A novel Microscope Illumination Modulation System is proposed that will conveniently attach to most microscope condenser or epi-illumination ports and provide complete spatio-temporal illumination control and the capability of using multiple light sources at fast alternation speeds. The specific aims of this proposal are to design and model the system and to build and test certain key sub-components necessary to prove its feasibility. Design and modeling methodologies will involve geometric and diffraction optical theory and ray-tracing. Photometric measurements will be made to verify the models, analyze aberrations, and test sub- component performance. This invention will uniquely offer the flexibility and control required for 1) spatial photolysis of "caged" compounds (including neurotransmitters, calcium, ATP, etc.), 2) modulated light pattern generation for fluorescence recovery after photobleaching (FRAP) and photoactivation of fluorescence (PAF), 3) high-speed ratio imaging (up to 1000 images/sec) for use with dual-wavelength fluorescent excitation indicators used to measure intracellular calcium, pH, sodium, etc. or for use in alternating polarization illumination, 4) enhanced diffraction- limited, uniform-field illumination for improved brightfield, polarized light, differential interference contrast (DIC), phase contrast, and fluorescence microscopy, and 5) computer control and straightforward integration with existing image and data acquisition systems.Proposed commercial application:The system should have a broad commercial sales potential in biomedical labs where it will have many diverse applications in basic and applied research within academia, industry, and the government. For example, its capabilities for pattern- selective photo-catalysis and high-speed ratio imaging make it ideal for applications in enzyme and pharmacological kinetics for drug testing and development. It will enable higher spatio-temporal resolution using fluorescent indicators that are widely used to measure critical intracellular second messengers, receptor densities, and other synaptic properties.National Institute of Mental Health (NIMH)
