Fluorescence Performance Standards for Confocal Microscopy Steffen Rüttinger 1 , Peter Kapusta 2 , Volker Völlkopf 2 , Felix Koberling 2 , Rainer Erdmann 2 and Rainer Macdonald 1 1 Physikalisch-Technische Bundesanstalt, Abbe Str. 2-12, 10587 Berlin 2 PicoQuant GmbH, Kekulé Str. 7, 12489 Berlin 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-Speck TM 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. Keywords: confocal microscopy, reference sample, performance standard, single molecule detection, FCS, FLIM PERFORMANCE STANDARD FOR OPTICAL RESOLUTION AND ALIGNMENT The Molecule Detection Function (MDF) determines the resolution of a confocal fluorescence microscope. The MDF quantifies the efficiency of detecting a fluorescence photon from a fluorescing molecule at a given position in the sample. If the emission dipole orientation is statistically independent of the absorption dipole orientation (rotational diffusion time much faster than the fluorescence lifetime), then the MDF is given by the direct product of the excitation probability distribution (EPD) and the Collection Efficiency Function (CEF). If the excitation intensity is much below saturation, the EPD is proportional to the Excitation Intensity Distribution (EID) and the MDF will be the product of the EID and the CEF. Usually the term confocal volume (V conf ) is used as a practical measure of the resolution of a confocal microscope. The borders of the confocal volume are defined by the 1/e 2 values of the MDF. The confocal volume is the volume in which the efficiency of detecting a fluorescence photon from a fluorophore exceeds 13.5 % of the maximum efficiency. Instead of the 1/e 2 values often (and also here) the FWHM of the confocal volume is used as a measure. Multiphoton Microscopy in the Biomedical Sciences X, edited by Ammasi Periasamy, Peter T. C. So, Karsten König, Proc. of SPIE Vol. 7569, 75692I · © 2010 SPIE · CCC code: 1605-7422/10/$18 · doi: 10.1117/12.840501 Proc. of SPIE Vol. 7569 75692I-1 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 11/19/2014 Terms of Use: http://spiedl.org/terms