Optimise Spatial Resolution in Infrared Microscopy & Imaging
Optimise Spatial Resolution in Infrared Microscopy & Imaging. Infrared microscopy is a technique that allows chemical identification of small particles. Infrared hyperspectral imaging is an extension of this technique that allows distribution of chemical moieties to be determined.
The utility of infrared microscopy for the chemical identification of small particles has lead to its widespread use over the past two decades. The recent addition of imaging detectors to infrared microscopes has opened up many more applications of the technique. As its name implies, infrared microscopy is a fusion of infrared spectroscopy and optical microscopy. In order to fully interpret the data that such microscopes provide, the user must understand both the spectroscopic aspects and the optical performance of the system.
The result of infrared microscopy is a spectrum that is correlated to a visual image of the sample. In many cases, the sample of interest is surrounded by a matrix that also generates an infrared spectrum. Standard practice is to spatially isolate the small area of interest (AOI) from the surrounding matrix in order to obtain a spectrum that is representative of the AOI with minimal contributions from the surrounding region. This spatial isolation, or masking, is achieved using apertures placed before, after, or both before and after the sample.
Theoretically it can be shown that the spatial resolution of an infrared microscope using a single aperture is given by 2.0 l/NA where l is the wavelength and NA is the numerical aperture of the lens. This resolution improves to 1.0 l/NA in a dual aperture system. The practical consequence of this is shown in Fig. 1. In a single aperture system, significant contributions are made to the spectrum by components outside the apertured area. This hinders spectral interpretation in the analysis of complex mixtures. A dual aperture system largely eliminates this stray light.
Infrared imaging systems differ from basic microscopes in that they employ detectors that contain a multitude of small elements, which correspond to pixels in the final image. The resolution of such systems is often incorrectly perceived as equal to the pixel element size at the sample.
The resolution of an imaging system is controlled primarily by wavelength and numerical aperture. Fig. 2 shows images collected with an imaging system having a pixel size of around six microns.
The actual resolution achieved, as determined from knife-edge intensity measurements, is 14 and 37 microns at 3000 cm-1 and 1000 cm-1 respectively. These values correspond well with the theoretical values obtained from 2.0l/NA but are much greater than the pixel element size of six microns. A dual aperture system achieves optimum spatial resolution in infrared microscopy. An imaging system provides the optimum data acquisition speed. Therefore, chemical identification of an inclusion in a complex matrix is best done with a dual aperture single point microscope. Spatial distribution of a compound within a matrix is best determined with an imaging system.
Thermo Electron Corporation
The Scott Partnership
Tel.: +44 1606 837787