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

Abstract

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.

Extension of the concept of an anomalous water component to images of T-cell organelles

“…Therefore, it is logical to assume that the component referred to as 4-water 2 somehow affects microscopic phase images of cells and is detectable in these objects. [7][8][9][10][11] However, we have not found any mention of such a phenomenon in the literature. This also applies to cell images obtained by various interference microscopy methods; no effect of 4-water on the images has been reported.…”

“…Details of the technique and specimen preparation have been described earlier. [8][9][10][11] The functional state of TCs was changed by the addition of compounds with a known mechanism of pharmacological action. We used the concentrations of compounds generally reported in the literature for treatment of whole cells and organelles.…”

“…1). We have previously shown the possibility of determining the physical parameters of structural elements at the boundaries of these zones 10,11 as a system of concentric spherical layers with the diameters d ij and volumes V ij ¼ πðd…”

“…Common for all interference methods is the representation of the images in the form of a two-dimensional distribution hðx; yÞ of optical path difference (OPD) in the image plane. The most important and the unique advantage of the interference microscopy is the measurement of OPD in absolute units of wavelength [7][8][9][10][11][12][13][14][15][16][17][18][19] with a very high precision, which is hardly achievable or is even impossible with other methods. Normally, the phase image is described as the function hðx; yÞ ¼ Z ðnðx; y; zÞ − n 0 Þdz in the image plane ðx; yÞ, where nðx; y; zÞ and n 0 are the refractive indices of the object and the environment, respectively.…”

“…[12][13][14][15] Quantitative analysis of phase images of various cell types [7][8][9][10][11][12] has shown that the changes in their optical parameters caused by external factors cannot be solely explained by alterations of their proteins. In solving this problem, we suggested [7][8][9][10][11][12] that not only the change in protein RI averaged by the cell volume should be taken into account, but the possibility that the RI of intracellular water is changed should also be assumed. To test this hypothesis, we took advantage of the unique properties of cell phase images 16 and original algorithms.…”