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Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue.
KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-5584-9170
KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. Karolinska Institutet, Sweden.ORCID iD: 0000-0003-0578-4003
2016 (English)In: Kidney International, ISSN 0085-2538, E-ISSN 1523-1755, Vol. 89, no 1, 243-247 p.Article in journal (Refereed) Published
Abstract [en]

The glomerular filtration barrier, consisting of podocyte foot processes with bridging slit diaphragm, glomerular basement membrane, and endothelium, is a key component for renal function. Previously, the subtlest elements of the filtration barrier have only been visualized using electron microscopy. However, electron microscopy is mostly restricted to ultrathin two-dimensional samples, and the possibility to simultaneously visualize multiple different proteins is limited. Therefore, we sought to implement a super-resolution immunofluorescence microscopy protocol for the study of the filtration barrier in the kidney. Recently, several optical clearing methods have been developed making it possible to image through large volumes of tissue and even whole organs using light microscopy. Here we found that hydrogel-based optical clearing is a beneficial tool to study intact renal tissue at the nanometer scale. When imaging samples using super-resolution STED microscopy, the staining quality was critical in order to assess correct nanoscale information. The signal-to-noise ratio and immunosignal homogeneity were both improved in optically cleared tissue. Thus, STED of slit diaphragms in fluorescently labelled optically cleared intact kidney samples is a new tool for studying the glomerular filtration barrier in health and disease.

Place, publisher, year, edition, pages
Nature Publishing Group, 2016. Vol. 89, no 1, 243-247 p.
Keyword [en]
glomerulus, podocyte, optical clearing, STED, super-resolution
National Category
Other Physics Topics Biological Sciences Cell Biology Biophysics
Identifiers
URN: urn:nbn:se:kth:diva-182108DOI: 10.1038/ki.2015.308ISI: 000368321300034PubMedID: 26444032Scopus ID: 2-s2.0-84943311622OAI: oai:DiVA.org:kth-182108DiVA: diva2:903290
Funder
Swedish Research Council, 2013-6041Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20160218

Available from: 2016-02-15 Created: 2016-02-15 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. 34 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2017:28
Keyword
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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