Total three-dimensional imaging of phase objects using defocusing microscopy: Application to red blood cells (original) (raw)
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Shape reconstruction and height fluctuations of red blood cells using defocusing microscopy
In this paper the bright-field defocusing microscopy (DM) technique is presented. DM is able to obtain quantitative information of each plane/surface of pure phase objects, as live unlabeled cells, and its application to red blood cells (RBCs) is demonstrated. Based on contrast, simple methods to obtain thickness profile and three dimensional (3D) total reconstruction of RBCs are proposed and the actual height profiles of upper and lower surface-membranes (lipid bilayer/cytoskeleton) of discocyte and stomatocyte red cells are presented as examples. In addition, using the mean square contrast fluctuation and modeling the RBC membranes fluctuations spectra as dependent of a bending modulus (κ c ), a surface tension (σ) and a confining potential (γ) term, slowly varying quantities along the cell radius, a genetic algorithm (GA) is used and the radial height fluctuations of each surface-membrane are accessed, separately. The radial behaviors of κ c , σ and γ are also obtained, allowing the discussion of physical aspects of the RBC membrane.
Measuring Optical and Mechanical Properties of a Living Cell with Defocusing Microscopy
Biophysical Journal, 2006
Defocusing microscopy (DM) is a recently developed technique that allows quantitative analysis of membrane surface dynamics of living cells using a simple bright-field optical microscope. According to DM, the contrast of defocused images is proportional to cell surface curvature. Although, until now, this technique was used mainly to determine size and amount of membrane shape fluctuations, such as ruffles and small random membrane fluctuations, in macrophages, its applications on cell biology extend beyond that. We show how DM can be used to measure optical and mechanical properties of a living macrophage, such as cell refractive index n, membrane bending modulus K c , and effective cell viscosity h for membrane-actin meshwork relaxation. Experimental data collected from defocused images of bone marrow-derived macrophages were used to evaluate these parameters. The obtained values, averaged over several different macrophages, are n ¼ (1.384 6 0.015), K c % 3.2 3 10 ÿ19 J, and h % 459 PaÁs. We also estimate the amplitude of the small fluctuations to be of the order of 3 nm, which is around the step size of a polymerizing actin filament.
Cell Biochemistry and Biophysics, 1989
The properties of an optical microscope are analyzed and analytically evaluated with a simple and effective model in order to understand the true meaning, limitations, and real capabilities of a defocusing technique. Major emphasis is given to the applications related to microscopic objects of biological interest using fluorescence and absorption light microscopy. A procedure for three-dimensional viewing is analyzed and discussed.
Confocal diffraction phase microscopy of live cells
Optics Letters, 2008
We present a new quantitative phase microscopy technique, confocal diffraction phase microscopy, which provides quantitative phase measurements from localized sites on a sample with high sensitivity. The technique combines common-path interferometry with confocal microscopy in a transmission geometry. The capability of the technique for static imaging is demonstrated by imaging polystyrene microspheres and live HT29 cells, while dynamic imaging is demonstrated by quantifying the nanometer scale fluctuations of red blood cell membranes.
Biomedical optics express, 2018
We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspension that are not during division can be assumed to be an ellipsoid. The entire shape of a cell in suspension can be assumed to be a sphere. Then, the cell and nucleus 3-D shapes can be evaluated based on their in-plain radii available from the 2-D phase and fluorescent measurements, respectively. Finally, the nucleus integral refractive index profile is calculated. We demonstrate the new techniq...
Subsurface imaging and cell refractometry using quantitative phase/ shear-force feedback microscopy
20th International Conference on Optical Fibre Sensors, 2009
Over the last few years, several novel quantitative phase imaging techniques have been developed for the study of biological cells. However, many of these techniques are encumbered by inherent limitations including 2π phase ambiguities and diffraction limited spatial resolution. In addition, subsurface information in the phase data is not exploited. We hereby present a novel quantitative phase imaging system without 2 π ambiguities, which also allows for subsurface imaging and cell refractometry studies. This is accomplished by utilizing simultaneously obtained shear-force topography information. We will demonstrate how the quantitative phase and topography data can be used for subsurface and cell refractometry analysis and will present results for a fabricated structure and a malaria infected red blood cell.
Diffraction phase microscopy for quantifying cell structure and dynamics
Optics Letters, 2006
We have developed diffraction phase microscopy as a new technique for quantitative phase imaging of biological structures. The method combines the principles of common path interferometry and single-shot phase imaging and is characterized by subnanometer path-length stability and millisecond-scale acquisition time. The potential of the technique for quantifying nanoscale motions in live cells is demonstrated by experiments on red blood cells.
Journal of Biomedical Optics, 2007
A method for the determination of the integral refractive index of living cells in suspension by digital holographic microscopy is described. Digital holographic phase contrast images of spherical cells in suspension are recorded, and the radius as well as the integral refractive index are determined by fitting the relation between cell thickness and phase distribution to the measured phase data. The algorithm only requires information about the refractive index of the suspension medium and the image scale of the microscope system. The specific digital holographic microscopy advantage of subsequent focus correction allows a simultaneous investigation of cells in different focus planes. Results obtained from human pancreas and liver tumor cells show that the integral cellular refractive index decreases with increasing cell radius.