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Confocal imaging of slit diaphragm proteins in expanded kidney tissue
KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
Karolinska Institutet.
Karolinska Institutet.
Karolinska Institutet.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The subtlest element of the kidney, such as the slit diaphragm, has historically only been spatially resolved using electron microscopy due to the nanometer-scale dimensions of these structures. Recently, it was shown that the nanoscale distribution of proteins in the slit diaphragm can be resolved by fluorescence based stimulated emission depletion (STED) microscopy, in combination with optical clearing. Fluorescence microscopy has advantages over electron microscopy in terms of multiplex imaging of different molecular species (i.e. epitopes), and also the amount of volumetric data that can be extracted from a thick sample. However, STED microscopy is still a costly technique commonly not available to all life science researchers. An image technique with which the slit diaphragm proteins in the kidney can be visualized using more standard fluorescence imaging techniques is thus desirable. Recent studies have shown that biological tissue samples can be isotropically expanded while optically cleared, revealing nanoscale localizations of multiple epitopes using confocal microscopy. Here we show that kidney samples can be expanded sufficiently to study the finest elements of the filtration barrier under both healthy and diseased conditions using confocal microscopy. This finding opens up the possibility for any researcher with access to a confocal microscope to study foot process protein distributions on the effective nanometer-scale. We also show that expansion microscopy can be combined with STED microscopy to further increase the effective spatial resolution down to below 20 nm.

Keywords [en]
Expansion microscopy, kidney, slit diaphragm, Super-resolution microscopy
National Category
Biophysics
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-207579OAI: oai:DiVA.org:kth-207579DiVA, id: diva2:1097101
Funder
Swedish Foundation for Strategic Research , RFI14-0091Swedish Research Council, 2013-6041
Note

QC 20170523

Available from: 2017-05-22 Created: 2017-05-22 Last updated: 2017-05-23Bibliographically approved
In thesis
1. High-resolution imaging of kidney tissue samples
Open this publication in new window or tab >>High-resolution imaging of kidney tissue samples
2017 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

The kidney is one of the most important and complex organs in the human body, filtering hundreds of litres of blood daily. Kidney disease is one of the fastest growing causes of death in the modern world, and this motivates extensive research for better understanding the function of the kidney in health and disease. Some of the most important cellular structures for blood filtration in the kidney are of very small dimensions (on the sub-200 nm scale), and thus electron microscopy has been the only method of choice to visualize these minute structures. In one study, we show for the first time that by combining optical clearing with STED microscopy, protein localizations in the slit diaphragm of the kidney, a structure around 75 nanometers in width, can now be resolved using light microscopy. In a second study, a novel sample preparation method, expansion microscopy, is utilized to physically expand kidney tissue samples. Expansion improves the effective resolution by a factor of 5, making it possible to resolve podocyte foot processes and the slit diaphragm using confocal microscopy. We also show that by combining expansion microscopy and STED microscopy, the effective resolution can be improved further. In a third study, influences on the development of the kidney were studied. There is substantial knowledge regarding what genes (growth factors, receptors etc.) are important for the normal morphogenesis of the kidney. Less is known regarding the physiology behind how paracrine factors are secreted and delivered in the developing kidney. By depleting calcium transients in explanted rat kidneys, we show that calcium is important for the branching morphogenesis of the ureteric tree. Further, the study shows that the calcium-dependent initiator of exocytosis, synaptotagmin, is expressed in the metanephric mesenchyme of the developing kidney, indicating that it could have a role in the secretion of paracrine growth factors, such as GDNF, to drive the branching.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. p. 34
Series
TRITA-FYS, ISSN 0280-316X ; 2017:28
Keywords
Super Resolution Microscopy, Kidney, Imaging, Fluorescence, Kidney development, Calcium
National Category
Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-207577 (URN)978-91-7729-456-6 (ISBN)
Presentation
2017-06-15, Air-Fire, Science for Life Laboratories, Tomtebodavägen 23A, Solna, 09:30 (English)
Opponent
Supervisors
Funder
Swedish Foundation for Strategic Research , RIF14-0091Swedish Research Council, 2013-6041
Note

QC 20170523

Available from: 2017-05-23 Created: 2017-05-22 Last updated: 2017-05-23Bibliographically approved

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