Anal. Chem. 2008, 80, 9589–9596
Transient State Imaging for Microenvironmental Monitoring by Laser Scanning Microscopy Tor Sande´n, Gustav Persson, and Jerker Widengren* Department of Applied Physics, Experimental Biomolecular Physics, Royal Institute of Technology, SE-106 91 Stockholm, Sweden. Photoinduced transient dark states are exhibited by practically all common fluorophores. These relatively longlived states are very sensitive to the local environment and thus highly attractive for microenvironmental imaging purposes. However, because of methodological constraints, their sensitivity has to date been very sparsely exploited. Here, a concept based on spatio-temporal modulation of the excitation intensity is presented that can image these states via their photodynamic fingerprints. With the use of a standard laser scanning microscope, it unites the outstanding environmental sensitivity of the transient state parameters with the high sensitivity of the fluorescence readout and is easily implemented. For demonstration, triplet state images of liposomes with different internal environments were generated. These images provide an example of how local environmental differences can be resolved, which are not clearly distinguishable via other fluorescence parameters. Having minor instrumental and sample constraints the concept can be foreseen to provide several new, useful, and independent fluorescence-based parameters in biomolecular imaging. Multiplexing by simultaneous registration of several independent parameters is an important means to increase information content in fluorescence-based biomolecular imaging, in wide-field and confocal fluorescence microscopy,1 readouts of DNA and protein microarrays,2 as well as in imaging of single molecules.3 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, the clear rationale for multiplexing in biomolecular imaging also makes it interesting to consider additional independent readout parameters. Parameters related to the population dynamics of photoinduced, long-lived, non- or weakly fluorescent transient states of fluorophores, generated by trans-cis isomerization, intersystem crossing, or photoinduced charge transfer are attractive in this context. While the fluorescence lifetime of a singlet excited-state of a fluorophore is ∼10-9 s, the lifetimes of these transient states * To whom correspondence should be addressed. E-mail: jerker@ biomolphysics.kth.se. (1) Suhling, K.; French, P. M. W.; Phillips, D. Photochem. Photobiol. Sci. 2005, 4, 13–22. (2) Scha¨ferling, M.; Nagl, S. Anal. Bioanal. Chem. 2006, 385, 500–517. (3) Kudryavtsev, V.; Felekyan, S.; Wozniak, A.; Ko ¨nig, M.; Sandhagen, C.; Ku ¨ hnemuth, R.; Seidel, C.; Oesterhelt, F. Anal. Bioanal. Chem. 2007, 387, 71–82. 10.1021/ac8018735 CCC: $40.75 2008 American Chemical Society Published on Web 11/14/2008
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, rendering them highly sensitive to the local environment. Their kinetics can thus change considerably due to small changes in accessibility of quencher molecules or microviscosities, reflecting, e.g., a biomolecular interaction. However, although attractive for biomolecular imaging, this information has only been exploited to a very limited extent, mainly due to methodological constraints. A range of techniques indeed exist to characterize these transient states and their population kinetics. Transient absorption spectroscopy is a well established technique that has been extensively used to characterize fluorophore photodynamics.4-6 However, the technique is relatively technically complicated, lacks the sensitivity for measurements at low (< micromolar) concentrations and is mainly restricted to cuvette experiments. Emission originating either directly (phosphorescence) or indirectly (delayed fluorescence) from the long-lived first excited triplet state can also be used for monitoring7,8 and has been exploited for microscopic imaging.9 However, coupled to the long-lived emission is also the susceptibility of the triplet state to dynamic quenching by oxygen and trace impurities, which can be circumvented only after elaborate and careful sample preparation. This artifactual quenching not only shortens the triplet lifetime but practically makes the luminescence undetectable. Biomolecular monitoring by this readout is thus largely restricted to deoxygenized, carefully prepared samples, which restricts its applicability to biological specimen. Alternatively, the population and kinetics of transient states of fluorophores can be followed by fluorescence correlation spectroscopy (FCS), via the fluorescence fluctuations generated as individual fluorophores transit to and from the (nonfluorescent) transient state.10-13 In FCS, the highly sensitive fluorescence readout is used to monitor the triplet state, rather than the faint, easily quenched phosphorescence signal from the triplet state itself. For isomerized or photo-oxidized states, there is normally (4) van Amerongen, H.; van Grondelle, R. Methods Enzymol. 1995, 246, 201– 226. (5) Chen, E.; Chance, M. R. Methods Enzymol. 1993, 226, 119–147. (6) Korobov, V. E.; Chibisov, A. K. Russ. Chem. Rev. 1983, 52, 27–42. (7) Jovin, T. M.; Vaz, W. L. Methods Enzymol. 1989, 172, 471–513. (8) Cioni, P.; Strambini, G. B. Biochim. Biophys. Acta 2002, 1595, 116–130. (9) Marriott, G.; Clegg, R. M.; Arndt-Jovin, D. J.; Jovin, T. M Biophys. J. 1991, 60, 1374–1387. ¨ . J. Fluoresc. 1994, 4, 255–258. (10) Widengren, J.; Rigler, R.; Mets, U ¨ .; Rigler, R. J. Phys. Chem. 1995, 99, 13368–13379. (11) Widengren, J.; Mets, U (12) Widengren, J.; Dapprich, J.; Rigler, R. Chem. Phys. 1997, 216, 417–426. (13) Widengren, J.; Schwille, P. J. Phys. Chem. A 2000, 104, 6416–6428.
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no alternative emission available. In this sense, FCS makes use of a uniquely favorable combination of a high signal level (given by the readout of fluorescence photons) and an outstanding environmental sensitivity (given by the long lifetimes of the transient states). One limitation however is that only a limited number of spots can be measured simultaneously. To partly overcome this, a compromise between the temporal analysis of FCS and fluorescence fluctuation analysis in the spatial domain14 can be obtained by exploiting the time structure of sample/laser scanning confocal microscope images.15,16 Thereby, spatial correlation analysis of the emitted fluorescence is combined with temporal characterization of the fluorescence emission from the serial data stream of subsequently scanned pixels. This means, however, that the temporal characterization refers to the average of a sample distributed over a relatively large part of the image recorded, rather than to individual pixels. Moreover, fluctuation approaches in general rely on spontaneous fluorescence fluctuations. Thus only very few molecules (
