Abstract
Dynamic phase microscopy (DPM) allows the monitoring of optical path difference (or phase height), h( x, y, t) ≈∫ n( x, y, z, t)d z, 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 bean 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 B) 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 ∼2 μm. The fluctuation intensity decreased in the presence of uncouplers (nigericin and valinomycin, 20 μM). A stronger (in comparison with 20 μM valinomycin) effect of 20 μM nigericin suggests that the light-induced generation of the transmembrane pH difference (ΔpH) 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.
Published Version
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