Interferometric SHG
Interferometric SHG (I-SHG) measures the phase of the second-harmonic signal to reveal the polarity of non-centrosymmetric structures such as collagen and microtubules.
Standard SHG microscopy shows where fibrillar collagen is, but two complementary measurements reveal how it is organised. Polarization-resolved SHG (Stoller et al., 2002) records the signal while the incident polarization is rotated: the resulting anisotropy encodes the in-plane orientation and the degree of alignment of the fibrils. To go further and recover the polarity — the actual direction in which fibrils point, which ordinary SHG cannot distinguish — one has to measure the phase of the second-harmonic field. Because SHG is a coherent process, two fibrils pointing in opposite directions emit fields in phase opposition (a π shift), so a phase measurement maps fibril polarity directly.
Polarization-resolved SHG: the signal is recorded while rotating the incident polarization (angle α). The anisotropy of the polar response encodes the orientation and alignment of the collagen fibrils.
Interferometric SHG (I-SHG) (Rivard et al., 2013) accesses this phase by letting the SHG from the sample interfere with a reference second-harmonic field generated by a quartz plate; a glass plate finely tunes the relative delay, and the interference fringes recorded while scanning the delay give the local SHG phase pixel by pixel. Applied to cartilage, I-SHG resolves domains of opposite collagen polarity that appear as a clear π phase shift in the phase map — and as two peaks in the phase histogram — information that is completely hidden in the intensity image.
I-SHG setup (a): the sample SHG interferes with the second harmonic from a quartz plate, the delay being tuned with a glass plate. The interference fringes (b) give the local phase; in cartilage (c–d) opposite collagen polarities show up as a π phase shift between domains (Couture et al., 2015).
The same principle extends beyond collagen: microtubules are also non-centrosymmetric and SHG-active. Using I-SHG on mitotic spindles in living embryos, we could follow the polarity of microtubule bundles in real time during cell division, distinguishing the two antiparallel halves of the spindle through their relative phase.
