Sherry G. Clendenon, Heather H. Ward, Kenneth W. Dunn, Robert Bacallao

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Presentation transcript:

High resolution 4-dimension imaging of metanephric embryonic kidney morphogenesis Sherry G. Clendenon, Heather H. Ward, Kenneth W. Dunn, Robert Bacallao Kidney International Volume 83, Issue 4, Pages 757-761 (April 2013) DOI: 10.1038/ki.2012.464 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Diagram of system for live imaging of embryonic kidney at high resolution. A × 25 water immersion objective with numerical aperture of 1.05 and working distance of 2mm, optimized to pass infrared excitation wavelengths, is mounted on an inverted Olympus FV1000 microscope equipped for multiphoton imaging. Oil with the refractive index of water optically couples the objective to the coverslip bottom dish. Coverglass thickness is 170μm. The reservoir of the medium above the glass is 1000μm in thickness. The clear transwell filter thickness is 10μm. The embryonic kidney maximum thickness is about 100μm. The total thickness of the embryonic kidney, filter, media, and coverslip is 1280μm, which is within the working distance of the objective. Stage, stage heater, and objective heater are not illustrated. Kidney International 2013 83, 757-761DOI: (10.1038/ki.2012.464) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Live embryonic kidneys imaged at high resolution in 3D over time. Bodipy ceramide–labeled (a, b and d–J) and Hoechsts-labeled (c) live embryonic kidneys imaged beginning at day 0 (a, b), day 1 (d–j), and day 2 (c) in culture. The embryonic kidneys were imaged at intervals of 30min (a, b) or one hour (d–j). Time after initiation of imaging is shown in hours. All images are single planes. Bodipy ceramide–labeled embryonic kidneys could be imaged in 3D over time (a, b and d–j). Hoechsts-labeled embryonic kidneys (c) could only be imaged one to two time points before the fragmentation of nuclei was observed. Images taken 30μm from the dorsal side over time (d–f) show condensation of the mesenchyme at bud tips (f, arrowhead), and branched ureteric buds (f, arrows). Images taken 50μm from the dorsal side (g–j) show cross-sections of ureteric bud branches, renal vesicles, S-shaped bodies (i), and formation of S-shaped bodies (j). (a–f) Bars=100μm. (g) Bar=200μm. (j) Bar=50μm. RV, renal vesicle; SSB, S-shaped body. Kidney International 2013 83, 757-761DOI: (10.1038/ki.2012.464) Copyright © 2013 International Society of Nephrology Terms and Conditions