Microspectroscopy apertures

FTIR Microspectroscopy.3 A microscope accessory coupled to a liquid-nitrogen-cooled mercury-cadmium-telluride (MCT) detector can be used to obtain an IR spectrum. This is possible in both the transmission and reflectance modes. Several beads are spread on an IR-transparent window (NaCl, KBr, diamond) and possibly flattened via a hand-press or a compression cell. The IR beam is focused on a single bead using the view mode of the microscope. The blank area surrounding the bead is isolated using an adjustable aperture, and a spectrum is recorded using 32 scans (<1 min). A nearby blank area of the same size on the IR transparent window is recorded as the background. 

The selection of the microscopic area for FTIR microspectroscopy is achieved by a remote aperture located between the objective and detector. The remote aperture commonly has a rectangular opening with two pairs of knife-edged blades. The blades are often made from a material that is transparent to visible light but opaque to infrared light. 

FTIR microspectroscopy is a microanalytical technique, which interfaces an FTIR spectrometer to an optical microscope. Regions of interest in the sample are spatially isolated using the microscope s apertures. It enables the IR spectrum of sampling regions down to about 10 pm resolution to be taken. Consequently, FTIR microscopy is ideal for compositional mapping and analysis of heterogeneous samples whose domain sizes are in the tens of micrometre range. 

FTIR imaging suffers from low signal to noise compared to other types of FTIR spectroscopy and microspectroscopy [106,107]. However, the major sources of error in an imaging system arise from detector noise and not from optical components of the imaging system (for example, apertures). The major factors in this regard are ... 

Figure 6.5 Spectral features as a function of the apodisation function in mid-IR microspectroscopy of tissues (colon tissue cryo-section, same sample position as in Figure 6.4). Transmission type spectra were acquired using Bruker s IRScope II microscope and an IFS28/B spectrometer. Further measurement parameters aperture diameter 900 pm, Cassegrain objective (36 x, NA 0.5), 128 scans, optical substrate CaF2 pm of 1 mm thickness. Spectral resolution 6 cm zero-filling factor (ZFF) 4. Transmission spectra were processed with a first derivative Savitzky-Golay filter with nine smoothing points.

In general, the goal for synchrotron-based FTIR microspectroscopy is to deliver images at the diffraction limit. This usually means setting one (or both) of the microscope s apertures to define a region somewhat smaller than the diffraction limit for the respective objective. For convenience, we use d= XjNA to define this diffraction-limited dimension. This is consistent with the calculated Full Width at Half Maximum (FWHM) of the Schwarzschild diffraction pattern and is also confirmed by experimental resolution studies on test specimens (to be shown later in this section). 

To summarize, in this section we have shown how single-point microspectroscopy using the synchrotron IR source and small, confocal aperturing can deliver improved resolution and contrast for biological imaging when compared with various array detector microscopes using a thermal source, but these improvements come with a serious handicap - extremely long measurement times. 

Figure 11.4 Infrared spectrum of hamster peripheral nervous tissue in the spectral range from 4000-900 cm measured at a synchrotron source by FTIR microspectroscopy through a lOxlOpm aperture.

Arguably the most important advantage of many microscopes used for Raman microspectroscopy is the fact that they have a confocal design. In a typical confo-cal design, see Figure 1.9, the laser beam is focused on a small aperture (to clean... 

Figure 14.2. Optics of a typical microscope with a single aperture used for FT-IR microspectroscopy. (Courtesy of PerkinElmer Corporation.)...

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