Medical imaging is advancing rapidly through the development of novel laser sources and non-linear imaging methodologies. These developments are boosting deep tissue imaging allowing researchers to study diseases deep in the body enabling early diagnosis and better treatment. To help with the testing and optimization of these imaging systems and to aid in this process of deep tissue imaging, it's important to have robust, stable and reproducible standards and phantoms. Herein we present the design and fabrication of robust, multi-layered, hydrogel-based standards. The hydrogel used is a double network hydrogel consisting of two interpenetrating networks agarose and polyacrylamide. Thin layers of tough double network hydrogels are stacked to form multilayered depth standards having modality specific signaling markers embedded in between. Standard design and assembly ensured long term stability and easy transport. These proved useful in-depth imaging studies, utilizing multiple imaging modalities, including one photon fluorescence (1PEF), two photon fluorescence (2PEF), coherent anti-Stokes Raman imaging (CARS) and second harmonic generation imaging (SHG).
SignificanceRapid 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.AimWe aim to design and fabricate flexible, multi-layered hydrogel-based optical standards and evaluate advanced optical imaging techniques at depth.ApproachStandards 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.ResultsThese 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 1240 μm, respectively. Once fabricated, the phantoms were found to be stable for many months.ConclusionsThe 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.
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