High performance imaging spectrometer for biomedical applications

The overall goal of this research is to develop a high dynamic range snapshot hyperspectral microscope for imaging live biological samples The proposed system offers faster frame rates and higher dynamic range than current methods for multi spectral imaging of live cells while still permitting diffraction limited resolution with low photobleaching The system is fully parallel so data at all wavelengths is collected simultaneously from the entire field of view Thus this approach has great potential to overcome limitations of existing hyperspectral systems and enable real time imaging of multiple signaling processes in living cells Towards this goal our first aim will focus on the developments of a high performance Image Mapping Spectrometer IMS The IMS transforms a D spatial spectral object cube to a D mapped image which allows acquisition in the snapshot mode The core of the system is the custom made redirecting mirror required for this mapping operation For low noise imaging the proposed IMS will employ a Mpix sCMOS camera The system will be optimized for throughput and will permit imaging at up to frames second rates and up to bit dynamic range In addition we will develop a synchronized structured illumination mode to provide optical sectioning capability similar to that of confocal systems For the initial systems we will provide for datacube dimensions spanning from x x with spectral resolution of nm over spectral range and effective spatial resolution of micron In Aim we will also test the IMS spectrometer against currently available spectral imaging systems in several living cell imaging applications These experiments will focus on tests of dynamic biological systems which are routinely being used in the lab of Dr David Piston s lab collaborator on the project In the Piston lab quantitative imaging measurements are being use specifically to study many cell signaling features including cellular metabolism membrane potential Ca i cAMP dynamics and ATP ADP ratio We will also quantitatively evaluate the results from the IMS against the Zeiss LSM META and Optical Insights Spectral DV systems System evaluations will utilize variety of fluorophore combinations starting with two color pairs moving to more complex combinations such as CFP GFP YFP Fluo and mCherry SNARF Fura Red In the second aim we will develop high performance calibration and real time linear unmixing procedures This aim will lead to optimized performance in regard of resolution spectral unmixing and data collection for dimensional optically sectioned imaging x y This aim will also provide software capable of displaying both data cubes and pseudo color unmixed images in real time This last feature is critical for live cell imaging as it will provide immediate feedback during real time experiments Aim focusses on development of a new technique of mapping mirror fabrication based on polishing of stacks of thin glass plates of appropriate angles to make process more robust and production efficientThe project targets the development of a modern spectrometer called high sampling Image Mapping Spectrometer enabling high resolution spectral imaging in real time In consequence researchers will be able to rapidly advance the investigation of live cells with multiple fluorescent contrasts The instrument s principle allows obtaining spectral information for entire image without scanning and thus improve signal to noise ratio and limit photo bleaching effects It also allows more efficient investigation of transient biological events Technologies applied in the project and their low cost may potentially allow access of larger group of scientists to spectral imaging instrumentation