Cell surface fluctuations studied with defocusing microscopy (original) (raw)
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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.
Scientific reports, 2017
Dramatic and rapid changes in cell shape are perhaps best exemplified by phagocytes, such as neutrophils. These cells complete the processes of spreading onto surfaces, and phagocytosis within 100 s of stimulation. Although these cell shape changes are accompanied by an apparent large increase in cell surface area, the nature of the membrane "reservoir" for the additional area is unclear. One proposal is that the wrinkled cell surface topography (which forms micro-ridges on the neutrophil surface) provides the resource for neutrophils to expand their available surface area. However, it has been problematic to test this proposal in living cells because these surface structures are sub-light microscopic. In this paper, we report the development of a novel approach, a variant of FRAP (fluorescent recovery after photo-bleaching) modified to interrogate the diffusion path-lengths of membrane associated molecules. This approach provides clear evidence that the cell surface topog...
Biophysical Journal, 2009
We investigate the dynamic response of single cells to weak and local rigidities, applied at controlled adhesion sites. Using multiple latex beads functionalized with fibronectin, and each trapped in its own optical trap, we study the reaction in real time of single 3T3 fibroblast cells to asymmetrical tensions in the tens of pN $ mm À1 range. We show that the cell feels a rigidity gradient even at this low range of tension, and over time develops an adapted change in the force exerted on each adhesion site. The rate at which force increases is proportional to trap stiffness. Actomyosin recruitment is regulated in space and time along the rigidity gradient, resulting in a linear relationship between the amount of recruited actin and the force developed independently in trap stiffness. This time-regulated actomyosin behavior sustains a constant and rigidity-independent velocity of beads inside the traps. Our results show that the strengthening of extracellular matrix-cytoskeleton linkages along a rigidity gradient is regulated by controlling adhesion area and actomyosin recruitment, to maintain a constant deformation of the extracellular matrix.
Journal of Innovative Optical Health Sciences, 2011
Optical magnetic twisting cytometry and traction force microscopy are two advanced cell mechanics research tools that employ optical methods to track the motion of microbeads that are either bound to the surface or embedded in the substrate underneath the cell. The former measures rheological properties of the cell such as cell sti®ness, and the latter measures cell traction force dynamics. Here we describe the principles of these two cell mechanics research tools and an example of using them to study physical behaviors of the living cell in response to transient stretch or compression. We demonstrate that, when subjected to a stretchÀunstretch manipulation, both the sti®ness and traction force of adherent cells promptly reduced, and then gradually recover up to the level prior to the stretch. Immuno°uorescent staining and Western blotting results indicate that the actin cytoskeleton of the cells underwent a corresponding disruption and reassembly process almost in step with the changes of cell mechanics. Interestingly, when subjected to compression, the cells did not show such particular behaviors. Taken together, we conclude that adherent cells are very sensitive to the transient stretch but not transient compression, and the stretch-induced cell response is due to the dynamics of actin polymerization.
Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 2008
The motility of living eukaryotic cells is a complex process driven mainly by polymerization and depolymerization of actin filaments underneath the plasmatic membrane (actin cytoskeleton). However, the exact mechanisms through which cells are able to control and employ 'actin-generated' mechanical forces, in order to change shape and move in a well-organized and coordinated way, are not quite established. Here, we summarize the experimental results obtained by our research group during recent years in studying the motion of living cells, such as macrophages and erythrocytes. By using our recently developed defocusing microscopy technique, which allows quantitative analysis of membrane surface dynamics of living cells using a simple bright-field optical microscope, we were able to analyse morphological and dynamical parameters of membrane ruffles and small membrane fluctuations, study the process of phagocytosis and also measure values for cell refractive index, membrane bending modulus and cell viscosity. Although many questions still remain unanswered, our data seem to corroborate some aspects of recent physical models of cell membranes and motility.
Real-time actin-cytoskeleton depolymerization detection in a single cell using optical tweezers
Optics Express, 2007
The cytoskeleton provides the backbone structure for the cellular organization, determining, in particular, the cellular mechanical properties. These are important factors in many biological processes, as, for instance, the metastatic process of malignant cells. In this paper, we demonstrate the possibility of monitoring the cytoskeleton structural transformations in optically trapped yeast cells (Saccharomyces cerevisiae) by tracking the forward scattered light via a quadrant photodiode. We distinguished normal cells from cells treated with latrunculin A, a drug which is known to induce the actin-cytoskeleton depolymerization. Since the proposed technique relies only on the inherent properties of the optical trap, without requiring external markers or biochemical sensitive spectroscopic techniques, it can be readily combined with existing optical tweezers setups.
Fluorescence microscopic imaging and image analysis of the cytoskeleton
2010 44th Asilomar Conference on Signals, Systems and Computers, 2010
Cell stability and motility depends on a complex dynamic cytoplasmic scaffolding called the cytoskeleton. It is composed of actin filaments, intermediate filaments and microtubules, and interacts with neighbouring cells and the extracellular matrix via specialized adhesion sites-multimolecular complexes responsible for the transmission of mechanical force and regulatory signals. The dynamic behaviour of these subcellular structures in living cells can be analysed by fluorescence microscopy yielding series of 2D or 3D images. Towards a quantitative analysis, we present methods for the segmentation and motion estimation of cytoskeletal filaments as well as for the tracking of adhesion sites, allowing the quantification of cytoskeletal dynamics under different conditions.
A spatiotemporal characterization method for the dynamic cytoskeleton
Cytoskeleton (Hoboken, N.J.), 2016
The significant gap between quantitative and qualitative understanding of cytoskeletal function is a pressing problem; microscopy and labeling techniques have improved qualitative investigations of localized cytoskeleton behavior, whereas quantitative analyses of whole cell cytoskeleton networks remain challenging. Here we present a method that accurately quantifies cytoskeleton dynamics. Our approach digitally subdivides cytoskeleton images using interrogation windows, within which box-counting is used to infer a fractal dimension (Df ) to characterize spatial arrangement, and gray value intensity (GVI) to determine actin density. A partitioning algorithm further obtains cytoskeleton characteristics from the perinuclear, cytosolic, and periphery cellular regions. We validated our measurement approach on Cytochalasin-treated cells using transgenically modified dermal fibroblast cells expressing fluorescent actin cytoskeletons. This method differentiates between normal and chemically...
International Journal of Molecular Sciences, 2021
It is well known that living cells interact mechanically with their microenvironment. Many basic cell functions, like migration, proliferation, gene expression, and differentiation, are influenced by external forces exerted on the cell. That is why it is extremely important to study how mechanical properties of the culture substrate influence the cellular molecular regulatory pathways. Optical microscopy is one of the most common experimental method used to visualize and study cellular processes. Confocal microscopy allows to observe changes in the 3D organization of the cytoskeleton in response to a precise mechanical stimulus applied with, for example, a bead trapped with optical tweezers. Optical tweezers-based method (OT) is a microrheological technique which employs a focused laser beam and polystyrene or latex beads to study mechanical properties of biological systems. Latex beads, functionalized with a specific protein, can interact with proteins located on the surface of the...