Craigie, Eilidh

Devuyst, Olivier

Haenni, Dominik

Hall, Andrew M.

Schuh, Claus Dieter

Weber, Bruno

Ziegler, Urs

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ACUTE KIDNEY INJURY –EXPERIMENTALFP174 INTRAVITALMULTIPHOTON KIDNEY IMAGING USING ACUSTOMDESIGNEDMICROSCOPE AND EXTENDEDWAVELENGTH EXCITATION LASERClaus Dieter Schuh1, Dominik Haenni2, Eilidh Craigie1, Urs Ziegler2, Bruno Weber3,Olivier Devuyst4 and Andrew M Hall11University of Zurich, Institute of Anatomy, Zurich, Switzerland, 2University ofZurich, Center for Microscopy & Image Analysis, Zurich, Switzerland, 3University ofZurich, Institute of Pharmacology & Toxicology, Zurich, Switzerland, 4University ofZurich, Institute of Physiology, Zurich, SwitzerlandIntroduction and Aims: Intravital multiphoton microscopy is a powerful tool toinvestigate structure and function in the intact kidney at high resolution, but severalissues have constrained its usage to date. Imaging depth is limited to the outer cortexwith standard femtosecond excitation laser sources (680-1050 nm) due to strongexcitation light scattering, whilst fluorescent probes with emission maxima in the greencan be masked by auto-fluorescence (AF) signals. Moreover, recording of dynamicevents in vivo can be complicated by movement artefacts. Thus, there is a need for newapproaches to overcome these practical hurdles to fully realise the potential of intravitalimaging in investigating kidney physiology and disease mechanisms. Newly availableextended wavelength excitation lasers can work in the far red range where thescattering is less pronounced thereby offering the possibility to image deeper intotissues and spectrally away from AF signals.Methods:We have constructed a custom built multiphoton microscope for in vivokidney imaging, coupled to an extended wavelength excitation laser (680-1300nm).The microscope is modularly designed rendering it flexible for adaption e.g.concerning objectives and detectors. C57Bl/6N mice were used for experiments andwere anaesthetised with inhaled isoflurane. All procedures were conducted inaccordance with Swiss Cantonal regulations. A jugular vein catheter was used forapplication of dyes. Kidneys were externalized via a lateral flank incision. Animals wereplaced on a stage specially designed to reduce transmission of respiratory movements,and kidneys were embedded in agarose to further dampen movements. Fluid-phaseand receptor-mediated endocytosis in proximal tubules (PTs) were imaged usingfluorescent-labelled small dextrans and/or proteins. Lysosomes and mitochondria werelabelled with established probes (Lysotracker and Rhodamine 123). Acute kidneyinjury (AKI) was induced with sodium maleate (600 mg/kg).Results:We gained several distinct advantages with this novel set-up. First, we couldexcite far red probes such as Albumin-Alexa 647, away from masking AF signals, thusincreasing the signal to background ratio. Second, with far red probes we were able toimage deeper into the kidney than with comparable blue/green/red dyes, enhancing thevisualisation of key structures such as glomeruli. Third, we could perform label-freeimaging of structures such as collagen and lipid vesicles using second and thirdharmonic generation signals, respectively. Fourth, we could acquire 4 signalssimultaneously (blue, green, red, far red), and could thus image multiple differentstructures within cells (including endosomes, lysosomes and mitochondria). Fifth, dueto enhanced stability of the imaged organ, we could acquire images over prolongedperiods of time (up to 5 hours), and discovered that the intracellular environment inPT cells is highly dynamic, with rapid and directed movement of organelles. Real-timechanges in organelle function during AKI were also imaged following injections ofmaleate.Conclusions: In summary, using a custom-designed multiphoton microscope andextended wavelength excitation, we have made significant improvements to intravitalkidney imaging that will enhance the capabilities of this technique in the futureinvestigation of renal physiology and pathophysiology.© The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.Nephrology Dialysis Transplantation 30 (Supplement 3): iii125–iii139, 2015doi:10.1093/ndt/gfv172.2

FP174INTRAVITAL MULTIPHOTON KIDNEY IMAGING USING A CUSTOM DESIGNED MICROSCOPE AND EXTENDED WAVELENGTH EXCITATION LASER

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FP174INTRAVITAL MULTIPHOTON KIDNEY IMAGING USING A CUSTOM DESIGNED MICROSCOPE AND EXTENDED WAVELENGTH EXCITATION LASER

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