SBIR-STTR Award

In this grant, we seek to fully implement one aspect of our overall long term goal of developing advanced instrumentation and reagents for ion and second messenger imaging and analysis. Our instrumentation development efforts have-proceeded quite well in the area of hardware and software to support any combination of dual excitation and or dual emission fluorescent ratio imaging. We will apply our unique capabilities to simultaneous ration imaging of calcium ion and cyclic AMP in single living cells. For calcium we will use the fluorescent dual wavelength excitation-single wavelength emission dye fura-2, and for cyclic AMP the single wavelength excitation-dual wavelength emission fluorescence energy transfer dye "FlCRhR". We own the exclusive world-wide rights to this technology for small molecules developed at UC San Diego. In this phase I application we will first develop the reagent for cyclic AMP imaging in production quantities, establish methods of stabllization for its distribution and determine the proper hardware, software and calibration procedures to accurately utilize it for the dynamic measurement of cyclic AMP in cells. We will then develop techniques for the simultaneous measurement of cyclic AMP and calcium in single living cells using the fluorescent probes, "FlCRhR" and fura-2. Success at simultaneous measurement of these two major cell second messengers will be important in providing the tools to elucidate a myriad of cell functions which are interrezulated by cyclic AMP and calcium. Furthermore, success in this project will confirm the applicability of this generar approach to the dynamic analysis of other substances of intraceTlular regulatory importance, potentially implicated in a wide variety of diseases. Cancer, heart and certain neurological disease are the most prominent pathological states in which more detailed knowledge of the dynamics of cyclic AMP and calcium could help in an understanding of the basic cell biological changes associated with these diseases.Awardee's statement of the potential commercial applications of the research:Support in this project will enable us to optimize and make commercially available l)the FlCRhR reagent (the only fluorescent cyclic AMP sensor for single cell ratio imaging developed so far and 2) hardware/software technologies needed for the simultaneous ratio imaging of calcium ion (fura-2) and cyclic AMP ("FlCRhR") in living cells.National Institute of General Medical Sciences (NIGMS)

During Phase I we fully implemented one aspect of our overall goal of developing advanced instrumentation and reagents for ion and second messenger imaging and analysis. We successfully produced, in production quantities, the fluorescence energy transfer reagent for cyclic AMP imaging, FICRhR, originated at UCSD, of which Atto Instruments, Inc. owns the exclusive world-wide rights. We also developed the hardware, software, and methods to utilize it for the dynamic measurement of cyclic AMP in living cells and simultaneous cAMP and calcium imaging in conjunction with fura-2.In the phase II SBIR project, Atto Instruments plans to1) market FlCRhR in its current structure and develop improved reagents for imaging CAMP as well as other second messengers,2) develop a non-radioactive fluorescence assay for cAMP in cell extracts using FlCRhR, and3) develop the hardware and software for ultra high speed microscopic ratio imaging and photometry incorporating a novel excitation light source which can switch between two wavelengths at rates higher than 5 Khz.Basic and clinical research into a myriad of cell functions, both in health and disease, regulated by cyclic AMP, calcium, and other second messengers will be advanced by the commercial availability of these important investigative tools.National Institute of General Medical Sciences (NIGMS)