Microscope, infrared

The spatial resolution of the Raman microprobe is about an order of magnitude better than that obtainable using an infrared microscope. Measurement times, typically of a few seconds, are the same as for other Raman spectrographs. To avoid burning samples, low (5—50-mW) power lasers are employed. 

Infrared microscopes can focus the beam down to a 20-pm spot size for microprobing in either the transmission or reflection mode. Trace analysis, microparticle analysis, and spatial profiling can be performed routinely. 

In transmission mode a spatial resolution of about 15-20 pm can be achieved with infrared microscopes [32]. This is generally sufficient to properly identify such as small impurities, inclusions, gels or single components of multilaminate foils. Similar to Raman spectroscopy, line profiles or maps over larger sample areas can be performed. 

The value of infrared spectroscopy in archaeology and materials conservation has been greatly enhanced in the last ten years or so by the development of infrared microscopes (Kempfert et al. 2001). Especially when... 

Wang, J. A., Sun, S. Q., Zhou, Q., et al. (1999). Nondestructive identification of ballpoint writing inks with Fourier transform infrared microscope. Chinese Journal of Analytical Chemistry 27 697-700. 

After the experiment, the experimental charge is prepared for analysis of the diffusion component or species. The analytical methods include microbeam methods such as electron microprobe, ion microprobe, Rutherford backscatter-ing, and infrared microscope to measure the concentration profile, as well as bulk methods (such as mass spectrometry, infrared spectrometry, or weighing) to determine the total gain or loss of the diffusion component or species. Often, the analysis of the diffusion profile is the most difficult step in obtaining diffusivity. 

Figure 10.13—Computer controlled IR spectrometer. Many models of FTIR instruments permit the adaptation of an infrared microscope for the study of microsamples model NEXUS (11>W), reproduced by permission of Nicolet).

Mirabella, F. M., Jr. (1987) Applications of microscopic Fourier transform infrared spectrophotometry sampling techniques for the analysis of polymer systems. In The Design, Sample Handling and Applications of Infrared Microscopes (P. B. Rousch, ed.), American Society for Testing and Materials, Philadelphia, PA, pp. 74—83. 

Fourier transform infrared microscopes are equipped with a reflection capability that can be used under these circumstances. External reflection spectroscopy (ERS) requires a flat, reflective surface, and the results are sensitive to the polarization of the incident beam as well as the angle of incidence. Additionally, the orientations of the electric dipoles in the films are important to the selection rules and the intensities of the reflected beam. In reflectance measurements, the spectra are a function of the dispersion in the refractive index and the spectra obtained are completely different from that obtained through a transmission measurement that is strongly influenced by the absorption index, k. However, a complex refractive index, n + ik can be determined through a well-known mathematical route, namely, the Kramers-Kronig analysis. 

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. 

Ouyang, H, Sherman, P. J., Paschalis, E. P., Boskey, A. L. and Mendelsohn, R. (2004) Fourier transform infrared microscopic imaging effects of estrogen and estrogen deficiency on fracture healing in rat femurs. Appl. Spectrosc. 58, 1-9. 

Baeten, V., Michotte Renier, A., Fernandez Pierna, A. et al. (2004) Review of the possibilities offered by the near infrared microscope (NIRM) and near infrared camera (NIR Camera) for the detection of MBM, Oral Presentation, Intenational Symposium Food and Feed Safety in the Context of Prion Diseases, 16-18 June, Namur, Belgium. 

Our group was the first to report imaging with a diamond ATR accessory that provided a field of view of ca. 1 mm2 and the spatial resolution of ca. 15 pm without the use of an infrared microscope [18], The demonstration of the applicability of a diamond ATR accessory for FTIR imaging opened up a range of new opportunities in polymer research, from compaction of tablets [21-23] to studying phase separation in polymer blends subjected to supercritical fluids [24], This imaging approach was successfully utilised for the study of dissolution of tablets in aqueous solutions [25], We have also demonstrated macro... 

Figure 4.6-11 Selective reflection observed with an infrared microscope at different positions in a wedged sample as schematically outlined by the insert (see text), Zachmann and Kortc, unpublished.

