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Proceedings Paper

Fluorescence performance standards for confocal microscopy
Author(s): Steffen Rüttinger; Peter Kapusta; Volker Völlkopf; Felix Koberling; Rainer Erdmann; Rainer Macdonald
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Paper Abstract

State of the art confocal microscopes offer diffraction limited (or even better) spatial resolution, highest (single molecule) sensitivity and ps-fluorescence lifetime measurement accuracy. For developers, manufacturers, as well as users of confocal microscopes it is mandatory to assign values to these qualities. In particular for users, it is often not easy to ascertain that the instrument is properly aligned as a large number of factors influence resolution or sensitivity. Therefore, we aspire to design a set of performance standards to be deployed on a day-to-day fashion in order to check the instruments characteristics. The main quantities such performance standard must address are: • Spatial resolution • Sensitivity • Fluorescence lifetime To facilitate the deployment and thus promote wide range adoption in day-to-day performance testing the corresponding standards have to be ready made, easy to handle and to store. The measurement procedures necessary should be available on as many different setups as possible and the procedures involved in their deployment should be as easy as possible. To this end, we developed two performance standards to accomplish the mentioned goals: • Resolution reference • Combined molecular brightness and fluorescence lifetime reference The first one is based on sub-resolution sized Tetra-SpeckTM fluorescent beads or alternatively on single molecules on a glass surface to image and to determine quantitatively the confocal volume, while the latter is a liquid sample containing fluorescent dyes of different concentrations and spectral properties. Both samples are sealed in order to ease their use and prolong their storage life. Currently long-term tests are performed to ascertain durability and road capabilities.

Paper Details

Date Published: 26 February 2010
PDF: 10 pages
Proc. SPIE 7569, Multiphoton Microscopy in the Biomedical Sciences X, 75692I (26 February 2010); doi: 10.1117/12.840501
Show Author Affiliations
Steffen Rüttinger, Physikalisch-Technische Bundesanstalt (Germany)
Peter Kapusta, PicoQuant GmbH (Germany)
Volker Völlkopf, PicoQuant GmbH (Germany)
Felix Koberling, PicoQuant GmbH (Germany)
Rainer Erdmann, PicoQuant GmbH (Germany)
Rainer Macdonald, Physikalisch-Technische Bundesanstalt (Germany)

Published in SPIE Proceedings Vol. 7569:
Multiphoton Microscopy in the Biomedical Sciences X
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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