Using Correlative Imaging to Study Collagen Denaturation

Collagen is the key structural protein found in animal connective tissue and has a wide range of applications. Collagen in the form of gelatin is used in the cosmetic, food and pharmaceutical industries to name but a few.

Co-localization (overlay) of AFM topography and nanoscale infrared (IR) image of degraded collagen using MountainsMap^®.

In addition, this protein can be found in articles used by ancient civilizations such as bow strings, glue or objects made from leather (scrolls, clothes, etc.).

Recently, Researchers at the Université Paris-Sud (France)* presented the results of an innovative project to map denaturation of collagen using nanoscale infrared (IR) spectroscopy and nonlinear optical microscopy (NLO).

Our main aim in this study was to characterize gelatinization, the ultimate and irreversible alteration corresponding to collagen denaturation to gelatin. This process can be caused by UV radiation, or contact with water or heat for example. In our study, we analyzed its effects on a 17^th century parchment but our findings could of course be applied to the study of other materials where collagen is present including biological tissues (skin for example).

Professor Alexandre Dazzi, Author of the study

Professor Dazzi then added, "Two complementary imaging techniques were used to probe alterations at various structural levels. Both these techniques have the advantage of being non-invasive and non-destructive, which means even fragile or precious objects may be studied, as was the case here."

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Firstly, nonlinear optical microscopy (NLO) was used to acquire 3D multimodal imaging of scattering samples with micrometer-scale resolution; however, this method cannot resolve nanometer structural features.

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Therefore, NLO was combined with infrared (IR) spectroscopy to detect the chemical origin of morphological changes. Moreover, given the scale at which spectroscopic analysis had to be performed (~100 nm), IR spectroscopy (nanoIR) was used.

This is the latest technique that combines an Atomic Force Microscope (AFM) with an IR pulsed tunable laser (commercialized by Anasys Instruments).

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This made it possible to acquire local IR spectra and chemical mapping and thus characterize images at the nanoscale.

Comparing Spectral Data in MountainsMap^®

The data obtained from pure collagen reference samples and the spectroscopic signatures acquired were compared together. The following results were provided by MountainsMap^® tools for spectral analysis:

Spectral signature of collagen reference sample (filtered using the Smooth the spectra operator).

Four extracted spectra corresponding to four different points on the degraded collagen surface (see above).

Comparison of these four points with collagen reference spectra (blue), showing phase shift to the right. Data displayed in new Normalized view.

Note: * Along with the French National Center for Scientific Research (CNRS), the Ministry of Culture and Communication, the École Polytechnique and the Université Pierre et Marie Curie.

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