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Multiphoton microscopy of cleared tissue has previously been demonstrated to generate large three-dimensional (3D) volumetric image data on entire intact mouse organs using intrinsic tissue fluorescence. This technique holds great promise for performing 3D virtual biopsies, providing unique information on tissue morphology, and guidance for subsequent traditional slicing and staining. Here, we demonstrate the use of fluorescence lifetime imaging in cleared organs for achieving molecular contrast that can reveal morphologically distinct structures, even in the absence of knowledge of the underlying molecular source. In addition, we demonstrate the power of multimodal imaging, combining multiphoton fluorescence, second harmonic generation, and lifetime imaging to reveal exceptional morphological detail in an optically cleared mouse knee.

Multiphoton fluorescence, second harmonic generation, and fluorescence lifetime imaging of whole cleared mouse organs