Reflectivity and topography of cells grown on glass-coverslips measured with phase-shifted laser feedback interference microscopy (original) (raw)
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Phase-modulation laser interference microscopy: an advance in cell imaging and dynamics study
Journal of Biomedical Optics, 2008
This paper describes how phase-modulation laser interference microscopy and wavelet analysis can be applied to non-invasive non-stained visualization and study of the structural and dynamical properties of living cells. We show how phase images of erythrocytes can reveal the difference between various erythrocyte forms and stages of hemolysis and how phase images of neurons reveal their complex intracellular structure. Temporal variations of the refractive index are analysed to detect cellular rhythmic activity on different time scales as well as to uncover interactions between the cellular processes.
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.
Scanning angle interference microscopy reveals cell dynamics at the nanoscale
Nature Methods, 2012
Emerging questions in cell biology necessitate nanometer-scale imaging in live cells. Here we present scanning angle interference microscopy, capable of localizing fluorescent objects with nanometer-scale precision along the optical axis in motile cellular structures. We use this approach to resolve nano-topographical features of the cell membrane and cytoskeleton, as well as the temporal evolution, three-dimensional architecture, and nano-scale dynamics of focal adhesion complexes.
Journal of Anatomy, 2001
Fast intracellular motion (FIM) was first revealed by back scattered light (BSL) imaging in video rate confocal scanning laser microscopy (VRCSLM), beyond the limits of spatial and temporal resolution obtainable with conventional optical microscopy. BSL imaging enabled visualisation of intra and extracellular motion with resolution in space down to 0n2 µm and in time to 1\25th of a second. Mapping the cell space at 0n2 µmi0n2 µm (XY l in instantaneous best focal plane)i0n5 µm (Z l height\depth, optic axis direction) volume steps revealed a communication layer above the known contact layer and an integrated dynamic spatial network (IDSN) towards the cell centre. FIM was originally observed as localised quasichaotic dancing (dithering) or reflecting patches\spots in the cell centre, faster in the darker nuclear space. Later, a second type of FIM was recognised which differed by the presence of a varied proportion of centrifugal and centripetal directional movements and\or jumping of patches\spots in the cell centre and outside the nuclear space. The first type is characteristic for cells in slightly adverse conditions while the second type has so far only been found in eutrophic cells. Temporal speeding up and coarsening of FIM, followed by slowing and eventually cessation at cell death, was found on exposure to strong stressors. It was concluded that the state of FIM provides instantaneous information about individual cell reactions to actual treatment and about cell survival. A putative switch between the first and second type FIM could be considered as an indicator of timing of cellular processes. The significance of FIM for the biology of the cell is seen in the rapid assessment of the condition of an individual live cell investigated by combination of various methods. Requirements for further development of this approach are outlined.
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.
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...
Cell surface fluctuations studied with defocusing microscopy
Physical Review E, 2003
Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom used as a useful tool. We revisited the theory of defocusing and apply it to our optical microscope with optics corrected at infinity. In our approximation, we obtain that the image contrast is proportional to the two-dimensional ͑2D͒ Laplacian of the phase difference introduced by the phase object. If the index of refraction of the phase object is uniform the image obtained from defocusing microscopy is the image of curvature ͑Laplacian of the local thickness͒ of the phase object, while standard phase-contrast microscopy gives information about the thickness of the object. We made artificial phase objects and measured image contrasts with defocusing microscopy. Measured contrasts are in excellent agreement with our theoretical model. We use defocusing microscopy to study curvature fluctuations ͑ruffles͒ on the surface of macrophages ͑cell of the innate immune system͒, and try to correlate mechanical properties of macrophage surface and phagocytosis. We observe large coherent propagating structures: Their shape, speed, density are measured and curvature energy estimated. Inhomogeneities of cytoskeleton refractive index, curvature modulations due to thermal fluctuations and/or periodic changes in cytoskeleton-membrane interactions cause random fluctuations in image contrast. From the temporal and spatial contrast correlation functions, we obtain the decay time and correlation length of such fluctuations that are related to their size and the viscoelastic properties of the cytoskeleton. In order to associate the dynamics of cytoskeleton with the process of phagocytosis, we use an optical tweezers to grab a zymosan particle and put it into contact with the macrophage. We then measure the time for a single phagocytosis event. We add the drug cytochalasin D that depolymerizes the cytoskeleton F-actin network: It inhibits the large propagating coherent fluctuations on the cell surface, increases the relaxation time of cytoskeleton fluctuations, and increases the phagocytosis time. Our results suggest that the methods developed in this work can be of utility to assess the importance of cytoskeleton motility in the dynamics of cellular processes such as phagocytosis exhibited by macrophages.
Optical Elastography and Tissue Biomechanics IV, 2017
There is an unmet need in tissue engineering for non-invasive, label-free monitoring of cell mechanical behaviour in their physiological environment. Here, we describe a novel optical coherence phase microscopy (OCPM) setup which can map relative cell mechanical behaviour in monolayers and 3D systems non-invasively, and in real-time. 3T3 and MCF-7 cells were investigated, with MCF-7 demonstrating an increased response to hydrostatic stimulus indicating MCF-7 being softer than 3T3. Thus, OCPM shows the ability to provide qualitative data on cell mechanical behaviour. Quantitative measurements of 6% agarose beads have been taken with commercial Cell Scale Microsquisher® system demonstrating that their mechanical properties are in the same order of magnitude of cells, indicating that this is an appropriate test sample for the novel method described.
Cytometry Part A, 2005
Background: The refractive index (RI) of cellular material provides fundamental biophysical information about the composition and organizational structure of cells. Efforts to describe the refractive properties of cells have been significantly impeded by the experimental difficulties encountered in measuring viable cell RI. In this report we describe a procedure for the application of quantitative phase microscopy in conjunction with confocal microscopy to measure the RI of a cultured muscle cell specimen. Methods: The experimental strategy involved calculation of cell thickness by using confocal optical sectioning procedures, construction of a phase map of the same cell using quantitative phase microscopy, and selection of cellular regions of interest to solve for the cell RI. Results: Mean cell thickness and phase values for six cell regions (five cytoplasmic and one nuclear) were deter-mined. The average refractive index calculated for cytoplasmic and nuclear regions was 1.360 6 0.004. The uncertainty in the final RI value represents the technique measurement error. Conclusions: The methodology we describe for viable cell RI measurement with this prototype cell has broad generic application in the study of cell growth and functional responses. The RI value we report may be used in optical analyses of cultured cell structure and morphology. q 2005 Wiley-Liss, Inc.