Infrared microspectroscopic imaging spatial resolution

Lasch, P. and Naumann, D. (2006) Spatial resolution in infrared microspectroscopic imaging of tissues. Biochim. Biophys. Acta, 1758, 814-29. 

Field of view (the sample area, the image of which is to be observed) and lateral spatial resolution are two important factors in infrared microspectroscopic imaging. These are given as follows ... 

Infrared microscopic imaging provides the significant advantages of direct spatially resolved concentration and molecular structure information for sample constituents. Raman microscopy (not further discussed in this chapter) possesses the additional benefit of confocal acquisition of this information and a 10-fold increase in spatial resolution at the expense of reduced signal-to-noise ratios compared with IR. The interested reader is urged to check the seminal studies of the Puppels group in Rotterdam,38 0 as well as our own initial efforts in this direction.41 The current section describes the initial applications of IR microspectroscopic imaging to monitor the permeation and tissue distribution of the dermal penetration enhancer, DMSO, in porcine skin as well as to track the extent of permeation of phospholipid vesicles. 

There are a number of approaches to allow infrared microspectroscopic investigations of polymers. While interferometer based approaches dominate the instrumentation in general, other approaches may be effective in specihc cases or when dedicated instrumentation examining a limited range of problems is desired. FPAs, employed to achieve spatial discrimination as in FTIR imaging or spatial and spectral discrimination as in dispersive and hlter approaches, provide flexibility and faster spectroscopy due to their multichannel detection advantage. Many new approaches to microspectroscopy are dedicated to achieving spatial resolution better... 

In order to enhance spatial resolution, it is necessary to make the NA of the objective larger, as is clear from Equation (17.3) that is, either n or 6, or both of them should be increased. Due to the optical geometry of a microscope, there is an upper limit for 0. On the other hand, it is possible practically to increase n by introducing an attenuated total reflection (ATR) accessory into a microscope (see Chapter 13 for the ATR method this is a frequently used accessory for many recent infrared microspectroscopy absorption measurements). The refractive index n of Ge, which is a commonly used material for an internal reflection element (IRE) in the ATR method, is about 4, and the NA when using a Ge IRE exceeds 2. This means that, if an ATR accessory with a Ge IRE is combined with a microscope, the theoretical spatial resolution is enhanced about four times that of the conventional reflection measurement. In fact, in the FT-IR microspectroscopic imaging measurement with a Ge ATR accessory, it has been confirmed that a spatial resolution comparable to the infrared wavelength used for the measurement is realized, and thus a higher spatial resolution may be attainable. 

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