Quantitative Reflection Interference Contrast Microscopy (RICM) in Soft Matter and Cell Adhesion

Limozin, Laurent; Sengupta, Kheya

doi:10.1002/cphc.200900601

Cited by 250 publications

(312 citation statements)

References 69 publications

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“…• rotation of the polarisation [67]. Here we introduce an alternative method for collecting reflected signals through cross polarisers: by gluing a mica sheet of controlled thickness to the bottom of the sample, we achieve polarisation control without using a specifically designed objective.…”

Section: Methodsmentioning

confidence: 99%

The Conformation of Thermoresponsive Polymer Brushes Probed by Optical Reflectivity

2016

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We describe a microscope-based optical setup that allows us to perform space-and time-resolved measurements of the spectral reflectance of transparent substrates coated with ultrathin films. This technique is applied to investigate the behavior in water of thermosensitive polymer brushes made of poly(N-isopropylacrylamide) grafted on glass. We show that spectral reflectance measurements yield quantitative information about the conformation and axial structure of the brushes as a function of temperature. We study how molecular parameters (grafting density, chain length) affect the hydration state of a brush, and provide one of the few experimental evidence for the occurrence of vertical phase separation in the vicinity of the lower critical solution temperature of the polymer. The origin of the hysteretic behavior of poly(N-isopropylacrylamide) brushes upon cycling the temperature is also clarified. We thus demonstrate that our optical technique allows for in-depth characterization of stimuli-responsive polymer layers, which is crucial for the rational design of smart polymer coatings in actuation, gating or sensing applications.

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Section: Methodsmentioning

confidence: 99%

The Conformation of Thermoresponsive Polymer Brushes Probed by Optical Reflectivity

2016

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“…Samples were then mounted on an inverted microscope (TE-2000, Nikon, Tokyo, Japan) equipped with a temperature and humidity controller (37 C). The surface was imaged via reflection interference contrast microscopy (RICM) using a 40Â oil-immersion objective (22). Focus was maintained using a Nikon Perfect Focus controller.…”

Section: Cell Culture and Fp Assaymentioning

confidence: 99%

Frustrated Phagocytic Spreading of J774A-1 Macrophages Ends in Myosin II-Dependent Contraction

Kovari

Wei

Chang

et al. 2016

Biophysical Journal

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Conventional studies of dynamic phagocytic behavior have been limited in terms of spatial and temporal resolution due to the inherent three-dimensionality and small features of phagocytosis. To overcome these issues, we use a series of frustrated phagocytosis assays to quantitatively characterize phagocytic spreading dynamics. Our investigation reveals that frustrated phagocytic spreading occurs in phases and is punctuated by a distinct period of contraction. The spreading duration and peak contact areas are independent of the surface opsonin density, although the opsonin density does affect the likelihood that a cell will spread. This reinforces the idea that phagocytosis dynamics are primarily dictated by cytoskeletal activity. Structured illumination microscopy reveals that F-actin is reorganized during the course of frustrated phagocytosis. F-actin in early stages is consistent with that observed in lamellipodial protrusions. During the contraction phase, it is bundled into fibers that surround the cell and is reminiscent of a contractile belt. Using traction force microscopy, we show that cells exert significant strain on the underlying substrate during the contraction phase but little strain during the spreading phase, demonstrating that phagocytes actively constrict during late-stage phagocytosis. We also find that latestage contraction initiates after the cell surface area increases by 225%, which is consistent with the point at which cortical tension begins to rise. Moreover, reducing tension by exposing cells to hypertonic buffer shifts the onset of contraction to occur in larger contact areas. Together, these findings provide further evidence that tension plays a significant role in signaling late-stage phagocytic activity.

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“…The threshold intensity I thresh was derived from equation 1 by setting the threshold membrane-to-substrate distance at 40 nm [25][26][27][28] for a wavelength of λ 1 = 593 nm or λ 2 = 546 nm and using the minimum and maximum intensities I m and I M on each cell image [29]. This is a semi-quantitative method [25,29] which distinguishes between adherent and nonadherent parts of the cell membrane. Adherent parts are defined as less than 40 nm away from the surface and non-adherent parts as further away.…”

Section: Data Analysis Of Ricm Imagesmentioning

confidence: 99%

Cell membrane topology analysis by RICM enables marker-free adhesion strength quantification

et al. 2013

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Reflection interference contrast microscopy (RICM) allows the visualization of the cell's adhesion topology on substrates. Here it is applied as a new label-free method to measure adhesion forces between tumor cells and their substrate without any external manipulation, i.e., the application of force or adjustments in the substrate elasticity. Malignant cancer transformation is closely associated with the down-regulation of adhesion proteins and the consequent reduction of adhesion forces. By analyzing the size and distribution of adhesion patches from a benign and a malignant human pancreatic tumor cell line, we established a model for calculating the adhesion strength based on RICM images. Further, we could show that the cell's spread area does not necessarily scale with adhesion strength. Despite the larger projected cell area of the malignant cell line, adhesion strength was clearly reduced. This underscores the importance of adhesion patch analysis. The calculated force values were verified by microfluidic detachment assays. Static and dynamic RICM measurements produce numerous adhesion-related parameters from which characteristic cell signatures can be derived. Such a cellular fingerprint can refine the process of categorizing cell lines according to their grade of differentiation.

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Quantitative Reflection Interference Contrast Microscopy (RICM) in Soft Matter and Cell Adhesion

Cited by 250 publications

References 69 publications

The Conformation of Thermoresponsive Polymer Brushes Probed by Optical Reflectivity

The Conformation of Thermoresponsive Polymer Brushes Probed by Optical Reflectivity

Frustrated Phagocytic Spreading of J774A-1 Macrophages Ends in Myosin II-Dependent Contraction

Cell membrane topology analysis by RICM enables marker-free adhesion strength quantification

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