Interference contrast 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-... 

Li, J. H., Guiltinan, M. J., and Thompson, D. B. 2006. The use of laser differential interference contrast microscopy for the characterization of starch granule ring structure. Starch-Starke 58 1-5. 

Powerful methods that have been developed more recently, and are currently used to observe surface micro topographs of crystal faces, include scanning tunnel microscopy (STM), atomic force microscopy (AFM), and phase shifting microscopy (PSM). Both STM and AFM use microscopes that (i) are able to detect and measure the differences in levels of nanometer order (ii) can increase two-dimensional magnification, and (iii) will increase the detection of the horizontal limit beyond that achievable with phase contrast or differential interference contrast microscopy. The presence of two-dimensional nuclei on terraced surfaces between steps, which were not observable under optical microscopes, has been successfully detected by these methods [8], [9]. In situ observation of the movement of steps of nanometer order in height is also made possible by these techniques. However, it is possible to observe step movement in situ, and to measure the surface driving force using optical microscopy. The latter measurement is not possible by STM and AFM. 

Figure 7.22 Microstructure of acidified mixed emulsions (20 vol% oil, 0.5 wt% sodium caseinate) containing different concentrations of dextran sulfate (DS). Samples were prepared at pH = 6 in 20 mM imidazole buffer and acidified to pH = 2 by addition of HCl. Emulsions were diluted 1 10 in 20 mM imidazole buffer before visualization by differential interference contrast microscopy (A) no added DS (B) 0.1 wt% DS (C) 0.5 wt% DS (D) 1 wt% DS. Particle-size distributions of the diluted emulsions determined by light-scattering (Mastersizer) are superimposed on the micrographs, with horizontal axial labels indicating the particle diameter (in pm). Reproduced with permission from Jourdain et al. (2008).

Nomarski differential interference contrast microscopy is an alternative to phase contrast microscopy which gives an almost three... 

Fig. 15 A Chemical structures of Thy- and DAP-functionalized polymers, and schematic illustration of vesicle formation. B Differential interference contrast microscopy (DIG), C AFM, and D fractured TEM images of resultant vesicular aggregates. Reprinted with permission from [88]...

Crane JM, Kiessling V, Tamm LK. Measuring lipid asymmetry in planar supported bilayers by fluorescence interference contrast microscopy. Langmuir 2005 21 1377-1388. 

Hie most important optical technique for examining semiconductor wafer surfaces is the differential interference contrast microscopy method of Nomarski (N-DIC). First described in 1952, DIG... 

Detection of Cryptosporidium oocysts in clinical specimens or in environmental samples can be performed using microscopy. Modified Ziehl-Nielsen stain, auramine, fluorescein-labeled specific antibody, differential interference contrast microscopy... 

Phase-Contrast and Differential Interference Contrast Microscopy Visualize Unstained Living Cells... 

To understand the release mechanism, cryomicrotomy was used to slice 10 m-thick sections throughout the matrices. Viewed under an optical microscope, polymer films cast without proteins appeared as nonporous sheets. Matrices cast with proteins and sectioned prior to release displayed areas of either polymer or protein. Matrices initially cast with proteins and released to exhaustion (e.g., greater than 5 months) appeared as porous films. Pores with diameters as large as 100 /xm, the size of the protein particles, were observed. The structures visualized were also confirmed by Nomarski (differential interference contrast microscopy). It appeared that although pure polymer films were impermeable to macromolecules (2), molecules incorporated in the matrix dissolved once water penetrated the matrix and were then able to diffuse to the surface through pores created as the particles of molecules dissolved. Scanning electron microscopy showed that the pores were interconnected (7). 

An even more refined method of study is interference-contrast microscopy [following Nomarski (767)] which converts minute topographical differences into color changes. Replica electron microscopy (77), gold decoration (see Section III,A,1), and scanning electron microscopy are also useful but less practicable. Occasionally it is profitable to follow the course of the reaction, in situ, using time-lapse cinematography (77). 

The surface topography of the partially oxidized specimens is examined, with low resolution, using interference-contrast microscopy and at high resolution by means of the gold-decoration technique 177, 180) in conjunction with transmission electron microscopy. 

Fig. 1. Wound healing and inflammation in Drosophila embryos, (a) A laser ablation wound to a moesinGFP expressing Drosophila embryo will, by a purse-string mechanism, (b) seal shut over a period of a few hours, (c) This same laser wound will also recmit hemo-cytes that, by driving GFP specifically in these cells, can be imaged by widefield microscopy. (d) Alternatively, the wound site can be imaged by Differential Interference Contrast microscopy to give a general idea of wound size (dotted line) and hemocyte presence ( ). (e) The insertion of a heparin bead into the wound site will also lead to the recruitment of hemocytes that will surround and encapsulate the bead, (f) A hemocyte undergoing developmental dispersal viewed by confocal microscopy.

Fig. 1. Outlining by complexity. In this case, the original Image (a) ot a D. discoideum amoeba was obtained with differential Interference contrast microscopy, (b) Initial complexity outlining, (c) Final outline, (d) Outer edge with Internal architecture subtracted. Dilation of 3 and erosion of 2 was used.

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. 

Fig. 4 Top row Successive (010) slices (200 p) of dyed potassium hydrogen phthalate crystals reveal the growth histories in their patterns of luminescence. Bottom row The orientation of the fast- and slow-moving steps of a growth hillock are shown on the (010) face in a comparison of images made by differential interference contrast microscopy and fluorescence microscopy. (View this art in...

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. 

Fig. 3 Cell spreading of RAW 264.7 macrophages cultured on polyacrylamide gel surfaces with varying stiffness. Differential interference contrast microscopy images (A) and con-focal images for actin staining (B) from RAW 264.7 macrophages on a less rigid (1.2 kPa) versus rigid (150 kPa) polyacrylamide gel coated with poly-L-lysine. Green color represents actin staining (phalloidin) with blue stained nuclei (Hoechst). Adapted from [39] with permission.

Specimens sufficiently small to be examined entire may also be examined alive, contrast being provided by optical devices such as phase contrast or interference contrast microscopy, or occasionally by means of vital dyes - nontoxic stains that have an affinity for certain components of cells. However, most organisms, their organs and tissues, and even their cells, are too large to provide high-resolution information with the conventional light microscope, and they must be killed and then (usually) dehydrated, embedded, sectioned, stained, and movmted before microscopical examination. 

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