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  • FTIR microspectroscopic analysis: future perspectives. Infrared spectroscopy

Molecular bonds are not stationary, but rather undergo motion such as twisting, bending, stretching, rotation and vibration. When irradiated with infrared radiation, these vibrational mo­tions absorb at specific wavelengths, characteristic of the over­all configuration of the atoms, and representative of specific functional groups. Moreover, through detailed analysis of the absorption wavelengths, information may be deduced on the subtle interactions with the surrounding atoms of a molecule. FTIR spectra provide information on all tissue components. The protein and mineral constituents produce intense, struc­ture sensitive IR modes.

IR spectroscopy has been extensively utilized in the analysis of bone mineral. Spectroscopic and mathematical analy­sis of the phosphate band by means of techniques such as de- convolution, second derivative spectroscopy, and curvefitting, spectral regions (underlying peaks) were identified and corre­lated with the various chemical environments present in biolog­ical apatites, enabling the monitoring of the calcium phosphate crystal maturity (ionic substitutions, stoichiometry).

The protein Amide I (peptide bond C=O stretch) and Amide II (mixed C-N stretch and N-H in-plane bend) modes near 1650 and 1550 wavenumbers (cm-1), undergo frequency and inten­sity changes as a result of changes in protein secondary struc­ture. The Amide I band is especially sensitive to secondary structures. In such studies, information on protein struc­tures is extracted from broad envelopes consisting of compo­nent bands arising from the Amide I modes of various sec­ondary structures by applying a technique of resolution en­hancement such as Fourier self-deconvolution, second deriva­tive spectroscopy, and difference FTIR. Although detailed information on mineral maturity and protein secondary structure was obtainable utilizing these techniques, homogenized bone tissue and / or proteins in solution had to be used, thus it was not possible to correlate the findings with the metabolic activity of bone surfaces (tissue age).

Infrared microspectroscopy

The coupling of an optical microscope with an infrared spec­trometer in the early 1990’s offered the unique opportunity of studying thin bone tissues with a spatial resolution of ~ 10 |jm, and to select the anatomical areas to be analyzed based on parallel histologically stained sections thus enabling the corre­lation of the spectroscopic result with bone surface metabolic activity (tissue age). The pioneering work of Drs Mendelsohn and Boskey was later followed and expanded by them and others, resulting in a wealth of new in­formation about the mineral component of bone as a function of cellular activity, tissue age, disease, and therapeutic inter­vention. Beat the drug companies and buy cialis professional online

A major breakthrough was the development of spectroscopic parameters that enabled for the first time the monitoring of two of the major collagen cross-links (pyr and deH-DHLN) in thin, histologically stained bone sections, allowing the monitoring of the variation in their spatial distribution as a function of anatomical location, cellular activity, and tissue age. As informative as it may be, FTIR microspectroscopic analysis on instruments equipped with a single infrared detector was a time-consuming proposition as analysis of a single section re­quired 2-3 days. The fairly recently available combination of an infrared focal-plane array (FPA) detector and a FTIR micro­scope is a powerful one for obtaining spectroscopic images with unprecedented image fidelity. The advantage of this technique lies in the fact that the spectra acquisition and processing time is shortened at least 1000-fold compared with conventional IR microspectroscopy. Use of a step-scanning FTIR spectrometer with an MCT array detector placed at an image focal plane of an IR microscope enables areas 400×400 |m2 to be collected in less than 3-4 minutes at a spatial resolu­tion of ~6.3 jim. To date, it has been successfully applied in the analysis of cell cultures, and bones from animal models and humans.

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