PAPER www.rsc.org/pps | Photochemical & Photobiological Sciences Characterization of new fluorescent labels for ultra-high resolution microscopy Andriy Chmyrov, a Jutta Arden-Jacob, b Alexander Zilles, c Karl-Heinz Drexhage c and Jerker Widengren a Received 17th June 2008, Accepted 11th September 2008 First published as an Advance Article on the web 30th September 2008 DOI: 10.1039/b810991p Photo-induced switching of dyes into dark, long-lived states, such as a triplet state, has recently gained increasing interest, as a means to achieve ultra-high optical resolution. Additionally, these long lived states are often highly environment-sensitive and their photodynamics can thus offer additional independent fluorescence-based information. However, although providing a useful mechanism for photo-induced switching, the triplet state often appears as a precursor state for photobleaching, which potentially can limit its usefulness. In this work, a set of rhodamine and pyronin dyes, modified by substitution of heavy atoms and nitrogen within or close to the central xanthene unit of the dyes, were investigated with respect to their triplet state dynamics and photostabilities, under conditions relevant for ultra-high resolution microscopy. Out of the dyes investigated, in particular the rhodamine and pyronin dyes with a sulfur atom replacing the central oxygen atom in the xanthene unit were found to meet the requirements for ultra-high resolution microscopy, combining a prominent triplet state yield with reasonable photostability. Introduction Photophysical and photochemical properties of fluorescent dyes as well as their photostability are of great importance for all applications using fluorescence for the read-out of information, in particular when a high read-out rate or high sensitivity is required. The presence of various long-lived transient states of fluorophores generally has been considered disadvantageous, as they reduce the fluorescence quantum yield and render the data analysis more complicated. Triplet states of fluorophores belong to this category of states. They are intrinsically present to various extents after excitation of all fluorophores. In fluorescence spectroscopy, and more particular in the context of ultra-sensitive fluorescence spectroscopy triplet states have been found to be precursor states, from which photobleaching can occur. 1,2 However, recent work has also demonstrated strategies to particularly exploit the information contained in the population dynamics of triplet states, 3 and transitions to and from triplet and other photo- induced states have been used as a means to achieve ultra- high resolution microscopy. 4–6 Fundamental ideas utilizing photo- induced switching phenomena to circumvent the diffraction limit of resolution in fluorescence-based light microscopy have been formulated already in the 1990s. 7 However, the experimental break-through has occurred only in the last few years. 5,6,8–10 Based on the overall strategy to minimize the volume from which fluo- rescence is detected, stimulated emission, within the STimulated Emission by Depletion (STED) concept, 7 has proven to be a useful means to generate photoswitching for the purpose of ultra-high resolution microscopy. However, the use of long-lived transient state generation, referred to as Ground State Depletion (GSD), 4 offers several advantages compared to STED. In particular, for a Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of Technology, SE-10691, Stockholm, Sweden b ATTO-TEC GmbH, Am Eichenhang 50, D-57076, Siegen, Germany c Department of Chemistry, University of Siegen, D-57068, Siegen, Germany the switching mechanism lower irradiances are required. This may be important e.g. for investigations of live cells. For optimal performance of the GSD strategy, the fluorophores used should be readily photo-switchable into dark states. High triplet state populations of most dyes can be easily achieved by use of certain additives that enhance photo-induced triplet state generation, or by removal of dissolved oxygen, whereby the re- generation rate of singlet state dye molecules from their triplet state counterparts is strongly reduced. However, addition of these chemical compounds or de-oxygenation is not always compatible with biological applications. Consequently, high yield of triplet state formation, sufficient for GSD, and high photostability are typically contradictory requirements. The photophysical properties of dyes and other fluorescence emitters are major limiting factors for all types of fluorescence- based sub-diffraction limit imaging techniques based on photo- switching, also including imaging techniques based on stochastic photoactivation and localization of single molecules. 9,10 In this context, much effort has been spent on both development 11,12 and characterization 13 of various fluorescent proteins. In this work, we present investigations of a set of dyes, specifically developed to combine an intrinsically high triplet quantum yield with a relatively high photostability. The dyes were characterized in detail by Fluorescence Correlation Spectroscopy (FCS), 14–17 combined with spectrofluorimetry and fluorescence lifetime measurements by Time-Correlated Single Photon Counting (TCSPC). 18,19 The outcome of the characterization leads us to conclude that this family of dyes can meet the requirements for GSD, as well as other fluorescence microscopy applications where the same combination of triplet properties and photostability is required. Experimental Excitation-emission spectra were recorded by a spectrofluorimeter with a time correlated single photon counting (TCSPC) option (FluoroMax3, Horiba Jobin Yvon, Longjumeau Cedex, France). 1378 | Photochem. Photobiol. Sci., 2008, 7, 1378–1385 This journal is © The Royal Society of Chemistry and Owner Societies 2008 Downloaded by Max Planck Institut fuer on 01 April 2011 Published on 30 September 2008 on http://pubs.rsc.org | doi:10.1039/B810991P View Online