Figure 2.

Visualization of 3D vessel trees and dendritic cells in entire ECi-cleared kidneys via LSFM and confocal/two-photon imaging. LSFM of specifically stained endothelial structures (CD31; red) allows three-dimensional reconstruction of whole kidneys. (A) Three-dimensional reconstructions of kidneys from a healthy control or an organ suffering from NTN (day 14). Note that the NTN kidney shows lower glomerular density, whereas two–dimensional optical sections reveal CD31-negative areas of corrupted glomeruli and the surrounding vasculature compared with a homogeneous field of equally sized glomeruli in healthy controls (white boxes). Furthermore, a higher heterogeneity of glomerular tuft sizes was observable in NTN kidneys at day 14. Many shrunken glomerular tufts (open arrowheads) were visible next to normally sized elements (white arrowheads). Scale bars, 50 μm. (B) Enhanced views of kidney structure (autofluorescence [gray] and fibrous tissue [second harmonic generation (SHG); blue]) and glomerular tuft (CD31; red) via combined confocal and two–photon laser scanning microscopy (LSM) compared with enhanced LSFM magnification. Compared with controls, NTN kidneys showed decreased tuft size (open arrowheads; as opposed to normal tuft size [white arrowheads]), decrease of endothelial CD31 label in their capillaries, and increased tissue fibrosis (indicated by higher SHG signal; white arrows) around damaged glomeruli. Scale bars, 20 μm in confocal/two-photon microscopy; 50 μm in LSFM. (C) Penetration depth of confocal LSM into ECi-cleared kidneys from CD11c-EYFP animals is enhanced compared with in uncleared samples. The maximum penetration depth of short wavelengths was increased from approximately 50 to 800 μm as shown via detection of CD11c-eYFP+ cells. The detected CD11c-eYFP+ cells still show the characteristic dendritic morphology after ECi clearing in the entire kidney tissue as emphasized at two exemplary focal planes of the shown Z stack (1 and 2). Longer wavelengths of the used endothelial marker CD31-Alexa Fluor-647 (red) can be detected even deeper in the tissue. The objective of the used confocal microscope allows a maximum penetration depth of 2500 μm because of the working distance. This penetration depth was reached in cleared kidneys and still allowed the discrimination of glomeruli from the surrounding tubular structures as indicated with the white lines at different focal planes (3–5).