Chlorophyll a fluorescence measurements of isolated spinach thylakoids obtained by using single-laser-based flow cytometry.

Flow cytometry data of spinach thylakoid membrane preparations indicate the presence of a homogeneous thylakoid population. Fluorescence data from a flow cytometer and comparison with data from two other fluorometers show that chlorophyll a fluorescence detected with a flow cytometer has the character of maximum fluorescence (Fmax), not of the constant component (Fo). This conclusion is important since Fo measures fluorescence that is affected mostly by changes in excitation energy transfer and Fmax-Fo (the variable fluorescence) by changes in photochemistry. This was demonstrated by: 1) The light intensity as well as diffusion rate dependence of the quenching effect of various quinones (p-benzoquinone, phenyl-benzoquinone, and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on fluorescence yield; quenching for the same concentration of these quinones was lower at the higher than at the lower light intensities. 2) Temperature dependence of the fluorescence yield; increasing the temperature from 20 to 70 degrees C did not show an increase in fluorescence yield using a flow cytometer in contrast to measurements with weak excitation light, but similar to those obtained for Fmax. 3) Addition of an inhibitor diuron up to 100 microM did not change the fluorescence intensity. A comparison of quenching of fluorescence by various quinones obtained by flow cytometry with those by other fluorometers suggests that the high intensity used in the cytometry produces unique results: the rate of reduction of quinones in much larger than the rate of equilibration with the bulk quinones.