Holographic second harmonic phase microscopy of collagen fibers

In the past decade, quantitative phase imaging gave a new dimension to optical microscopy, and the recent extension of digital holography techniques to nonlinear microscopy appears very promising, for the phase of nonlinear signal provides additional information, inaccessible to incoherent imaging schemes. Last year, we have demonstrated how the SHG phase makes possible real-time nanometric 3D-tracking of SHG emitters, such as nanoparticles (Shaffer et al., Opt. Express 18, p.17392-17403, 2010). Here, we investigate the phase of second harmonic generated by a label-free biological specimen—more precisely collagen fibers forming the connective tissue of a mouse dermis—and discuss of its interpretation. Notably, we show how the SHG phase, qualitatively acting as an indicator of phase-matching conditions, tends to indicate that second harmonic generation, in collagen, is dominated by coherent SHG scattering.