Reflected interference microscopy

Keywords Light interference Reflected interference microscopy Erythrocytes... 

From the point of view of having a well-defined system, an isolated vesicle freely floating in solution is almost perfect. For interesting applications or a higher level in complexity, interaction with other objects can be considered. The simplest case corresponds to adhesion to a rigid substrate [43]. For this case, a comprehensive set of experimental data can be obtained using reflection interference microscopy... 

The interest in vesicles as models for cell biomembranes has led to much work on the interactions within and between lipid layers. The primary contributions to vesicle stability and curvature include those familiar to us already, the electrostatic interactions between charged head groups (Chapter V) and the van der Waals interaction between layers (Chapter VI). An additional force due to thermal fluctuations in membranes produces a steric repulsion between membranes known as the Helfrich or undulation interaction. This force has been quantified by Sackmann and co-workers using reflection interference contrast microscopy to monitor vesicles weakly adhering to a solid substrate [78]. Membrane fluctuation forces may influence the interactions between proteins embedded in them [79]. Finally, in balance with these forces, bending elasticity helps determine shape transitions [80], interactions between inclusions [81], aggregation of membrane junctions [82], and unbinding of pinched membranes [83]. Specific interactions between membrane embedded receptors add an additional complication to biomembrane behavior. These have been stud-... 

While electron or ion beam techniques can only be applied under ultra-high vacuum, optical techniques have no specific requirements concerning sample environment and are generally easier to use. The surface information which can be obtained is, however, quite different and mostly does not contain direct chemical information. While with infra-red attenuated total reflection spectroscopy (IR-ATR) a deep surface area with a typical depth of some micrometers is investigated, other techniques like phase-measurement interference microscopy (PMIM) have, due to interference effects, a much better surface sensitivity. PMIM is a very quick technique for surface roughness and homogeneity inspection with subnanometer resolution. 

In interference microscopy the illumination is split into two beams [1,2]. The beam splitter is a half-silvered mirror. In reflection, one beam is reflected from the sample, while the other is reflected from a flat reference mirror [20-22]. Transmission is more complex as the beam splitter may be a double refracting crystal, and the two beams can be displaced horizontally or vertically [20-22], In all cases the two beams are recombined so that they interfere. The interference pattern can be used to measure the specimen thickness in transmission or the specimen roughness in reflection. 

Kojima, S. (1987). Interference fringes in reflection acoustic microscopy. Jap. J. Appl. Phys. 26 (Suppl. 26-1), 233-5. [255]... 

Two rather specialized methods are available to measure contact angles less than approximately 60". The method of interference microscopy makes use of fringe patterns reflected from the drop surface to calculate the contact angle [13]. Fisher [14] obtained contact angles less than 30 by simultaneously measuring the mass of the drop and the radius of the three-phase line. The contact angle was then derived from a semiempirical relationship involving these two quantities. 

As a consequence, researchers from different disciplines of the life sciences ask for efficient and sensitive techniques to characterize protein binding to and release from natural and artificial membranes. Native biological membranes are often substituted by artificial lipid bilayers bearing only a limifed number of components and rendering the experiment more simple, which permits the extraction of real quantitative information from binding experiments. Adsorption and desorption are characterized by rate constants that reflect the interaction potential between the protein and the membrane interface. Rate constants of adsorption and desorption can be quantified by means of sensitive optical techniques such as surface plasmon resonance spectroscopy (SPR), ellipsometry (ELL), reflection interference spectroscopy (RIfS), and total internal reflection fluorescence microscopy (TIRE), as well as acoustic/mechanical devices such as the quartz crystal microbalance (QCM)... 

The phase measurement interference microscopy (PMIM) is mainly used for surface analysis. A resolution depth of 0.6 nm can be achieved, whereas the lateral resolution is only in the range of about 1 ftm [White et al., 1990]. A laser beam in the microscope is reflected from the sample surface and simultaneously from a semitransparent smooth reference surface [Stamm, 1992]. The interference pattern is recorded which can be converted into a contour map of the sample surface, e.g., to determine the root-mean-square roughness of materials. 

Another exciting technique combines FRAP with total internal reflection fluorescence microscopy. This technique, called total internal reflection interference fringe fluorescence photobleaching... 

The optical anisotropy of cokes gives rise to a characteristic pattern of extinction contours when a polished surface is examined by polarized light microscopy using crossed polars, or reflection interference colours if the polars are parallel and a half wave plate is inserted into the optical system (, 12) ... 

Schindl, M., WaUraff, E., Deubzer, B. et al. (1995). Cell-substrate interactions and locomotion of Dictyostelium wild-type and mutants defective in three cytoskeletal proteins a study using quantitative reflection interference contrast microscopy. Biophys. ]. 68, 1177-1190. 

Alternatively to force-based approaches, cell adhesion can be quantified by measuring the intersurface distance between the cell and a planar transparent substrate (Fig. 4.30). Reflection interference contrast microscopy (RICM) is ideally suited for studying cell adhesion characteristics and dynamics in aqueous environment, as it allows for nanometer precise determination of intersurface distances with milliseconds time resolution [62]. It has the added... 

FIGURE 4.30 Optical principle of reflection interference contrast microscopy (RICM). The optical path (a) and the formation of constructive and destractive interference with the resulting reference pattern (b) are depicted on Ref. 61. 

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