Development of hydrogel-based standards and phantoms for non-linear imaging at depth

Rapid advances in medical imaging technology, particularly the development of optical systems with non-linear imaging modalities, are boosting deep tissue imaging. The development of reliable standards and phantoms is critical for validation and optimization of these cutting-edge imaging techniques. We aim to design and fabricate flexible, multi-layered hydrogel-based optical standards and evaluate advanced optical imaging techniques at depth. Standards were made using a robust double-network hydrogel matrix consisting of agarose and polyacrylamide. The materials generated ranged from single layers to more complex constructs consisting of up to seven layers, with modality-specific markers embedded between the layers. These standards proved useful in the determination of the axial scaling factor for light microscopy and allowed for depth evaluation for different imaging modalities (conventional one-photon excitation fluorescence imaging, two-photon excitation fluorescence imaging, second harmonic generation imaging, and coherent anti-Stokes Raman scattering) achieving actual depths of 1550, 1550, 1240, and , respectively. Once fabricated, the phantoms were found to be stable for many months. The ability to image at depth, the phantom's robustness and flexible layered structure, and the ready incorporation of "optical markers" make these ideal depth standards for the validation of a variety of imaging modalities.