Contrast mechanisms

There are two parts to a contrast mechanism. The first is the interaction between the incident radiation and the specimen. The second part is the optical system that makes the interaction produce an intensity change in an image. When imaging with lenses, the specimen may change the amphtude of the wave passing through it, or the direction or phase of the wave. Contrast in optical nucroscope images is rarely interpreted 

The constant B is determined by observing films of known thickness under the same standard conditions [7, 41, 42]. 

If the sample is crystalline, the scattered intensity depends very strongly on the orientation of the crystals and on their thickness. In bright field, a thin crystal will appear dark when it is correctly oriented for diffraction. If the crystal is not perfectly flat, the contours of correct orientation will appear as dark lines, called bend contours. Variation of intensity such as this in crystalline specimens is called crystallographic or diffraction contrast. Many types of defects in crystals cause localized distortion of the crystal lattice. These defects change the crystal orientation locally and so cause variations in the crystallographic contrast. Detailed information 

Any specimen will cause a phase change in the wavefront passing through it. The specimen has an optical thickness nt producing an optical path difference A = n — l)t and a phase retardation (27t/A)(w - l)f. To make the phase shift affect the image intensity, the retarded wave must be caused to interfere with another wave. The other wave must be coherent with that passing through the specimen. 

Deliberate defocusing enhances phase contrast at lower magnifications [45] but it must be used with caution. If there is only random structure in the specimen, defocus, deliberate or accidental, may induce clearly visible structure unrelated to 

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Computers will be integrated more and more into commercial SEMs and there is an enormous potential for the growth of computer supported applications. At the same time, related instruments will be developed and extended, such as the scanning ion microscope, which uses liquid-metal ion sources to produce finely focused ion beams that can produce SEs and secondary ions for image generation. The contrast mechanisms that are exhibited in these instruments can provide new insights into materials analysis. 

Papayannopoulou T, Craddock C, Nakamoto B, Priestley GV, Wolf NS. The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement... 

Bogdan, C., Paik, J., Vodovotz, Y. and Nathan, C. (1992) Contrasting mechanisms for suppression of macrophage cytokine release by transforming growth factor-beta and interleukin-10. Journal of Biological Chemistry 267, 23301-23308. 

It is anticipated that many of the catalytic Cp2Zr(II) reactions that might have been considered to proceed via oxidative addition and reductive elimination, such as hydrosila-tion [224] and hydrogenation [225], may actually proceed via a couple of o-bond metatheses, i. e. transmetallation and p-H abstraction, as exemplified by the two contrasting mechanisms for the hydrosilation of alkenes (Scheme 1.70). 

The second major contrast mechanism is extinction contrast. Here the distortion of the lattice arotmd a defect gives rise to a different scattering power from that of the surrotmding matrix. In all cases, it arises from a breakdown or change of the dynamical diffraction in the perfect ciystal. In classical structure analysis, the name extinction was used to describe the observation that the integrated intensity was less than that predicted by the kinematical theoiy. 

We can begin to understand the contrast mechanism by considering the ciystal to be made up of three distinct regions, namely the perfect crystal above the defect, the perfect crystal below the defect, and the deformed region around the defect (Figure 8.15(a)). We set the hmit of the deformed region as that where the effective misorientation ( ) arotmd a defect exceeds the perfect crystal... 

Nonlinear light-matter interactions have been successfully applied to create new visualization contrast mechanisms for optical microscopy. Nonlinear optical microscopy employs femtosecond and picosecond lasers to achieve a high photon flux density by focusing the beam onto a sample with a high numerical aperture (NA) microscope... 

This chapter introduces principles of three nonlinear contrast mechanisms— MPF, SHG, and THG—for microscopic imaging. The instrumentation of nonlinear multicontrast microscopy is described, and the methods of multicontrast image analysis are presented. The later parts of the chapter focus on several examples of multicontrast nonlinear microscopy applications in biological imaging. 

Two-photon excitation fluorescence is currently the most widely nsed nonlinear contrast mechanism for microscopic investigations. The first experimental demonstration of two-photon excitation fluorescence was provided in 1961 (Kaiser and Garrett 1961), even though the first theoretical description of two-photon excitation flnorescence stems back to 1931 (Goppert-Mayer 1931). Three-photon absorption was demonstrated a few years later by Singh and Bradley (1964). Two-photon absorption is a third-order nonlinear effect, whereas three-photon absorption is a fifth-order nonlinear effect. The transition rate for two-photon absorption, R, depends on the square of the intensity, /, as follows (see Boyd 1992) ... 

Parallel images, recorded using different contrast mechanisms, can be directly compared on a pixel-by-pixel basis. Althongh SHG, THG, and MPF images originate... 

Barzda, V. 2008. Non-linear contrast mechanisms for optical microscopy. In Biophysical Techniques in Photosynthesis. T. J. Aartsma and J. Matysik, Eds. 35-54. Urbana, IL, USA Springer. 

In regard to Equation (5.4), we have to note that without the above mentioned assumptions the nonlinearities will contain weighting factors that are proportional to the corresponding wave-vector mismatch and inversely proportional to refractive index, thus suggesting that the THG signal is sensitive to the refractive index interface(s) as well. In order to differentiate between contrast mechanisms in THG imaging of soft tissue materials it would be important to know the relationships of the corresponding linear and nonlinear optical parameters. A nonlinear optical... 

In this chapter we explore several aspects of interferometric nonlinear microscopy. Our discussion is limited to methods that employ narrowband laser excitation i.e., interferences in the spectral domain are beyond the scope of this chapter. Phase-controlled spectral interferometry has been used extensively in broadband CARS microspectroscopy (Cui et al. 2006 Dudovich et al. 2002 Kee et al. 2006 Lim et al. 2005 Marks and Boppart 2004 Oron et al. 2003 Vacano et al. 2006), in addition to several applications in SHG (Tang et al. 2006) and two-photon excited fluorescence microscopy (Ando et al. 2002 Chuntonov et al. 2008 Dudovich et al. 2001 Tang et al. 2006). Here, we focus on interferences in the temporal and spatial domains for the purpose of generating new contrast mechanisms in the nonlinear imaging microscope. Special emphasis is given to the CARS technique, because it is sensitive to the phase response of the sample caused by the presence of spectroscopic resonances. 

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