A dynamic phase microscopic study of optical characteristics of individual chloroplasts

Abstract

Dynamic phase microscopy (DPM) allows the monitoring of optical path difference (or phase height), h(x,y,t) approximately integraln(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 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 approximately 2 microm. The fluctuation intensity decreased in the presence of uncouplers (nigericin and valinomycin, 20 microM). A stronger (in comparison with 20 microM valinomycin) effect of 20 microM nigericin 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.

Study of optical parameters of the nucleoli under the effect of transcription inhibitors by coherent phase microscopy

“…3, b). These signs indicate the beginning segregation of the nucleolus components, a phenomenon characterizing actinomycin D effect [5]. By the 30th min of exposure dissociation of the structures inside the nucleoli progresses: the number and size of disorderly scattered dense areas increases (compact FC), alternating with rarefaction zones (Fig.…”

“…We developed a method of coherent phase microscopy (CPM) and demonstrated the possibility of using it for studies of metabolism in chloroplasts, spores, and isolated mitochondria [5,6]. Analysis of optically dense organelles in live cells is a new application of CPM [1].…”

“…V. P. Tychinsky Life-time imaging of biological objects is a most important requirement to evaluation of their structure and physiological status. We developed a method of coherent phase microscopy (CPM) and demonstrated the possibility of using it for studies of metabolism in chloroplasts, spores, and isolated mitochondria [5,6]. Analysis of optically dense organelles in live cells is a new application of CPM [1].…”

The metabolic component of cellular refractivity and its importance for optical cytometry

“…It was most evident in energy-transdu- [33] cing objects (mitochondria, chloroplasts, cyan bacteria, and spores) with relatively large surfaces of phospholipids membranes in their structure. Detailed description of this phenomenon can be found in [5,[14][15][16][17]. The refractivity decrease in de-energizing was less distinct in nuclei and nucleoli of human cells [21].…”

“…The origin of the refractivity component that was found to be dependent on the functional state, further referred to as the metabolic component of refractivity (MCR), remains mysterious. Later, MCR was found in bacteria spores [16], chloroplasts [17], cyan bacteria, and human cells [18][19][20][21]. The phenomenon, surprising in its universal character, led us to the hypothesis that the common factor might be water [5].…”