Application of multiphoton microscopy to study of collagen regeneration

Nonlinear optical microscopy (NLOM) is a new imaging technique that produces clear images of biological samples on the subcellular resolution level (Zipfel et al. ). The key subsystem of NLOM is an ultrashort-pulse laser whose radiation is tightly focussed in a biological specimen.  At focus, enormous optical intensity (10^17-fold that of the sunlight for a short time interval of 100 femtoseconds) elicits nonlinear optical response resulting in photon emission at a frequency twice that of the incident photons.  The process is called second harmonic generation (SHG).  Simultaneous absorption of two (or several) photons followed by fluorescence emission is called multiphoton fluorescence (MPF).  Under typical conditions, the infrared laser excitation is filtered, so that the SHG and MPF signals in the visible spectral range result in high-contrast en face imaging, if the focal spot is scanned across the specimen (as exemplified in Figure). This mechanism of NLOM image formation entails the most valuable property of MPM, "optical sectioning", i.e. clearing a micron-thin image slice from the turbidity of the rest of the specimen.

In this project, we intend to apply NLOM to image collagenous tissue, such as cartilage, bone, skin, etc., relying on a strong second-harmonic signal from collagen. In collaboration with the medical and laser physics researchers, we will investigate, yet mysterious, mechanisms of bone regeneration, and collagen regenration treated with laser light (Ignatieva et al.). Understanding these mechanisms will have tremendous impact on the current practice of treatment of osteoarthritis and the related disease.

W. R Zipfel, R. M. Williams, and W. W. Webb, Nature Biotechnology 21 , 1369 (2003)

N. Ignatieva, O. Zakharkina, G. Leroy , E. N. Sobol , N. Vorobieva, S. Mordon, "Molecular processes and structural alterations in laser reshaping of cartilage", Laser Phys. Lett. 1–5 (2007) / DOI 10.1002/lapl.200710042

Figure. NLOM (SHG) image of the unstained mouse bone fracture, showing the bone regrowth (centre).  Inset: An overlay image of SHG (red) and MPF (green) zoomed area (white square).  Red blood cells (arrow) are clearly observable.  Preliminary results of our collaborative work with the ANZAC Research Institute investigators.

For more details on this project, please contact us

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