PTI RatioMaster is a monochromator-based wide-field microscope system for measuring ratio-fluorescence or fluorescence dye intensity of labeled proteins in nanomolar concentrations. Ratio-fluorescence microscopy is the ultimate tool to study dynamic events in live cells and cellular structures. Unlike conventional intensity based fluorescence microscopy, ratio-fluorescence eliminates the effects of photo-bleaching, cell path length variation, non-uniformity of dye loading, non-uniform field of view, making ratio-fluorescence the most accurate method to determine intracellular ion concentrations. More importantly, PTI RatioMaster is fast enough to provide essential dynamic information about ion concentrations and interactions with other molecules, drugs or added reagents.
The PTI RatioMaster is capable of dynamic ratio fluorescence measurements on a millisecond timescale. A xenon arc lamp provides high intensity, continuous broadband illumination. Alternating excitation wavelengths are selected by a computer-controlled high-speed random access monochromator coupled to a (user specified/supplied) inverted or upright, fluorescence microscope with a liquid light guide. Emitted light is collected from the sample and passed through a photometer having a bilateral translatable iris and a viewing eyepiece to a switchable analog/photon counting photomultiplier detector. Analog detection is used when emitted light is relatively intense; low light levels are detected by photon counting. All system functions are under computer control. Data is collected and analyzed by proprietary Windows FelixGX based advanced fluorescence software.