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.

Abstract. We applied coherent phase microscopy to develop a method of quantitative evaluation of functional state of eukaryotic cells using the coordinates of characteristic points (CP) in the functions of the phase volume W and area S. In a fragment of a single cell image (HCT116 human colon carcinoma cell line) with detectable nucleolus, the values of the phase thickness, area, and volume were calculated. These values dramatically changed within the initial minutes of cell exposure to the transcriptional inhibitor actinomycin D. The positions of CP in the graphs of S and W functions allowed for monitoring the time-dependent decrease of nucleolar contrast, a major optical hallmark of "nucleolar stress." Given that the area and volume functions reflect optical heterogeneity of the cell and are independent of its optical model, these functions can be applicable as general mathematical tools for the analysis of cell morphology and physiology. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

The use of coherent phase microscopy for online quantitative registration of nucleolar reaction to transcription inhibition is validated. Reduction of phase thickness of the nucleoli was detected during the first minutes of the experiment; 30 min after addition of the drug rarefaction zones predominated and areas of condensation were seen. These changes reflect the dynamics of disorders in the nucleolar ultrastructure during transcription inhibition.

Optical parameters of human cell nucleoli (HCT116 colorectal cancer cells) in depolymerization of microtubules and depletion of intracellular ATP pool were studied by coherent phase microscopy. These influences were associated with a rapid (recorded within the first minutes) reduction of the phase thickness of the nucleoli. These changes are similar to the nucleolar response to direct inhibitors of transcription. Hence, quantitative parameters of coherent phase microscopy describe common reaction of the nucleolus to stress; reduction of optical thickness of the nucleolus is a component of this reaction.