Correlating Stimulated Emission Depletion Microscopy With Fluorescence Lifetime Imaging Microscopy to Study the TIE2...

The TIE2 transmembrane receptor protein, known for its role in vascular stability and blood vessel remodeling, has primarily been studied in endothelial cells. This receptor has also been found on several non-endothelial cell types including podocytes, although its presence on podocytes remains a matter of debate. Conventional immunofluorescence approaches applied to membrane proteins are often challenged by the strong tissue autofluorescence spanning green and red parts of the electromagnetic spectrum, sample thickness, and the antibody specificity. Here, we used stimulated emission depletion (STED) microscopy, a super-resolution microscopy method, to detect the TIE2 protein in complex biological tissue with a resolution of less than 50 nm. To further confirm the presence of TIE2 on podocytes and to investigate its localization, we used fluorescence lifetime imaging microscopy (FLIM) to more effectively distinguish between nonspecific autofluorescence and specific emission in cleared and antibody-labeled tissue samples. By correlating these two techniques, we mapped the subcellular localization of TIE2 on mouse podocytes and confirmed its presence on the cell surface facing the Bowman's space, albeit at lower expression levels. This study highlights the potential complementary power of STED and FLIM methods and provides additional evidence that the TIE2 receptor is present on podocytes.