content from each pixel into this space. Direct image re-mapping provides the final 3D (x, y, lambda) data cube. Fluorescent protein (FP)-based biosensors are increasingly valuable tools for identifying subcellular dynamic processes in live cells. Many biosensors are based on FP-FRET, and measurements of the resultant small changes in FRET require high quality data. Tracking intracellular free Ca2+ levels is also crucial to elucidating signaling events, but the best Ca2+ indicator dyes overlap in the spectral emission range of the common FP-FRET biosensors. We have used the IMS system to simultaneously image multicolored FPs (i.e., CFP, GFP, YFP) in combination with extrinsic indicator dyes, such as Fluo-4. To demonstrate the speed and resolution of the IMS approach, we monitored both intracellular Ca2+ oscillations and caspase-3 activity during hydrogen peroxide-induced apoptosis. Ca2+ activity was measured with Fluo-4 (emission peak at 517 nm) and caspase-3 activity was measured with SCAT3.1, a FRET biosensor based on the ECFP (emission peak at 484 nm) and EYFP (emission peak at 527 nm) pair. Using the IMS, the three fluorophores were imaged with sub-second temporal resolution and spectrally unmixed in real-time. This permits direct correlation of Ca2+ activity with other apoptotic signaling events and demonstrates the power of the IMS for measuring dynamic physiological processes.