Visualization of functional properties of individual cells and intracellular organelles still remains an experimental challenge in cell biology. The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this work, we report results of statistical analysis of CPM images of cyanobacterial cells (Synechocystis sp. PCC 6803) and spores (Bacillus licheniformis). It has been shown that CPM images of cyanobacterial cells and spores are sensitive to variations of their metabolic states. We found a correlation between one of optical parameters of the CPM image ('phase thicknesses' Deltah) and cell energization. It was demonstrated that the phase thickness Deltah decreased after cell treatment with the uncoupler CCCP or inhibitors of electron transport (KCN or DCMU). Statistical analysis of distributions of parameter Deltah and cell diameter d demonstrated that a decrease in the phase thickness Deltah could not be attributed entirely to a decrease in geometrical sizes of cells. This finding demonstrates that the CPM technique may be a convenient tool for fast and non-invasive diagnosis of metabolic states of individual cells and intracellular organelles.

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.

We present a concept for quantitative characterization of a functional state of an individual eukaryotic cell based on interference imaging. The informative parameters of the phase images of quiescent and mitogen-activated T lymphocytes included the phase thickness, phase volume, the area, and the size of organelles. These parameters were obtained without a special hypothesis about cell structure. Combinations of these parameters generated a "phase portrait" of the cell. A simplified spherical multilayer optic model of a T lymphocyte was used to calculate the refractivity profile, to identify structural elements of the image with the organelles, and to interpret the parameters of the phase portrait. The values of phase image parameters underwent characteristic changes in the course of mitogenic stimulation of T cells; thereby, the functional state of individual cells can be described using these parameters. Because the values of the components of the phase portrait are measured in absolute units, it is possible to compare the parameters of images obtained with different interference microscopes. Thus, the analysis of phase portraits provides a new and perspective approach for quantitative, real-time analysis of subcellular structure and physiologic state of an individual cell.

The assessment of metabolism of microobjects is a topical problem of biology. Studies of cells and subcellular structures and their responses to extracellular stress stimuli are of fundamental importance. Important practical applications of such studies include the selection of biologically active substances and the diagnostics of metabolic disturbances. The solution of these problems requires methods that allow quantitative analysis of physiological processes in real time using nonfixed single cells, without utilization of fluorescent dyes or other invasive approaches which may introduce experimental artifacts. The heterogeneity of animal cells and microorganisms stimulates the development of novel methods for the analysis of physiological state of single cells and recording of individual response to changed microenvironment [1]. The method of coherent phase microscopy (CPM) developed by our group [2, 3] is based on representation of a real biological object as a spatially heterogeneous distribution of optical refraction index. The phase images obtained by interference techniques represent two-dimensional distributions of optical path difference (or phase thickness). Therefore, the structures with higher optical density are clearly distinguished in such phase images. Hence, the intracellular structures that have different refraction indices will be present in the static phase images. The local changes in the refraction index may be a result of metabolic processes and can be measured as changes in the phase thickness of intracellular structures [2][3][4][5][6][7][8]. CPM was used to study the metabolic state of single large organelles (isolated mitochondria) [4 − 6], plant cells (bean chloroplasts) [7], cyanobacteria, and spores [3]. A decrease in the phase thickness induced by pharmacologic uncoupling of mitochondrial transmembrane potential of protons or inhibition of electron transport (deenergization) was detected [3 − 7]. These studies showed that it is possible to assess the response of living biological objects to changes in homeostasis by CPM.The purposes of this work were (1) to identify the optically dense structures in the whole (unfractionated) cells by the CPM method and (2) to determine optical parameters of the disturbances of the state of nucleoli induced by suppression of gene transcription.The experiments were performed with the cell lines NIH 3T3 (immortalized murine fibroblasts) and HCT116 (transformed human colon epithelium) cultured in Dulbecco's modified Eagle's medium supplemented with 10% bovine serum, 2 mM glutamine, and gentamicin. The cells were maintained at 37°ë in 5% ëé 2 humidified atmosphere. The cells were seeded on cover slips 24 h before experiments; the monolayer density at the day of experiments was 50-75%. The cells were incubated with pharmacological reagents; the cover slips were placed on a polished silicon substrate and analyzed by CPM. Single cells were first studied using the optical channel of CPM; then, their topograms and phase thickness profiles were measured. Abou...

We develop a method of coherent phase microscopy (CPM) for direct visualization of nonfixed, nonstained mammalian cells (both cultured cells and freshly isolated tumor biopsies) followed by computer-assisted data analysis. The major purpose of CPM is to evaluate the refractive properties of optically dense intracellular structures such as the nucleus and the nucleoli. In particular, we focus on quantitative real-time analysis of the nucleolar dynamics using phase thickness as an equivalent of optical path difference for optically nonhomogenous biological objects. Pharmacological inhibition of gene transcription leads to a dramatic decrease of the phase thickness of the nucleoli within the initial minutes of cell exposure. Furthermore, the acute depletion of intracellular ATP pool, depolymerization of microtubules and inhibition of DNA replication resulted in a rapid decrease of the nucleolar phase thickness. These optical effects were paralleled by segregation of nucleolar components as documented by electron microscopy. Thus, CPM detects early changes of nucleolar dynamics, in particular, the nucleolar segregation as part of general cellular response to cytotoxic stress, regardless of whether the nucleolus is or is not the primary target of the toxin. CPM is applicable for monitoring and quantitative analysis of the "nucleolar stress" in living mammalian cells.