Anal. Chem. 2007, 79, 3330-3341
Monitoring Kinetics of Highly Environment Sensitive States of Fluorescent Molecules by Modulated Excitation and Time-Averaged Fluorescence Intensity Recording Tor Sande´n, Gustav Persson, Per Thyberg, Hans Blom, and Jerker Widengren*
Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of Technology, Albanova University Center, 106 91 Stockholm, Sweden
In this work, a concept is described for how the kinetics of photoinduced, transient, long-lived, nonfluorescent or weakly fluorescent states of fluorophore marker molecules can be extracted from the time-averaged fluorescence by using time-modulated excitation. The concept exploits the characteristic variation of the population of these states with the modulation parameters of the excitation and thereby circumvents the need for time resolution in the fluorescence detection. It combines the single-molecule sensitivity of fluorescence detection with the remarkable environmental responsiveness obtainable from long-lived transient states, yet does not in itself impose any constraints on the concentration or the fluorescence brightness of the sample molecules that can be measured. Modulation of the excitation can be performed by variation of the intensity of a stationary excitation beam in time or by repeated translations of a CW excitation beam with respect to the sample. As a first experimental verification of the approach, we have shown how the triplet-state parameters of the fluorophore rhodamine 6G in different aqueous enviroments can be extracted. We demonstrate that the concept is fully compatible with low time-resolution detection by a CCD camera. The concept opens for automated transient-state monitoring or imaging on a massively parallel scale and for high-throughput biomolecular screening as well as for more fundamental biomolecular studies. The concept should also be applicable to the monitoring of a range of other photoinduced nonfluorescent or weakly fluorescent transient states, from which subtle changes in the immediate microenvironment of the fluorophore marker molecules can be detected. In the last years, much effort has been spent on developing and applying fluorescence-based methods, for monitoring biomolecules and their interactions, and for high-throughput screening (HTS) purposes, as well as for more thorough fundamental studies. Fluorescence-based readouts typically offer high specificity and sensitivity and lend themselves well for miniaturization and automation as well as for so-called homogeneous assays, * Corresponding author. biomolphysics.kth.se.
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3330 Analytical Chemistry, Vol. 79, No. 9, May 1, 2007
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requiring no separation or purification prior to the readout (mix and measure).1,2 The sensitivity also makes fluorescence spectroscopy one of the major means by which to perform singlemolecule detection (SMD) and analysis for fundamental biomolecular studies (see refs 3-5 for reviews). In fluorescence applications, where molecules are to be monitored at low concentrations or within short interrogation times, multiplexing by registration of several independent parameters is an important means to increase the information content per amount of sample and time. For both HTS1 and SMD-based fundamental biomolecular studies,6,7 multiplexing has proven to be very useful to minimize reagent consumption and increase throughput, and to increase accuracy and precision, respectively. Multiplexing has also been successfully applied to a range of other fluorescence-based techniques, including readout of DNA and protein microaarrays,8 to flow cytometry as an analysis platform for high-throughput, high-content biological testing and drug discovery,9 as well as within both wide-field and confocal fluorescence microscopy.10 In most applications, multiplexing is based on the recording of two or more of the traditional fluorescence parameters: intensity, emission wavelength, lifetime, and polarization. However, given the broad application and clear rationale for multiparameter fluorescence recording for biomolecular interaction monitoring, it is interesting to note that additional dimensions of fluorescence information seem not to have been exploited to their full potential. In particular, it seems as if the information contained in the population dynamics of photoinduced, long-lived, nonfluorescent or weakly fluorescent transient states, e.g., states generated by (1) de Jong, L. A.; Uges, D. R.; Franke, J. P.; Bischoff, R. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2005, 829, 1-25. (2) Eggeling, C.; Brand, L.; Ullmann, D.; Jager, S. Drug Discovery Today 2003, 8, 632-641. (3) Weiss, S. Nat. Struct. Biol. 2000, 7, 724-729. (4) Haustein, E.; Schwille, P. Curr. Opin. Struct. Biol. 2004, 14, 531-540. (5) Moerner, W. E.; Fromm, D. P. Rev. Sci. Instrum. 2003, 74, 3597-3619. (6) Eggeling, C.; Berger, S.; Brand, L.; Fries, J. R.; Schaffer, J.; Volkmer, A.; Seidel, C. A. M. J. Biotechnol. 2001, 86, 163-180. (7) Widengren, J.; Kudryavtsev, V.; Antonik, M.; Berger, S.; Gerken, M.; Seidel, C. A. M. Anal. Chem. 2006, 78, 2039-2050. (8) Scha¨ferling, M.; Nagl, S. Anal. Bioanal. Chem. 2006, 385, 500-517. (9) Edwards, B. S.; Oprea, T.; Prossnitz, E. R.; Sklar, L. A. Curr. Opin. Chem. Biol. 2004, 8, 392-398. (10) Suhling, K.; French, P. M. W.; Phillips, D. Photochem. Photobiol. Sci. 2005, 4, 13-22. 10.1021/ac0622680 CCC: $37.00
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trans-cis isomerization, intersystem crossing, or photoinduced charge transfer within fluorescent marker molecules, can be exploited further in the framework of HTS and for fundamental biomolecular studies. An attractive feature of these states is their long lifetimes, rendering them highly sensitive to the immediate environment of their fluorescent host molecules. While the fluorescence lifetime of a singlet excited state of a fluorophore is ∼10-9 s, the lifetimes of these transient states are ∼10-6-10-3 s. Consequently, these states have a factor of ∼103-106 more time to interact with the immediate environment of the fluorophore. Their kinetics can thus change considerably, also due to small changes in, for example, accessibility of quencher molecules or microviscosities, caused by a biomolecular interaction. In the past few years, photoinduced transient states of fluorophores and fluorescent proteins have attracted a large interest primarily due to applications of photoswitching, for protein transport and localization studies in cells,11 and as a means to increase resolution in fluorescence-based light microscopy.12-14 In this work, we focus on the environment responsiveness of these states, combined with their photoswitchability, and present a concept for how photoinduced, long-lived, nonfluorescent or weakly fluorescent transient states can be monitored without the need of a time-resolved detection. This opens for parallel monitoring of these states, to an extent not possible with present techniques. The concept is verified experimentally, by monitoring the triplet-state kinetics of the fluorophore rhodamine 6G (Rh6G) in different aqueous solutions. Triplet-state imaging or spectroscopy for biomolecular studies has in the past mainly been performed by transient absorption spectroscopy, via phosphorescence recording, or more recently by fluorescence correlation spectroscopy (FCS). Transient absorption spectroscopy is a well-established technique,15,16 where various states are monitored via their absorption by a separate probing beam, following an excitation pulse. However, the absorption spectra of these transient states can often overlap with those of other photoinduced states, making it difficult to separate them from each other. Moreover, this technique is relatively technically complicated, lacks the sensitivity for measurements at low (
