IR Mapping and Imaging

Since the genome sequence of A. thaliana became available in 2000, a multitude of studies made use of this model plant in order to combine functional genomics and spectroscopic analysis, especially to elucidate the complex biosynthetic pathways involved in the buildup of the cell wall. Numerous mutants were designed 

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Wheat and maize are the two plants in between all agricultural crops, which are most intensively studied by FT-IR and FT-Raman mapping and imaging. 

The introduction of microspectrosopy by coupling IR and Raman spectroscopic studies with confocal microscopes directed the research focus away from the chemical analysis of macroscopic assemblies (biofilms, whole algae communities) or extracts [138,139] to the point of studies on single individuals [140, 141] and even to the (sub)cellular level of individual cells [142, 143]. The introduction of mapping and imaging techniques enlarged the scientific view to distributional analysis in addition to the classical qualitative and quantitative determination. 

Figure 8.6 (a) Visible Image of a silicone oil In water emulsion (b) and (c) schematic comparison of the FT-iR mapping and FT-iR/FPA imaging techniques (d) two single-pixel spectra (e) color-coded Images based on the water-specific (top) and siiicone oii-specific (bottom) absorption bands. 

Figure 6.15. Comparison of IR mapping and IR imaging of a thin tissue section measured in transmission. Integrated intensities of a band typical of sugar polymers in tissues (at 1,155 cm ) are plotted at the top and those for methylene (at 2,850cm ) at the bottom. Mapping with 12 ttm aperture and lO/tm stepping is shown on the left (32 x 32) IR imaging (64 x 64) on the right. Images are 250/im square. (From Schultz [307] reproduced with permission.)...

In the end, what matters obviously is whether the features of interest distinctly show up in the analytical data, e.g., in a spectrum or a map or image. Thus, even though IR is not ultimately a surface specific technique, when an outermost surface layer reveals characteristic absorption bands that are related to say a property such as adhesion, the technique can be a very valuable one and is extensively applied. 

Mapping Historically, mapping [11] was the first method used to acquire hyperspectral data cubes, in particular with Raman spectroscopy and infrared (IR) microscopy. The image is created pixel by pixel in a step-and-acquire mode A spectrum is measured at one point of the sample, and then the sample moves to the next measurement position and another spectrum is acquired. The process is iterative for all positions in the area that define the image. 

In principle, operation of the FTIR microspectroscopy is the same as for a conventional FTIR instrument except the spectrum is obtained from a microscopic area or intensity distribution is mapped in the sample plane. A spectrum from an area in the order of 10 x 10 /im can be obtained. Mapping or FTIR imaging at micro-level resolution can be achieved by scanning a sample using a motorized sample stage. The resolution is primarily determined by the size of the focused IR beam and precision of motorized stage. Reflectance microspectroscopy is more widely used than the transmittance mode in FTIR microscopy because minimal sample preparation is required. 

Although contemporary FT-IR spectrometers and microscopes are well matched, for spectra measured when using sample apertures that approach the diffraction limit (<20gm) even a 30 s collection may result in a spectrum with a rather poor SNR. It may be noted that if the measurement of each spectrum takes 30 s and a 64x64 map is required at 20 pm spatial resolution, it would take over 34h to acquire all the spectra required for the image ... 

Krafft, C., Kirsch, M., Beleites, C., Schackert, G. and Salzer, R. (2007) Methodology for fiber-optic Raman mapping and FT-IR imaging of metastases in mouse brains. Anal. Bioanal. Chem., 389, 133-1142. 

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