Browsing by Author "Bedard, Noah"
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Item 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.; BioengineeringFluorescent 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.Item Image mapping spectrometry: calibration and characterization(Society of Photo-Optical Instrumentation Engineers, 2012) Bedard, Noah; Hagen, Nathan; Gao, Liang; Tkaczyk, Tomasz S.; BioengineeringImage mapping spectrometry (IMS) is a hyperspectral imaging technique that simultaneously captures spatial and spectral information about an object in real-time. We present a new calibration procedure for the IMS as well as the first detailed evaluation of system performance. We correlate optical components and device calibration to performance metrics such as light throughput, scattered light, distortion, spectral image coregistration, and spatial/spectral resolution. Spectral sensitivity and motion artifacts are also evaluated with a dynamic biological experiment. The presented methodology of evaluation is useful in assessment of a variety of hyperspectral and multi-spectral modalities. Results are important to any potential users/ developers of an IMS instrument and to anyone who may wish to compare the IMS to other imaging spectrometers.Item Multi-Modal Imaging Techniques for Early Cancer Diagnostics(2012-09-05) Bedard, Noah; Tkaczyk, Tomasz S.; Richards-Kortum, Rebecca Rae; Merenyi, Erzsebet; Gillenwater, Ann M.Cancer kills more Americans under the age of 75 than any other disease. Although most cancers occur in epithelial surfaces that can be directly visualized, the majority of cases are detected at an advanced stage. Optical imaging and spectroscopy may provide a solution to the need for non-invasive and effective early detection tools. These technologies are capable of examining tissue over a wide range of spatial scales, with widefield macroscopic imaging typically spanning several square-centimeters, and high resolution in vivo microscopy techniques enabling cellular and subcellular features to be visualized. This work presents novel technologies in two important areas of optical imaging: high resolution imaging and widefield imaging. For subcellular imaging applications, new high resolution endomicroscope techniques are presented with improved lateral resolution, larger field-of-view, increased contrast, decreased background signal, and reduced cost compared to existing devices. A new widefield optical technology called multi-modal spectral imaging is also developed. This technique provides real-time in vivo spectral data over a large field-of-view, which is useful for detecting biochemical alterations associated with neoplasia. The described devices are compared to existing technologies, tested using ex vivo tissue specimens, and evaluated for diagnostic potential in a multi-patient oral cancer clinical trial.Item Multimodal snapshot spectral imaging for oral cancer diagnostics: a pilot study(Optical Society of America, 2013) Bedard, Noah; Schwarz, Richard A.; Hu, Aaron; Bhattar, Vijayashree; Howe, Jana; Williams, Michelle D.; Gillenwater, Ann M.; Richards-Kortum, Rebecca; Tkaczyk, Tomasz S.; Bioengineering; Electrical and Computer EngineeringOptical imaging and spectroscopy have emerged as effective tools for detecting malignant changes associated with oral cancer. While clinical studies have demonstrated high sensitivity and specificity for detection, current devices either interrogate a small region or can have reduced performance for some benign lesions. We describe a snapshot imaging spectrometer that combines the large field-of-view of widefield imaging with the diagnostic strength of spectroscopy. The portable device can stream RGB images at 7.2 frames per second and record both autofluorescence and reflectance spectral datacubes in < 1 second. We report initial data from normal volunteers and oral cancer patients.Item 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.; BioengineeringThe 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.Item Real-time video mosaicing with a high-resolution microendoscope(Optical Society of America, 2012) Bedard, Noah; Quang, Timothy; Schmeler, Kathleen; Richards-Kortum, Rebecca; Tkaczyk, Tomasz S.; Bioengineering; Electrical and Computer EngineeringMicroendoscopes allow clinicians to view subcellular features in vivo and in real-time, but their field-of-view is inherently limited by the small size of the probe's distal end. Video mosaicing has emerged as an effective technique to increase the acquired image size. Current implementations are performed post-procedure, which removes the benefits of live imaging. In this manuscript we present an algorithm for real-time video mosaicing using a low-cost high-resolution microendoscope. We present algorithm execution times and show image results obtained from in vivo tissue.Item Snapshot spectrally encoded fluorescence imaging through a fiber bundle(Society of Photo-Optical Instrumentation Engineers, 2012) Bedard, Noah; Tkaczyk, Tomasz S.; BioengineeringFiber optic endomicroscopy is a valuable tool for clinical diagnostics and animal studies because it can capture images of tissue in vivo with subcellular resolution. Current configurations for endomicroscopes have either limited spatial resolution or require a scanning mechanism at the distal end of the fiber, which can slow imaging speed and increase the probe size. We present a novel configuration that provides high contrast 350 × 350 pixel images at 7.2 frames per second, without the need for mechanical scanning at the proximal or distal end of the fiber. The proofof- concept benchtop system is tested in fluorescence mode and can resolve 1.5 μm features of a high resolution 1951 USAF target.