Construction and performance of a rapid scan monochromator for multiwavelength fluorimetry

A rapid scan monochromator was constructed for multiwavelength illumination of biological samples. A light weight holographic grating (2400 grooves/mm) was driven by a rapid stepper motor with 0.014 degrees angular resolution in an f/4 monochromator. Angular velocity and acceleration kinetics were optimized to minimize the time for grating rotation and stabilization. The times required for shifting output wavelengths by 20, 100, and 300 nm were 6, 7, and 9 ms, respectively. Wavelength selection and dwell times were software controlled and reproducible over extended data collections. Instrument utility was demonstrated by measurement of intracellular calcium in fura-2-labeled Chinese hamster lung fibroblasts at excitation wavelengths of 340, 360, and 380 nm. Compared to available filter wheel and chopper devices, the rapid scan monochromator has advantages of rapid and software-selectable wavelength control, excellent optical alignment, small size, and low cost. Applications of this device include illumination optics for multiwavelength microfluorimetry and scanning absorption spectroscopy, and detection optics for microspectroscopy.