A DIFFUSED-OPTICS FLASH KINETIC SPECTROPHOTOMETER (DOFS) FOR MEASUREMENTS OF ABSORBENCY CHANGES IN INTACT PLANTS IN THE STEADY-STATE

Measurements of steady-state light-induced absorbance changes in intac t plants are often hindered by interference from large changes in the light-scattering properties of the chloroplasts. In this work we prese nt a new instrument, the diffused-optics flash spectrophotometer (DOFS ), which reduces the magnitude of light scattering interference to man ageable levels. In this spectrophotometer, the conventional light path is replaced with a set of light-scrambling chambers formed from a hig hly light-scattering plastic. The main scrambling chamber acts both to homogeneously diffuse as well as to split the measuring beam into sam ple and reference channels. Since the measuring beam has no defined in cident angle, it is essentially 'pre-scattered', and further scatterin g changes that occur in the sample have minimal effect on the apparent absorbance changes. The combination of a pulsed probe light and diffe rential optics and electronics provides a high signal-to-noise ratio, stable baseline and high time resolution. We also introduce a techniqu e to account for residual scattering changes. Sets of measurements are made with the instrument in optical configurations that are different ially sensitive to light-scattering changes but yield nearly identical absorbance changes. The difference in apparent absorbance spectra tak en with the two configurations reveals the spectral shape of the scatt ering changes without interference from absorbance signals. Spectra of the scattering contributions are then used to eliminate residual scat tering interference from kinetic traces. We suggest that DOFS is ideal ly suited for study of steady-state electron transfer reactions in int act plants.