Microscopy microspectroscopy

Figures 21(a) and 21(b) show the SEM micrographs of the freeze-fractured cross-section of the film used in the construction of the bag. There are two distinct layers and possibly a third very much thinner tie layer. The outside layer is a layer of nominal thickness 13 pm. The inside layer is much thicker and is approximately 70 pm thick. At the interface between the outer and inner layers the apparent very thin tie layer is about 1 pm thick. This is too thin to be identified by FUR microscopy on a cross-section of the sample, since the technique is diffraction-limited, which means that layers of about 10 pm thickness or greater can only be readily identified [1]. The tie layer thickness is also probably too thin for fingerprinting by Raman microspectroscopy on a cross-section the lateral spatial resolution of Raman microspectroscopy is about 1-2 pm.

Infrared microscopy is well suited for in situ analysis of contaminants fount in pharmaceutical processes. Due to the nondestructive nature of the analysis further experiments such as energy dispersive x-ray analysis may be performer on the same sample once IR investigations are complete. To illustrate the potentia of IR microspectroscopy, one application from the Bristol-Myers Squibl laboratories is presented. 

Cherry RJ. New Techniques of Optical Microscopy and Microspectroscopy, CRC Press, Boca Raton, FL, 1991. 

Microscopy technologies, polymer analysis using, 79 567-568 Micro-Sect formulation, 7 564t Microsilica, world demand for, 22 497 Microspectrometers, 76 484-485 Microspectroscopy, infrared, 76 486... 

Hilfiker et al. at Solvias used carbamazepine (CBZ) as a model compound to describe the use of Raman microscopy to characterize crystal forms, including during solvent evaporation experiments [228], The spectra were processed into clusters by spectral similarity. The authors note that all published and several new crystal forms were identified during the study. Solvias HTS uses a specific set of crystallization protocols that have tended to produce new polymorphs. Hilfiker notes that Raman microspectroscopy is an ideal analytical tool for high-throughput discrimination between crystal structures. [229], The ability to collect spectra directly and automatically in a microtiter plate with or without solvent and during evaporation is a major advantage over many other techniques. 

Raman microspectroscopy is readily performed on multiple locations inside each well. As in other instances, the results might not be representative of the whole sample because of the small sample volume probed. Polarization effects can be pronounced, but may be mitigated by averaging the results from additional locations. An alternative is rotating the sample, but this usually is not practical for multiwell plates. Both options increase analysis time. Such problems appear to be minimized when handling bulk powders [222,223,230], Several vendors sell systems preconfigured for automated analysis of microtiter plates and are typically integrated with optical microscopy. 

Volkmer, A. 2005. Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering microscopy. J. Phys. D-Appl. Phys. 38 (5) R59-R81. 

The outline of this chapter is as follows. First, we discuss the methods of THG microscopy and CARS microspectroscopy and outline the major developments over the past years, emphasizing the application aspect of this work. Then, we discuss the application of these spectroscopy tools for several microfluidic problems, such as live-cell imaging and protein crystallization. 

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. 

Written by an international panel of experts, this volume begins with a comparison of nonlinear optical spectroscopy and x-ray crystallography. The text examines the use of multiphoton fluorescence to study chemical phenomena in the skin, the use of nonlinear optics to enhance traditional optical spectroscopy, and the multimodal approach, which incorporates several spectroscopic techniques in one instrument. Later chapters explore Raman microscopy, third-harmonic generation microscopy, and nonlinear Raman microspectroscopy. The text explores the promise of beam shaping and the use of a broadband laser pulse generated through continuum generation and an optical pulse shaper. 

Similar approaches were adopted by Ganikhanov (Chapter 5), who developed a state-of-the-art laser system, benefiting simultaneous third-harmonic and nonlinear Raman microscopy, and Yakovlev et al. (Chapter 6), who applied third-harmonic generation microscopy and nonlinear Raman microspectroscopy for biochemical analysis in microfluidic devices. 

Beyond imaging, the combination of CRS microscopy with spectroscopic techniques has been used to obtain the full wealth of the chemical and the physical structure information of submicron-sized samples. In the frequency domain, multiplex CRS microspectroscopy allows the chemical identification of molecules on the basis of their characteristic Raman spectra and the extraction of their physical properties, e.g., their thermodynamic state. In the time domain, time-resolved CRS microscopy allows the recording of the localized Raman free induction decay occurring on the femtosecond and picosecond time scales. CRS correlation spectroscopy can probe three-dimensional diffusion dynamics with chemical selectivity. 

A series of advances over the past decade have made CRS microscopy a highly sensitive tool for label-free imaging and vibrational microspectroscopy that is capable of real-time, non-perturbative studies of complex biological samples based on molecular Raman spectroscopy. In particular, biomedical applications where fluorescent labeling of small molecules represents a severe pertur-... 

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