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Pulsed interleaved excitation-based line-scanning spatial correlation spectroscopy (PIE-lsSCS)

Gao, X. 1; Gao, P. 1,2; Prunsche, B. 1; Nienhaus, K. 1; Nienhaus, G. U. ORCID iD icon 1,2,3
1 Institut für Angewandte Physik (APH), Karlsruher Institut für Technologie (KIT)
2 Institut für Nanotechnologie (INT), Karlsruher Institut für Technologie (KIT)
3 Institut für Toxikologie und Genetik (ITG), Karlsruher Institut für Technologie (KIT)


We report pulsed interleaved excitation (PIE) based line-scanning spatial correlation spectroscopy (PIElsSCS), a quantitative fluorescence microscopy method for the study of dynamics in free-standing lipid bilayer membranes. Using a confocal microscope, we scan multiple lines perpendicularly through the membrane, each one laterally displaced from the previous one by several ten nanometers. Scanning through the membrane enables us to eliminate intensity fluctuations due to membrane displacements with respect to the observation volume. The diffusion of fluorescent molecules within the membrane is quantified by spatial correlation analysis, based on the fixed lag times between successive line scans. PIE affords dual-color excitation within a single line scan and avoids channel crosstalk. PIE-lsSCS data are acquired from a larger membrane region so that sampling is more efficient. Moreover, the local photon flux is reduced compared with single-point experiments, resulting in a smaller fraction of photobleached molecules for identical exposure times. This is helpful for precise measurements on live cells and tissues. We have evaluated the method with experiments on fluorescently labeled giant unilamellar vesicles (GUVs) and membrane-stained live cells.

Verlagsausgabe §
DOI: 10.5445/IR/1000087861
DOI: 10.1038/s41598-018-35146-4
Zitationen: 5
Zitationen: 7
Cover der Publikation
Zugehörige Institution(en) am KIT Institut für Angewandte Physik (APH)
Institut für Nanotechnologie (INT)
Institut für Toxikologie und Genetik (ITG)
Universität Karlsruhe (TH) – Interfakultative Einrichtungen (Interfakultative Einrichtungen)
Karlsruhe School of Optics & Photonics (KSOP)
Publikationstyp Zeitschriftenaufsatz
Publikationsjahr 2018
Sprache Englisch
Identifikator ISSN: 2045-2322
KITopen-ID: 1000087861
HGF-Programm 43.23.01 (POF III, LK 01) Advanced Optical Lithography+Microscopy
Erschienen in Scientific reports
Verlag Nature Research
Band 8
Heft 1
Seiten Art. Nr.: 16722
Bemerkung zur Veröffentlichung Gefördert durch den KIT-Publikationsfonds
Nachgewiesen in Dimensions
Web of Science
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