Quantitative light microscopy is a valuable technique for studies of cellular function. New data concerning interactions of intracellular proteins, ion concentrations, membrane potential and additional forms of cellular activity are obtained from quantitative measurement of light from exogenous and endogenous optical probes. The accuracy of these measurements relies heavily on well calibrated and characterized microscope light detection systems. This proposal describes the development of a versatile calibration standard for quantitative light microscopy. A reliable and easily used high precision standard is not commercially available and would greatly facilitate reproducible measurements. Technologies currently used as light standards for microscopy present some practical difficulties. The calibrated light output of these devices is affected by illuminator lamp noise and aging. The light output of the electronic standard described in this proposal is independent of the microscope's illuminator output. It provides repeatable flat-field illumination at multiple user selectable wavelengths and illumination levels from a single aperture. Additionally, the instrument can be used to monitor and record the intensity level of the microscope's illuminator. The instrument is uniquely versatile, having operating modes unavailable in any competing product. This technology represents a significant technical innovation in the field of light microscopy.
The versatile electronic standard described in this proposal represents a new approach for the calibration of light microscopy equipment. It has significant advantages compared to competing technologies, such as neutral density filters or fluorescent beads. Because of these advantages, this technology could capture a significant portion of the $50 million microscope calibration accessory market.
