LED array spectrophotometer for measurement of time resolved difference spectra in the 530–600 nm wavelength region

Abstract

A new type of computer controlled spectrophotometer is described which is based on an array of independent, monochromatic pulsed light sources consisting of light emitting diodes (LED) equipped with narrow band interference filters. The LEDs are sequentially pulsed at a high repetition rate. The absorbance information at specific wavelengths is sampled in the μs-time range, using a computer-controlled, highly selective technique of synchronous amplification. A first prototype of this LED Array Spectrophotometer allows simultaneous recording of kinetic changes at 16 different wavelengths in the range from 530 to 600 nm, with a time resolution of 1 ms/point. Special features of the new type of spectrophotometer are: Weak integrated measuring light intensity, high signal/noise ratio even with scattering samples like intact leaves, active baseline adjustment by LED current regulation, computer control of system operation and data analysis. To deconvolute the complex absorbance changes in the cytochrome α-band region, ‘standard spectra’ of the major components are stored in computer memory and used for curve fitting of difference spectra and kinetic changes. As an example of application, the light-induced absorbance changes in a heat-pretreated spinach leaf are analysed. The system effectively separates specific absorbance changes of C550, cyt f, cyt b ₅₅₉ and cyt b ₅₆₃ from a large background of non-specific changes.