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.

Collagen ultrastructure plays a central role in the function of a wide range of connective tissues. Studying collagen structure at the microscopic scale is therefore of considerable interest to understand the mechanisms of tissue pathologies. Here, we use second harmonic generation microscopy to characterize collagen structure within bone and articular cartilage in human knees. We analyze the intensity dependence on polarization and discuss the differences between Forward and Backward images in both tissues. Focusing on articular cartilage, we observe an increase in Forward/Backward ratio from the cartilage surface to the bone. Coupling these results to numerical simulations reveals the evolution of collagen fibril diameter and spatial organization as a function of depth within cartilage.

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 object's 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.

Conventional second harmonic generation (SHG) microscopy might not clearly reveal the structure of complex samples if the interference between all scatterers in the focal volume results in artefactual patterns. We report here the use of interferometric second harmonic generation (I-SHG) microscopy to efficiently remove these artifacts from SHG images. Interfaces between two regions of opposite polarity are considered because they are known to produce imaging artifacts in muscle for instance. As a model system, such interfaces are first studied in periodically-poled lithium niobate (PPLN), where an artefactual incoherent SH signal is obtained because of irregularities at the interfaces, that overshadow the sought-after coherent contribution. Using I-SHG allows to remove the incoherent part completely without any spatial filtering. Second, I-SHG is also proven to resolve the doubleband pattern expected in muscle where standard SHG exhibits in some regions artefactual single-band patterns. In addition to removing the artifacts at the interfaces between antiparallel domains in both structures (PPLN and muscle), I-SHG also increases their visibility by up to a factor of 5. This demonstrates that I-SHG is a powerful technique to image biological samples at enhanced contrast while suppressing artifacts.

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