Coherent phase microscopy in cell biology: visualization of metabolic states

Abstract

Visualization of functional properties of individual cells and intracellular organelles still remains an experimental challenge in cell biology. The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this work, we report results of statistical analysis of CPM images of cyanobacterial cells (Synechocystis sp. PCC 6803) and spores (Bacillus licheniformis). It has been shown that CPM images of cyanobacterial cells and spores are sensitive to variations of their metabolic states. We found a correlation between one of optical parameters of the CPM image ('phase thicknesses' Deltah) and cell energization. It was demonstrated that the phase thickness Deltah decreased after cell treatment with the uncoupler CCCP or inhibitors of electron transport (KCN or DCMU). Statistical analysis of distributions of parameter Deltah and cell diameter d demonstrated that a decrease in the phase thickness Deltah could not be attributed entirely to a decrease in geometrical sizes of cells. This finding demonstrates that the CPM technique may be a convenient tool for fast and non-invasive diagnosis of metabolic states of individual cells and intracellular organelles.

The metabolic component of cellular refractivity and its importance for optical cytometry

“…The hypothesis is discussed in detail below in the light of new data on water structure [22][23][24][25][26][27][28]. The aforementioned works [5][6][7][8][9][10][11] and the results of our measurements [14][15][16][17][18][19][20][21] lead to the conclusion that MCR is of fundamental importance for biophysics and optical cytometry.…”

“…The microscope provided spatial resolution up to 50 nm, time resolution 1-10 ms, sensitivity in OPD measurements 0.5 nm. A more indepth discussion of device parameters and description of measurement technique can be found in [14][15][16][17][18][19][20][21].…”

“…However our measurements [5,[14][15][16][17][18] have shown that the metabolic component dn is usually sufficiently large and exceeds the possible error in refractivity assessments by many times. As a rule, the refractive index of an immersion medium (n 1 ffi 1.33-1.34) does not differ very much from n 0 ¼ 1.333 in bulk water, which allows us to interpret measurement results in terms of refractivity Dn ¼ n À n 0 in relation to the well known constant (n 0 ).…”

“…Interference methods are highly sensitive and therefore enable to register changes in the functional state of the bio-object. In the mitochondrion we discovered a regular dependence of phase thickness and refractive index on the composition of incubation medium that determined its functional state [14,15]. For example, refractivity fell when the activity of enzymes in the mitochondrion's respiratory chain was inhibited, and it rose when the enzymes were stimulated.…”

Nanoscale surface characterization using laser interference microscopy

“…This class brings together methods of white light interferometry [1,2], phase shift interferometry [3][4][5], digital holographic microscopy [6,7], laser interference microscopy [8,9], coherent phase microscopy [10], coherent correlation interferometry [11,12], etc.…”