pubs.acs.org/JPCL
Fast Fluorescence Photoswitching in a BODIPY-Oxazine Dyad with Excellent Fatigue Resistance Erhan Deniz,† Salvatore Sortino,*,‡ and Franc- isco M. Raymo*,† †
Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146-0431, and Dipartimento di Scienze Chimiche, Universit a di Catania, viale Andrea Doria 8, Catania I-95125, Italy
‡
ABSTRACT We synthesized a photoswitchable fluorescent probe incorporating a BODIPY fluorophore and an oxazine photochrome within the same molecular skeleton. The selective excitation of the BODIPY component at visible wavelengths is accompanied by the emission of light in the form of fluorescence. However, the illumination of the sample at ultraviolet wavelengths opens reversibly the oxazine ring and activates the intramolecular transfer of energy from the fluorophore to the photochrome with concomitant fluorescence quenching. As a result, the emission of this fluorophore-photochrome dyad can be modulated on a microsecond time scale for hundreds of switching cycles, relying on the interplay of two exciting beams. Our operating principles for fast fluorescence photoswitching with excellent fatigue resistance can lead to the development of valuable probes for the super-resolution imaging of biological samples. SECTION Kinetics, Spectroscopy
However, STED requires high irradiation intensities at λOFF to deplete the excited state of the fluorophores, while ISC results in the population of reactive triplet states and the sensitization of singlet oxygen. These complications can have deleterious consequences on biological samples. In principle, the limitations associated with STED and ISC can be overcome with the design of RESOLFT based on photoisomerizations.4 Indeed, operating principles to switch fluorescence relying on the isomerization of photochromic compounds have already been demonstrated.17-22 Nonetheless, the need for fast switching speeds and excellent fatigue resistances for RESOLFT imaging have restricted the use of photochromic systems only to few representative examples.23-26 In search of viable structural designs to switch fluorescence with fast and stable photochromic compounds, we envisaged the possibility of connecting a boron dipyrromethene (BODIPY) fluorophore to an oxazine photochrome. In particular, we synthesized the BODIPY-oxazine dyad 1 (Figure 1) in five steps, with an overall yield of 30%, starting from commercial and known precursors (Supporting Information). Specifically, we prepared a 3H-indole with a 4-dimethylaminostyryl appendage in three steps first and then connected this compound covalently to a preformed BODIPY. Finally, we condensed the resulting molecule with 2-chloromethyl-4-nitrophenol to assemble the benzooxazine fragment of the target dyad 1. The steady-state absorption spectrum (a in Figure 2) of 1, recorded in acetonitrile at 20 °C, shows a band centered at 524 nm, which can be assigned to the fluorescent
luorescence microscopy1 offers the opportunity to image noninvasively biological samples in real time. As a result, this convenient technique, in combination with appropriate labeling protocols,2 has become an indispensable analytical tool in the biomedical laboratory for the investigation of cells and tissues. However, the phenomenon of diffraction3 limits the resolution of conventional fluorescence microscopes to submicrometer dimensions in both the horizontal and vertical directions. These stringent limitations prevent the visualization of biological samples with nanoscaled resolution and, hence, the elucidation of the factors regulating cellular functions at the molecular level. Impressive strategies to record images of biological samples with subdiffraction resolution have recently been designed around the properties of photoswitchable fluorescent probes.4-16 These imaging protocols are based either on the stochastic localization of individual fluorophores or the patterned illumination of the fluorescent sample. The latter approach requires probes able to undergo reversible saturable optically linear fluorescence transitions (RESOLFT). Specifically, illumination at one wavelength (λON) should excite the fluorophores to encourage their luminescence, and irradiation at another (λOFF) should switch their emission off. Under these conditions, a pair of lasers generating overlapping circular and doughnut-shaped spots on the focal plane of the sample at λON and λOFF, respectively, can be exploited to confine fluorescence in the nanoscaled hole of the doughnut. The synchronous scanning of the two beams across the sample can then offer the opportunity to acquire images with subdiffraction resolution. RESOLFT can be implemented on the basis of stimulated emission depletion (STED) or intersystem crossing (ISC).4
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Received Date: April 21, 2010 Accepted Date: May 10, 2010 Published on Web Date: May 12, 2010
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Figure 1. Photoinduced and reversible interconversion of 1 and 2.
Figure 3. Absorbance evolution at 450 (a) and 560 nm (b) after the excitation of 1 (0.01 mM, MeCN, 20 °C) at 355 nm (λOFF).