Ueno, T., Furukawa, D. and Matsumoto, A. (2008) Thermally-induced polymerization of muconic esters in the solid state studied by infrared microscope spectroscopy under temperature control. Macromol. Chem. Phys., 209, 357-365 See also the cover picture for the issue of Vol. 209,... 

The optical layout for the measurement of biological samples (cells) is shown in Figure 29.3b. The sample was irradiated with co-linear IR and visible light beams. The transient fluorescence from the sample was collected from the opposite side by an objective lens. In this optical layout, the spatial resolution was determined by the objective numerical aperture (NA) and the visible fluorescence wavelength IR superresolution smaller than the diffraction limit of IR light was achieved. Here, Arabidopsis thaliana roots stained with Rhodamine-6G were used as a sample. We applied this super-resolution infrared microscope to the Arabidopsis thaliana root cells, and also report the results of time-resolved measurements. 

One of the important functions of this infrared microscope is the measurement of the IR spectrum from a spatial region smaller than the diffraction limit. This possibility is already illustrated in Figure 29.4e. The TFD-IR spectrum, that corresponds to the IR absorption spectrum, was measured from a fluorescence region smaller than the IR diffraction limit. Infrared spectroscopy in a sub-micron region will be possible by using a high NA objective lens with the confocal optical system. 

We have performed super-resolution infrared microscopy by combining a laser fluorescence microscope with picosecond time-resolved TFD-IR spectroscopy. In this chapter, we have demonstrated that the spatial resolution of the infrared microscope improved to more than twice the diffraction limit of IR light. It should he relatively straightforward to improve the spatial resolution to less than 1 pm by building a confocal optical system. Thus, in the near future, the spatial resolution of our infrared microscope will be improved to a sub-micron scale. 

Therefore, by using this super-resolution infrared microscope, we will be able to carry out space- and time-resolved vibrational microspectroscopy in the IR super-resolved region. Given that IR absorption is regarded as the fingerprint of a molecule, the new super-resolution infrared microspectroscopy will become an extremely important tool, not only in microscopy but also in spectroscopy. 

We have also demonstrated picosecond time-resolved TFD-IR imaging of the vibration relaxation of Rhodamine-6G in Arabidopsis thaliana roots, and found an abnormally long-lived component of vibrational relaxation in a cell. This may result from a site dependence of vibrational relaxation within whole cells. These results indicate the possible utility of the two-color super-resolution infrared microscope in mapping specific IR absorptions with high spatial resolution, and the observation of dynamics in a non-uniform environment, such as a cell. By using this infrared super-resolution microscope, we will be able to visualize the structure and reaction dynamics of molecules in a wide range of non-uniform environments. 

Yano, K., Ohoshima, S., Gotou, Y., Kumaido, K., Moriguchi, T. and Katayama, H. (2000) Direct measurement of human lung cancerous and noncancerous tissues by Fourier transform infrared microscopy Can an infrared microscope be used as a clinical tool Anal. Biochem., 287, 218-225. 

Specialised sampling techniques such as attenuated total reflectance (ATR) and diffuse reflectance (DR) have been found to be exhemely effective and hence have gained considerable popularity. Microsampling, for measuring very small samples, has become a common technique over the last decade as beam condensers and infrared microscopes (plus accessories) have been improved. 

The manufacturers of infrared microscopes are split almost evenly between those that produce infinity-corrected and those producing non-infinity-corrected microscopes. Infinity correction effectively refers to a coUimation of the beam throughout the microscope (other than at the condenser and objective outputs), and is frequently used in research-grade optical microscopes. Despite the added... 

Figure 13.13 Spectral image of a cross-sectioned photographic film recorded using a prism-based PA IR spectrograph coupled to an infrared microscope. Reproduced with permission from Ref [8].

Infrared microscopes are commercially available from a number of companies worldwide, and operate much like conventional visible light microscopes. The IR radiation follows the same path as the sample iUumination Ught, so that IR micro-spectroscopy can be performed on the sample at the center of the viewing field. Because of their design, they are also equipped with a number of convenient methods for enhanced sample visualization. These include polarized light (visible and IR), fluorescence illumination and differential interference contrast (DIG), all of which are well known and frequently used to identify biological sample histology. 

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