Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution.

We present cleared-tissue axially swept light-sheet microscopy (ctASLM), which enables isotropic, subcellular resolution imaging with high optical sectioning capability and a large field of view over a broad range of immersion media. ctASLM can image live, expanded, and both aqueous and non-aqueous chemically cleared tissue preparations. Depending on the optical configuration, ctASLM provides up to 260 nm of axial resolution, a three to tenfold improvement over confocal and other reported cleared-tissue light-sheet microscopes. We imaged millimeter-scale cleared tissues with subcellular three-dimensional resolution, which enabled automated detection of multicellular tissue architectures, individual cells, synaptic spines and rare cell-cell interactions.

Heterozygous Mutation of Vegfr3 Reduces Renal Lymphatics without Renal Dysfunction.

BACKGROUND: Lymphatic abnormalities are observed in several types of kidney disease, but the relationship between the renal lymphatic system and renal function is unclear. The discovery of lymphatic-specific proteins, advances in microscopy, and available genetic mouse models provide the tools to help elucidate the role of renal lymphatics in physiology and disease. METHODS: We utilized a mouse model containing a missense mutation in Vegfr3 (dubbed Chy ) that abrogates its kinase ability. Vegfr3 Chy/+ mice were examined for developmental abnormalities and kidney-specific outcomes. Control and Vegfr3 Chy/+ mice were subjected to cisplatin-mediated injury. We characterized renal lymphatics using tissue-clearing, light-sheet microscopy, and computational analyses. RESULTS: In the kidney, VEGFR3 is expressed not only in lymphatic vessels but also, in various blood capillaries. Vegfr3 Chy/+ mice had severely reduced renal lymphatics with 100% penetrance, but we found no abnormalities in BP, serum creatinine, BUN, albuminuria, and histology. There was no difference in the degree of renal injury after low-dose cisplatin (5 mg/kg), although Vegfr3 Chy/+ mice developed perivascular inflammation. Cisplatin-treated controls had no difference in total cortical lymphatic volume and length but showed increased lymphatic density due to decreased cortical volume. CONCLUSIONS: We demonstrate that VEGFR3 is required for development of renal lymphatics. Our studies reveal that reduced lymphatic density does not impair renal function at baseline and induces only modest histologic changes after mild injury. We introduce a novel quantification method to evaluate renal lymphatics in 3D and demonstrate that accurate measurement of lymphatic density in CKD requires assessment of changes to cortical volume.