We describe a protocol to create tissue phantoms with hydrophobic nanoporous particles. The nanopores of the particles are loaded with biological molecules at the desired compositions. Tissue phantoms are prepared by immersing dried particles into aqueous biological matrixes. The hydrophobicity of the pore surface prevents the solution from penetrating into the nanopores, thus preserving the designed molecular composition inside the particles. This protocol provides a unique approach to preparing biological systems in small domains, at micrometer and nanometer dimensions, with well-defined boundaries and tailored biological and optical properties. The nanoporous particle approach is easy when compared to the common preparation methods such as with polymers and vesicles as it involves direct loading of the biological molecules into the pores and does not require complex synthetic steps. The method is adaptable, with tunable pore and particle sizes, and robust, with a rigid boundary to protect the designed biological domain. In addition to tissue phantom preparation, this approach is applicable in systems where a well-defined biological domain is desired.
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