Microscopy contrast

Fig. 4. CHO cells on a microcarrier a optical microscopy (contrast enhancement by Crystal Violet staining) and b scanning electron microscopy...

The microscope is an invaluable tool to the materials scientist. There are two quantities that enable microscopy contrast and resolution. Sensitivity is not inherently an issue in microscopy signal level is not limiting because it is now possible to count single photons and electrons. Contrast and resolution determine one s ability to see at all scales. Contrast is the ability to measure changes in signal with a detector. The detector can be your eye, a CCD camera, or an... 

M. Labardi, V. Likodimos, and M. Allegrini, Force microscopy contrast mechanisms in ferroelectric domain imaging, Phys. Rev. B 61, 14390 14398 (2000). 

Each analysis typically requires a specific sample preparation procedure. In the case of massive samples, if requested, it is only necessary to deposit a carbon (or gold) coating on the observed surface to improve the electron microscopy contrast and resolution. In contrast, for the micro- and nanopowders, the preparation procedure is more complex. In fact, generally, the following steps are needed ... 

K., Yamada, H., and Matsushige, K. (2004) Molecular-scale noncontact atomic force microscopy contrasts in topography and energy dissipation on c(4 X 2) superlattice structures of alka-nethiol self-assembled monolayers. J. Appl. Phys., 95, 1222 1226. 

One more significant aspect of modem microscopy is the quantitative interpretation of the images in terms of the microstmcture of the object. Although most microscopes include or can be combined with powerful image processing systems, the interpretation of the contrast is still the main problem. On the other hand, reliable micromorpbological information could be easily obtained from a set of thin flat cross sections which reveal only density information, from which case accurate two- and three-dimensional numerical parameters of the internal microstmcture could be calculated. 

Considering existing microscopical techniques, one can find that non-destmctive information from the internal stmcture of an object in natural conditions can be obtained by transmission X-ray microscopy. Combination of X-ray transmission technique with tomographical reconstmction allows getting three-dimensional information about the internal microstmcture [1-3]. In this case any internal area can be reconstmcted as a set of flat cross sections which can be used to analyze the two- and three-dimensional morphological parameters [4]. For X-ray methods the contrast in the images is a mixed combination of density and compositional information. In some cases the compositional information can be separated from the density information [5]. Recently there has been a... 

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-... 

The effect is more than just a matter of pH. As shown in Fig. XV-14, phospholipid monolayers can be expanded at low pH values by the presence of phosphotungstate ions [123], which disrupt the stmctival order in the lipid film [124]. Uranyl ions, by contrast, contract the low-pH expanded phase presumably because of a type of counterion condensation [123]. These effects caution against using these ions as stains in electron microscopy. Clearly the nature of the counterion is very important. It is dramatically so with fatty acids that form an insoluble salt with the ion here quite low concentrations (10 M) of divalent ions lead to the formation of the metal salt unless the pH is quite low. Such films are much more condensed than the fatty-acid monolayers themselves [125-127]. 

Frank J and Penczek P 1995 On the correction of the contrast transfer function in bioiogicai eiectron microscopy Optik 38 125-9... 

Light microscopy allows, in comparison to other microscopic methods, quick, contact-free and non-destmctive access to the stmctures of materials, their surfaces and to dimensions and details of objects in the lateral size range down to about 0.2 pm. A variety of microscopes with different imaging and illumination systems has been constmcted and is conunercially available in order to satisfy special requirements. These include stereo, darkfield, polarization, phase contrast and fluorescence microscopes. 

This overview will first deal with the optical aspects of conventional microscopes and the various means to improve contrast. Confocal microscopy, which in the last decade has become an important tool, especially for biology, is discussed in the final section. 

At this point it is worth comparing the different techniques of contrast enliancements discussed so far. They represent spatial filtering teclmiques which mostly affect the zeroth order dark field microscopy, which eliminates the zeroth order, the Schlieren method (not discussed here), which suppresses the zerotii order and one side band and, finally, phase contrast microscopy, where the phase of the zeroth order is shifted by nil and its intensity is attenuated. 

B1.18.5.5 CONTRAST ENHANCEMENT AND PRACTICAL LIMITS TO CONFOCAL ONE-PHOTON-EXCITATION FLUORESCENCE MICROSCOPY... 

Tdrdk P, Sheppard C J R and LaczikZ 1996 Dark field and differential phase contrast imaging modes in confocal microscopy using a half aperture stop Optik 103 101-6... 

Brabury S and Everett B 1996 Contrast Techniques In Light Microscopy, Microscopy Handbooks 34 (Oxford BIOS Scientific Publishers)... 

Thundat T, Warmack R J, Allison D P, Bottomley L A, Lourenco A J and Ferrell T L 1992 Atomic force microscopy of deoxyribonucleic acid strands adsorbed on mica the effect of humidity on apparent width and image contrast J. Vac. Sol. Technol. A 10 630... 

A completely new method of determining siufaces arises from the enormous developments in electron microscopy. In contrast to the above-mentioned methods where the surfaces were calculated, molecular surfaces can be determined experimentally through new technologies such as electron cryomicroscopy [188]. Here, the molecular surface is limited by the resolution of the experimental instruments. Current methods can reach resolutions down to about 10 A, which allows the visualization of protein structures and secondary structure elements [189]. The advantage of this method is that it can be apphed to derive molecular structures of maaomolecules in the native state. 

Figure 1.1 is a rather remarkable photograph which shows individual polystyrene molecules as spherical blobs having average diameters of about 20 nm. The picture is an electron micrograph in which a 10" % solution of polystyrene was deposited on a suitable substrate, the solvent evaporated, and the contrast enhanced by shadow casting. There is a brief discussion of both electron microscopy and shadowing in Sec. 4.7. Several points should be noted in connection with Fig. 1.1 ...

There ate four main approaches to x-ray imaging contact radiography, scanning x-ray microscopy, holographic x-ray microscopy, and shadow projection x-ray microscopy. In the future, there will likely be phase-contrast imaging and photoelectron x-ray microscopy. 

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