Kinetic studies of force activation and relaxation in the single myofibril preparation have enhanced our understanding of mechanisms that underlie regulation of muscle force. Likewise, stop-flow and steady-state measurement using fluorescently labeled TnC upon Ca2+ activation in reconstituted protein systems have provided data regarding the kinetics of thin filament regulation. However, to develop a detailed understanding and fully characterize muscle regulation, an integration of mechanical, biochemical, and structural information is necessary. We present the development of a new apparatus which allows simultaneous measurement of florescent probe signals from labeled contractile proteins (such as TnC), and force kinetics under active isometric force development to allow direct correlation between these parameters in a single myofibril. Single guinea-pig cardiac myofibrils were prepared in relaxing solution (PCa8.0) by mechanical homogenization; endogenous TnC was exchanged for recombinant mutant labeled human TnC (2.5 mg/ml; overnight 4°C; Alexa-350 label conjugated to cys-84). Myofibrils were attached to glass micro-tools with one serving as a cantilever of known stiffness to assess force (painted black for improved contrast) and the other attached to a high speed motor. Myofibrils were activated with a Ca2+ pulse delivered via a double barreled perfusion pipette attached to a stepper motor (∼5 ms). Excitation UV-light (270-350 nm), chopped at 52 kHz using a photo-elastic modulator, was projected (400nm DC mirror) onto the attached myofibril. Force probe deflection was measured in bright-field using red light (670nm) while fluorescence emission was measured (420nm LP filter) using a photomultiplier. Data was sampled by a high-speed (2MHZ) A/D converter and demodulated using custom designed software. Preliminary results indicate that using this apparatus and techniques, ultra-low intensity TnC fluorescence signals can be obtained at high temporal resolution in parallel with bright-field optical measurement of force development.