A dynamic phase microscopic study of optical characteristics of individual chloroplasts

摘要

Dynamic phase microscopy (DPM) allows the monitoring of optical path difference (or phase height), It (x,y,t) approximate to h (x,y,z,t)dz, an integral refractive index projection of the medium, n(x,y,z,t), in optically transparent biological specimens at high spatial and temporal resolutions. In this study, DPM was used for the analysis of fluctuations in the optical characteristics of individual heart chloroplasts in various metabolic states. A "phase image" of an individual chloroplast, which represents a three-dimensional plot of the "phase height", was obtained for the first time, and the frequency spectra of the fluctuations of h(x,y,t) were investigated. The fluctuation patterns, i.e., the intensity and the frequency spectra of phase height fluctuations in bean chloroplasts (Class 13) were found to depend on their metabolic state. Under conditions of noncyclic (or pseudocyclic) electron transport, the fluctuations displayed characteristic frequencies in the range of 0.25-0.6 Hz and were space-time-correlated in the chloroplast domains with the cross sizes of similar to2 mum. The fluctuation intensity decreased in the presence of uncouplers (nigericin and valinomycin, 20 muM). A stronger (in comparison with 20 muM valinomycin) effect of 20 muM nigeticin suggests that the light-induced generation of the transmembrane pH difference (DeltapH) makes the main contribution to the increment of space-correlated fluctuations of h(x,y,t). Studies of chloroplasts incubated in media of various osmolarity (50-500 mM sucrose) have shown that structural changes in thylakoids are among other factors responsible for phase height fluctuations. (C) 2004 Elsevier B.V. All rights reserved.