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 3D spatial-spectral object cube to a 2D 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 5.6 Mpix sCMOS camera. The system will be optimized for throughput and will permit imaging at up to 100 frames/second rates, and up to 16-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 250x250x60 with spectral resolution of 5 nm over 470 ? 670 spectral range, and effective spatial resolution of 0.5 micron.
In Aim 1, 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, [Ca2+]i, cAMP dynamics, and [ATP]/[ADP] ratio. We will also quantitatively evaluate the results from the IMS against the Zeiss LSM710 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-4, and mCherry/SNARF-1/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 3- 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 3 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 efficient
The 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.
