Jarno N. van der Kolk

The Impact of Collagen Fibril Polarity on Second Harmonic Generation Microscopy

In this work, we report the implementation of interferometric second harmonic generation (SHG) microscopy with femtosecond pulses. As a proof of concept, we imaged the phase distribution of SHG signal from the complex collagen architecture of juvenile equine growth cartilage. The results are analyzed in respect to numerical simulations to extract the relative orientation of collagen fibrils within the tissue. Our results reveal large domains of constant phase together with regions of quasi-random phase, which are correlated to respectively high- and low-intensity regions in the standard SHG images. A comparison with polarization-resolved SHG highlights the crucial role of relative fibril polarity in determining the SHG signal intensity. Indeed, it appears that even a well-organized noncentrosymmetric structure emits low SHG signal intensity if it has no predominant local polarity. This work illustrates how the complex architecture of noncentrosymmetric scatterers at the nanoscale governs the coherent building of SHG signal within the focal volume and is a key advance toward a complete understanding of the structural origin of SHG signals from tissues.

Effects of refractive index mismatch on SRS and CARS microscopy.

An inhomogeneous linear refractive index profile, such as that occurring in biological tissues, is shown to significantly alter stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy images. Our finite-difference time-domain simulations show that near-field enhancement and microlensing can lead to an increase of an objects perceived molecular density by a factor of nine and changes in its perceived position by 0.4 μm up to 1.0 μm. Thus the assumption that SRS scales linearly and CARS quadratically with density does not always hold. Furthermore, the inhomogeneous linear index can cause false CARS and AM-SRS signals, even for a homogeneous nonlinear susceptibility.

High Contrast Interferometric Second Harmonic Generation Microscopy Probes the Polarity of Fibrils in Complex Collagenous Tissues

We implemented for the first time Interferometric SHG microscopy, using femtosecond pulses, to image the relative fibrils polarity in cartilage and show that SHG intensity depends on the local ratio of fibrils with opposite polarities.