Three-dimensional difference correlation spectroscopy

Photon cross-correlation spectroscopy (PCCS) uses a novel three-dimensional cross-correlation technique which completely suppresses the multiple scattered fractions in a special scattering geometry. In this setup two lasers A and B are focused to the same sample volume, creating two sets of scattering patterns, as shown in Fig. 21-14. Two intensities are measured at different positions but with identical scattering vectors. 

Beyond imaging, CARS microscopy offers the possibility for spatially resolved vibrational spectroscopy [16], providing a wealth of chemical and physical structure information of molecular specimens inside a sub-femtoliter probe volume. As such, multiplex CARS 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 CARS microscopy allows recording of ultrafast Raman free induction decays (RFIDs). CARS correlation spectroscopy can probe three-dimensional diffusion dynamics with chemical selectivity. We next discuss the basic principles and exemplifying applications of the different CARS microspectroscopies. 

We have investigated peptides whose structures were known beforehand from NMR or x-ray spectroscopy and related these structures to 2D-IR spectroscopy. Ultimately, one would like to deduce the structure of an unknown sample from a 2D-IR spectrum. In the case of 2D NMR spectroscopy, two different phenomena are actually needed to determine peptide structures. Essentially, correlation spectroscopy (COSY) is utilized in a first step to assign protons that are adjacent in the chemical structure of the peptide so that J coupling gives rise to cross peaks in these 2D spectra. However, this through-bond effect cannot be directly related to the three-dimensional structure of the sample, since that would require quantum chemistry calculations, which presently cannot be performed with sufficient accuracy. The nuclear Overhauser effect (NOE), which is an incoherent population transfer process and has a simple distance dependence, is used as an additional piece of information in order to measure the distance in... 

Multidimensional NMR spectra are not restricted to cases where the separate frequency axes encode signals from different nuclear types. Indeed, much of the early work on the development of 2D NMR was performed on cases where both axes involved chemipal shifts. The main value in such spectra comes from the information content in cross peaks between pairs of protons. In COSY-type spectra (COSY = Correlation SpectroscopY) cross peaks occur only between protons that are scalar coupled (i.e., within 2 or 3 bonds) to each other, whereas in NOESY (NOE Spectroscopy) spectra cross peaks occur for protons that are physically close in space (<5 A apart). A combination of these two types of 2D spectra may be used to assign the NMR signals of small proteins and provides sufficient information on internuclear distances to calculate three-dimensional structures. Figure 12.3 includes a panel showing the COSY spectrum of cyclosporin and highlights the relationships between ID H-NMR spectra and corresponding 2D homonuclear (COSY) and heteronuclear (HSQC) spectra. 

While three-dimensional imaging is likely to have the most dramatic impact, there are opportunities to carry out fast radiography to image two-dimensional processes in thin foils at submillisecond rates. Such imaging can also be correlated with chemical information, including X-ray fluorescence maps. X-ray absorption spectroscopy, microdifffaction, and small-angle scattering. The spatial distribution of different chemical species is important for the development of robust corrosion... 

Two-dimensional NOESY allows detection of spatial proximity between protons that are separated by less than 4.5 A. By the correlated spectroscopy method (COSY) scalar couplings between protons, which are separated by at most three (in some exceptions four) chemical bonds, are used. In the case of ribooligonucleotides such couplings consequently can only be observed between the protons of the ribose rings and the C5 and C6 protons of the pyrimidine bases of a given nucleotide. The NOESY method, however, permits the detection of contacts between the protons of different nucleotides. 

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