Quantitative real-time analysis of nucleolar stress by coherent phase microscopy.
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.
Tychinsky
Vladimir P
VP
Moscow Institute of Radioengineering, Electronics and Automation, 78 Vernadsky Avenue Moscow 119454, Russia. vtych@yandex.ru
Kretushev
Alexander V
AV
Klemyashov
Ivan V
IV
Vyshenskaya
Tatyana V
TV
Filippova
Natalya A
NA
Raikhlin
Natan T
NT
Shtil
Alexander A
AA
D000465:Algorithms; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology* / Q000648:ultrastructure*; D003199:Computer Systems; D006801:Humans; D007089:Image Enhancement / Q000379:methods; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D008858:Microscopy, Phase-Contrast / Q000379:methods*; D018384:Oxidative Stress; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D041623:Tomography, Optical Coherence / Q000379:methods*