Browsing by Author "Ustione, Alessandro"

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    Hyperspectral imaging for simultaneous measurements of two FRET biosensors in pancreatic β-cells
    (Public Library of Science, 2017) Elliott, Amicia D.; Bedard, Noah; Ustione, Alessandro; Baird, Michelle A.; Davidson, Michael W.; Tkaczyk, Tomasz; Piston, David W.
    Fluorescent protein (FP) biosensors based on Förster resonance energy transfer (FRET) are commonly used to study molecular processes in living cells. There are FP-FRET biosensors for many cellular molecules, but it remains difficult to perform simultaneous measurements of multiple biosensors. The overlapping emission spectra of the commonly used FPs, including CFP/YFP and GFP/RFP make dual FRET measurements challenging. In addition, a snapshot imaging modality is required for simultaneous imaging. The Image Mapping Spectrometer (IMS) is a snapshot hyperspectral imaging system that collects high resolution spectral data and can be used to overcome these challenges. We have previously demonstrated the IMS’s capabilities for simultaneously imaging GFP and CFP/YFP-based biosensors in pancreatic β-cells. Here, we demonstrate a further capability of the IMS to image simultaneously two FRET biosensors with a single excitation band, one for cAMP and the other for Caspase-3. We use these measurements to measure simultaneously cAMP signaling and Caspase-3 activation in pancreatic β-cells during oxidative stress and hyperglycemia, which are essential components in the pathology of diabetes.
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    Real-time hyperspectral fluorescence imaging of pancreatic b-cell dynamics with the image mapping spectrometer
    (The Company of Biologists Ltd, 2012) Elliott, Amicia D.; Gao, Liang; Ustione, Alessandro; Bedard, Noah; Kester, Robert; Piston, David W.; Tkaczyk, Tomasz S.
    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 a 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 signal capture 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 fluorescent protein (FP)-FRET and [Ca2+]i biosensors to measure simultaneously intracellular cAMP and [Ca2+]i signaling in pancreatic b-cells. Additionally, we have compared quantitatively the IMS detection efficiency with a laser-scanning confocal microscope.