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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.
Highlights
The development of methods of interference microscopy opened new perspectives in living cell imaging, largely due to high sensitivity and realtime recording.[1,2,3,4,5,6,7,8,9,10,11,12] The phase imaging provides unique opportunity to dissect the biological object using normalized values of optical path difference (OPD)
The coherent phase microscopy (CPM)[9,10,11,12] has been proved to be informative for quantitative real-time monitoring of a variety of processes in living pro- and eukaryotic cells.[9,12,13,14]. Using this method and a simplified spherical model of a T-lymphocyte, we reported the numerical values of key physical parameters of the nucleus and organelles.[13]
We have shown that nucleolar stress can be detected by CPM.[14]
Summary
The development of methods of interference microscopy (dSLIT, SLIM, DHM, WFDI, CPM) opened new perspectives in living cell imaging, largely due to high sensitivity and realtime recording.[1,2,3,4,5,6,7,8,9,10,11,12] The phase imaging provides unique opportunity to dissect the biological object using normalized values of optical path difference (OPD). The coherent phase microscopy (CPM)[9,10,11,12] has been proved to be informative for quantitative real-time monitoring of a variety of processes in living pro- and eukaryotic cells.[9,12,13,14] Using this method and a simplified spherical model of a T-lymphocyte, we reported the numerical values of key physical parameters of the nucleus and organelles.[13] In this study, we applied the developed algorithms to the analysis of a more complex object, that is, a human colon carcinoma cell (HCT116 cell line) exposed to the antitumor drug actinomycin D (Act D) This treatment is known to cause an inhibition of gene transcription and segregation of components of the nucleoli, a highly dynamic system whose major functions are ribosomal biogenesis and regulation of extracellular stress responses.[16,17,18] The phenomenon of “nucleolar stress” has been demonstrated to be one of the earliest hallmarks of cellular response to many toxins. We demonstrated that integral functions derived from phase images of intact and Act D-treated cells, namely, the area SðhÞ and the phase volume WðhÞ, are valuable instruments for quantitative evaluation of physical parameters of subcellular structures
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