Dissecting eukaryotic cells by coherent phase microscopy: quantitative analysis of quiescent and activated T lymphocytes

Abstract

We present a concept for quantitative characterization of a functional state of an individual eukaryotic cell based on interference imaging. The informative parameters of the phase images of quiescent and mitogen-activated T lymphocytes included the phase thickness, phase volume, the area, and the size of organelles. These parameters were obtained without a special hypothesis about cell structure. Combinations of these parameters generated a "phase portrait" of the cell. A simplified spherical multilayer optic model of a T lymphocyte was used to calculate the refractivity profile, to identify structural elements of the image with the organelles, and to interpret the parameters of the phase portrait. The values of phase image parameters underwent characteristic changes in the course of mitogenic stimulation of T cells; thereby, the functional state of individual cells can be described using these parameters. Because the values of the components of the phase portrait are measured in absolute units, it is possible to compare the parameters of images obtained with different interference microscopes. Thus, the analysis of phase portraits provides a new and perspective approach for quantitative, real-time analysis of subcellular structure and physiologic state of an individual cell.

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

“…The estimation of absolute nucleolar refractivity Δn 4 ¼ Δn 3 þ δn 34 ≈ 0.05 − 0.055 came from the values of refractivity of the nucleus (Δn 3 ) or the dense part (Δn 2 ) of the cell. 13 The values Δn 2 ≈ Δn 3 ¼ 0.035 − 0.04 were close to the respective numbers for the dense part of a T-lymphocyte. thickness profile [ Fig.…”

“…The set of CP parameters (h ij , d ij , S ij , and W ij ) has been termed by us a "phase portrait" of the cell. 13 In this study, we performed quantitative analysis of a fragment of the cell's image, not of the entire cell. The indices of parameters h ij , d ij , S ij , and W ij (Figs.…”

“…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.…”

“…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).…”

Application of Phase Images for Estimation of Peritoneal Macrophages State

“…There are a lot of various type of cell were estimated using different methods of interference microscopy: erythrocytes (see e.g. Popescu et al, 2005;Popescu et al, 2006;Rappaz et al, 2007;Rappaz et al, 2008;Rappaz et al, 2009;Yusipovich, I et al, 2009;Yusipovich et al, 2011), fibroblasts (Dunn & Zicha, 1995), lymphocytes (Tychinsky et al, 2012), neurons (Rappaz et al, 2005;Yusipovich et al, 2006), nerve fibers (see e.g. Bibineyshvili et al, 2013;Brazhe et al, 2008;LaPorta & Kleinfeld, 2012;Maksimov et al, 2001), mast cells (Bondarenko et al, 2013), any microbiological objects (Tychinskii et al, 2007;Yusipovich et al, 2012;Zhurina et al, 2013) and others.…”