Design and application of a high-precision, broad spectral range CCD-based absorption spectrometer with millisecond time resolution

Abstract

We report the design, construction and performance of a CCD-based spectrometer system developed to study electronic absorbance spectral changes associated with electron transfer processes in biological systems. The instrument was designed to operate effectively over the 180-1100 nm wavelength range. The temperature of the sample can be controlled between 5 K and above 300 K. When operating between 500 nm and 1000 nm the resolution is ∼1.7-2.4 nm and stability better than 0.001 nm, whilst maintaining close to shot-noise-limited noise performance and minimal actinic fluence. Fully reproducible spectra can be taken in a single, gated exposure as short as 100 νs. Illustrative data taken of photosystem II core complexes are presented to demonstrate the spectrometer's ability to monitor complex multi-component kinetics with precision and speed. Laser-flash-induced spectral features, previously requiring laborious point-by-point accumulation and considerable signal averaging, can now be easily seen in a single measurement.