Elimination of imaging artifacts in second harmonic generation microscopy using interferometry

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 double-band 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.