Detection of trace crystallinity in amorphous pharmaceutical formulations by complementary harmonic imaging

Precise determination of crystalline content within amorphous formulations is crucial for ensuring drug product quality, stability and bioavailability. Despite the widespread use of powder X-ray diffraction and Differential scanning calorimetry, these techniques often lack the sensitivity to quantify trace crystallinity. We propose a multimodal approach based on polarization-controlled second-and third harmonic generation (SHG and THG) microscopy for detection and quantitative analysis of trace crystals in nominally amorphous samples. While SHG microscopy has demonstrated remarkable sensitivity, it remains fundamentally limited to non-centrosymmetric materials within the dipole approximation. By exploiting the symmetry-sensitive contrast SHG with THG, we successfully differentiated different solid forms of indomethacin, namely α,γ and amorphous form. Under circular polarization, THG is forbidden in isotropic amorphous regions, while persisting in all anisotropic phases, providing a universal detection mechanism. As such, THG microscopy enabled quantitative determination of crystalline fraction by simple comparison of THG images acquired under linear versus circular polarization, allowing straightforward thresholding and subsequent binary classification of amorphous versus crystalline regions. This approach addresses critical symmetry limitations of existing nonlinear optical techniques and provides a foundation for universal pharmaceutical crystallinity detection.