Aspectos geográficos Orografía

The first reports of infrared microscopy appeared during the late 40's and early 50's (Barer et al 1949, Fraser 1951, 1950, Coates et al 1953). Fraser (1950) introduced a reflecting infrared microscope attachment of high numerical aperture and predicted that, in biological applications, areas of potential use included analysis o f natural crystals and oriented natural fibres.

Infrared microspectroscopy and measurements of molecular orientation using the phenomenon of infrared linear dichroism played a role in elucidating molecular

conformation in fibrous proteins (Bradbury and Elliott 1963 and Beer et al 1959) and an especially early role in determining the conformation of collagen (Fraser 1950; Ambrose and Elliott 1951, Bradbury et al 1958). Dichroic studies provided, for example, firm evidence, in support of X-ray diffraction studies, that peptides in the collagen molecule are not coiled in the form of an a helix (Fraser 1950), but, instead, have a distinct helical structure. Instrumental limitations of the period included low radiation throughput in the microscopes due to poor radiation utilization of the early sodium chloride prism

spectrometers and significant polarization scrambling in the transmission-type infrared radiation polarizers (multiple plates of silver chloride, AgCl) available at the time. These

contributed to long measurement times in microspectroscopic work generally, and significant errors in measurements of dichroism. Ultimately, there was a loss of interest in the technique, and analysis of microscopic samples continued to involve.. ."the use of what is referred to as "traditional" microsampling techniques.. .[including], for example, pinhole masking or micro potassium bromide [KBr] pellets" (Messerschmidt and

Harthcock 1988).

The introduction of Fourier transform infrared (FTIR) spectroscopy (Griffiths 1975) and the publication of the first book on microspectroscopy (Roush 1987) have been followed by the appearance of several commercial infrared microscope attachments and renewed interest in the technique. Some of the more significant technical

developments, in addition to FTIR, include-(l) the development o f sensitive mercury- cadmium-telluride (MCT) infrared detectors, (2) the development o f zinc selenide grids which polarize the infrared at a high level of purity (3) spherical reflective objectives and (4) redundant aperturing at sample image planes before and after the sample plane in the optical path. Current instruments can analyse picogram quantities of samples with pathlengths generally below 10pm (Katon et al 1990).

3.1.1 Infrared Spectroscopy of Protein

Infrared spectra are produced by passing the radiation thorough a sample

(transmission mode) and recording energy absorption as a function of infrared frequency. Absorption is dependent on frequency because it occurs only when there is a strong enough interaction between radiation and sample to induce a transition to a higher

vibrational energy state (a greater vibrational amplitude) in molecular structure; frequency is related to energy through—

E = hv (3.1)

where E is energy, h is Planck's constant (6.63 X 10'^^ J/s) and v is frequency. The frequency, or energy, required to produce a transition is dependent on the bond type but also on neighbouring bonds and the local structural (bonding) environment. In proteins, and other polymeric materials with repeating structural subunits and conformation, the recorded transitional frequency is further dependent on vibrational coupling along a macromolecular chain and between adjacent chains through hydrogen bonds.

Absorbance is therefore rather sensitive to conformation in the highly structured fibrous proteins, of which collagen is probably the most ubiquitous (Urban (1993), Susi et al (1971), Fraser and MacRae 1973, Susi (1969)).

3.1.2 Infrared Linear Dichroism of Collagen Fibres

The high degree of order in collagen molecular and fibrillar structure is depicted best in infrared spectra obtained using linearly polarized radiation. Infrared absorbance band intensity is dependent, in part, on the degree of coincidence between the vector o f the transition dipole moment o f the molecular vibration causing the band and the electric field vector of the incident radiation (Colthrup et al 1990, Campbell and Dwek 1984, Siesler and Holland-Moritz 1980). Therefore, the electric field vector of the linearly

polarized radiation incident upon collagen molecular structure serves to determine, by way o f absorbance intensity studies, the direction of transition dipole moments within the structure.

The axial orientation of collagen molecular and fibrillar structure in fibres and the molecular symmetry which occurs radially around this axis produce infrared absorbing properties which resemble those of uniaxial crystals (Hukins 1984 p.214; Fraser and MacRae 1973 [p. 108]; Elliott 1969). Zbinden (1964) uses the term "axial orientation" for these properties. Because of the radial symmetry, collagen fibres exhibit the same absorption coefficients in unpolarized radiation transmitted in all directions perpendicular to the fibre axis. In practice, when collagen fibres are placed onto the sample plane of the microscope, the axes of the individual fibres are automatically oriented perpendicular to the incident infrared beam. All perpendicular transmission vectors through the fibres are then equally suitable for the experiments using polarized radiation.

When absorbance of a polymeric fibre or stretched polymer film is measured in two directions using linearly polarized radiation, any change in spectral band intensities between the two orientations is due to a preferred, non-random orientation o f transition [dipole] moments within the molecular system. In studies o f collagen fibres, and for most natural fibres, the preferred axial orientation of molecular and fibrillar structure dictates that the electric field vector of the polarized radiation is successively aligned parallel to the fibre axis to produce the so-called parallel absorbance measurement (A,) and then perpendicular to the fibre axis to produce the other measurement (A J. The intensities of corresponding absorbance bands in the two spectra are then used to produce a dichroic

ratio R, defined as-

R = A/Aj^ (3.2)

which is used to determine the specific conformation of macromolecules and for the present purposes to define the degree of molecular order in collagen fibres. Variations of this ratio are used (Chase 1990). When there is no preferred orientation to molecular structure, all absorption bands in the parallel and perpendicular spectra have similar intensities and the dichroic ratios of the bands approximate one.

If dichroism defines molecular organization within polymeric materials then any loss o f dichroism reflects a decrease in this organization. The various mechanisms at work in the deterioration of protein fibres are listed in Chapter One (see section 1.2), but despite their variety all are expected to alter the native molecular conformation of collagen in essentially the same way.

Deterioration results in losses of collagen molecular thermal stability, as evidenced by lowered dénaturation temperatures in fibres (Young 1990, see Chapter Two) and changes in other thermodynamic properties (see Chapter Four). Multiple coordinate hydrogen bonds in collagen molecules are largely responsible for the thermal stability, and losses or weakening of these bonds due to the disruption of molecular conformation by deterioration reduce thermal stability. Measurements o f infrared linear dichroism were therefore investigated to detect the disruption of native molecular conformation in collagen fibres resulting from deterioration. The work was undertaken

to complement the other two methods of study but also to develop the method for research and routine analysis of microscopic samples.

Dichroic measurements were also taken over the dénaturation temperature range. Dichroic profiles of dénaturation were produced from these measurements in an

investigation of the effects o f deterioration on denaturational phenomena.

Analysis of single collagen fibres by microspectroscopy, as opposed to the analysis of many in transmission experiments, reduces sample requirements (Okada et al

1990) and minimizes problems of orientation and radiation scatter due to

differences in optical density among regions in a sample consisting o f many oriented fibres (Chase 1988). Analysis of single collagen fibres should therefore provide greater photometric accuracy and thus greater absorption accuracy in spectra. The technique is essentially non-destructive to museum artifacts because of the minuscule sample requirements, but, as for other micro-analytical methods, care must be taken to ensure that representative samples are analysed.

3.2 METHOD