Successful translation of the use of nanoparticles from laboratories to clinics requires exhaustive and elaborate studies involving the biodistribution, clearance and biocompatibility of nanoparticles for in vivo biomedical applications. We report here the use of multimodal organically modified silica (ORMOSIL) nanoparticles for in vivo bioimaging, biodistribution, clearance and toxicity studies. We have synthesized ORMOSIL nanoparticles with diameters of 20-25 nm, conjugated with near infra-red (NIR) fluorophores and radiolabelled them with 124 I, for optical and PET imaging in vivo. The biodistribution of the non targeted nanoparticles was studied in non-tumored nude mice by optical fluorescence imaging, as well by measuring the radioactivity from harvested organs. Biodistribution studies showed a greater accumulation of nanoparticles in liver, spleen and stomach than in kidney, heart and lungs. The clearance studies carried out over a period of 15 days indicated hepatobiliary excretion of the nanoparticles. Selected tissues were analyzed for any potential toxicity by histological analysis, which confirmed the absence of any adverse effect or any other abnormalities in the tissues. The results demonstrate that these multimodal nanoparticles have potentially ideal attributes for use as biocompatible probes for in vivo imaging. KeywordsORMOSIL Nanoparticles; optical and PET Imaging; NIR fluorophore; 124 I radiolabeling; Biodistribution; clearance and toxicity Nanomaterials promise to address the current limitations of sensitivity and specificity of medical diagnostics, as well as significantly improve the outcome of existing and emerging therapeutics, via the introduction of new generation of multimodal nanoprobes. [1][2][3][4][5][6] In this regard, it is critical to design nanoprobes with desired composition, size and surface functionalities, and rigorously test them in vitro and in vivo for their safety and efficiency. Also biocompatibility and biodegradation of the nanoprobe materials play an important role in the use of the nanoprobes in the field of diagnostic and therapeutic applications.The rise in nanoprobes development has bolstered the prospects of in vivo optical imaging through the development of a variety of NIR-luminescent nanoformulations, which include quantum dots, 7 upconverting nanophosphors, 8 Herein, we report the synthesis of ultrafine ORMOSIL nanoparticles (diameter ∼20 nm), conjugated with a near-infra-red (NIR) fluorophore, as optical probes. The resulting NIRnanoparticles will facilitate optical bioimaging in the NIR window, with maximum tissue penetration of light and minimum background signal. 23, 24 Furthermore, we have also conjugated the well-known positron emission tomographic (PET) imaging probe Iodine-124 with the nanoparticle, which will allow bioimaging independent of tissue-depth, as well as more accurate quantification of accumulation of nanoparticles in various major organs in vivo. These multimodal nanoprobes have been injected systemically in mice, and their in v...