Quantitative real-time analysis of nucleolar stress by coherent phase microscopy

Abstract

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.

Quantitative phase imaging of living cells: application of the phase volume and area functions to the analysis of “nucleolar stress”

“…In this study, we introduced the SðhÞ and WðhÞ functions and demonstrated their applicability for the analysis of the previously reported phenomenon of nucleolar stress. 14 We believe that the significance of SðhÞ and WðhÞ functions is not limited to this particular model. This tool can provide valuable quantitative information largely due to independence of these functions on an optical model of the micro-object.…”

“…The fact that phase image parameters reflect cellular responses to external cues has been demonstrated earlier. [1][2][3]6,[9][10][11][12][13][14]19 Importantly, the S and W functions emerged as an important tool for monitoring local dynamics of nucleolar components. Such a dissection of phase portrait by high-resolution CPM is significant because the changes induced by Act D are neither monotonous nor are they uniformly distributed across the nucleus; general trend is a decrease of phase thickness and refractivity (Ref.…”

“…We have shown that nucleolar stress can be detected by CPM. 14 Here, 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.…”

“…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).…”

“…Measurement of this parameter, as well as refractivity, with exceptionally high accuracy allowed for registering the phase thickness (h) and volume-averaged refractive index of erythrocytes, [3][4][5][6][7][8] local fluctuations of cell dynamics, 1,2 and functional responses to extracellular stimuli. [3][4][5]7,[9][10][11][12][13][14] Still, the literature data on physical parameters of individual cellular compartments remain scarce (see Ref. 15 as an early study).…”