Figure 2. Steady-state absorption (a) and emission (b) spectra of 1 (0.05 mM, MeCN, 20 °C, 437 nm). Transient absorption spectrum (c) recorded 0.1 μs after the excitation of 1 (0.01 mM, MeCN, 20 °C) at 355 nm (λOFF).
with the photoinduced transformation of 1 into 2 and returns to the original value in few microseconds with the reisomerization of 2 back to 1. In fact, the fluorescence (a in Figure 4) of this particular system can be modulated by turning on and off an exciting beam at λOFF while illuminating the sample at λON, relying on the reversible photoactivation of an intramolecular quenching pathway. In agreement with these operating principles, the emission intensity (b in Figure 4) of the model fluorophore 3, lacking the photoswitchable component, remains essentially unaffected upon illumination at λOFF, under otherwise identical conditions. Our results demonstrate that a BODIPY fluorophore and an oxazine photochrome can be integrated within the same molecular skeleton to construct a photoswitchable fluorescent dyad. Indeed, the photoisomerization of the oxazine component activates an intramolecular energy-transfer pathway, which culminates in the effective quenching of the BODIPY fluorescence. As a result, the emission intensity of this particular dyad can be modulated with microsecond switching times for hundreds of switching cycles with no sign of degradation. Thus, our mechanism and choice of functional components for fluorescence photoswitching can evolve into the generation of valuable RESOLFT probes for the super-resolution imaging of biological samples. First, however, it is necessary to identify strategies to operate these fluorophore-photochrome dyads in aqueous environments and, possibly, improve their fluorescence quantum yield and contrast ratio.
component.27 Upon excitation within this band, the characteristic BODIPY fluorescence appears at 541 nm in the steady-state emission spectrum (b in Figure 2) with a quantum yield of 0.12. Instead, the illumination of the sample at 355 nm (λOFF) opens the [1,3]oxazine ring of the photochromic component after the cleavage of a [C-O] bond, in agreement with the behavior of similar oxazines.28-35 This photoinduced process occurs within the laser pulse (6 ns) and generates the zwitterion 2 (Figure 1) with a quantum yield of 0.05. Consistently, the transient absorption spectrum (c in Figure 2), recorded 0.1 μs after excitation at λOFF, shows the appearance of bands centered at 450 and 560 nm, corresponding to ground-state absorptions28 of the 4-nitrophenolate and 3H-indolium fragments of 2, respectively. Both bands decay monoexponentially on a microsecond time scale with the spontaneous reisomerization of 2 back to 1. Curve fitting of the temporal absorbance profiles (a and b in Figure 3) indicates the lifetime of 2 to be ∼1 μs. Thus, a full switching cycle, from 1 to 2 and back, is completed within few microseconds from illumination at λOFF. Furthermore, this system tolerates hundreds of switching cycles with no sign of degradation, even in the presence of molecular oxygen. Indeed, the steady-state and transient absorption spectra of 1 remain unaltered even after 400 cycles. The 3H-indolium cation of 2 absorbs (c in Figure 2) in the same range of wavelengths where the BODIPY fluorophore emits (b in Figure 2). As a result, the selective excitation of the fluorescent component at 532 nm (λON) within the photogenerated isomer 2 is followed by the transfer of energy to the photochromic component with concomitant fluorescence quenching. Indeed, the emission intensity at 580 nm decreases
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SUPPORTING INFORMATION AVAILABLE Experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 4. Emission intensity at 580 nm of solutions (0.01 mM, MeCN, 20 °C) of 1 (a) and 3 (b) recorded by turning on and off an excitation source at 355 nm (λOFF) while illuminating the sample at 532 nm (λON).
AUTHOR INFORMATION
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Corresponding Author: *To whom correspondence should be addressed. E-mail: ssortino@ unict.it (S.S.);
[email protected] (F.M.R.).
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ACKNOWLEDGMENT We thank the NSF (CAREER Award CHE0237578 and CHE-0749840) and MIUR (PRIN 2008) for financial support.
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REFERENCES (1) (2)
(3) (4) (5) (6) (7)
(8)
(9) (10)
(11) (12)
(13)
(14)
Pawley, J. B., Ed. Handbook of Biological Confocal Microscopy; Springer: New York, 2006. Haugland, R. P. The Handbook: A Guide to Fluorescent Probes and Labeling Technologies; Molecular Probes: Eugene, OR, 2005. Born, M.; Wolf, E. Principles of Optics; Cambridge University Press: Cambridge, U.K., 2002. Hell, S. W. Toward Fluorescence Nanoscopy. Nat. Biotechnol. 2003, 21, 1347–1355. Hell, S. W. Far-Field Optical Nanoscopy. Science 2007, 316, 1153–1158. Hell, S. W. Microscopy and Its Focal Switch. Nat. Methods 2009, 6, 24–32. Hell, S. W.; Schmidt, R.; Egner, A. Diffraction-Unlimited Three-Dimensional Optical Nanoscopy with Opposing Lenses. Nat. Photonics 2009, 3, 381–387. Bates, M.; Huang, B.; Zhuang, X. Super-Resolution Microscopy by Nanoscaled Localization of Photo-Switchable Fluorescent Probes. Curr. Opin. Chem. Biol. 2008, 12, 505–514. Huang, B.; Bates, M.; Zhuang, X. Super-Resolution Fluorescence Microscopy. Annu. Rev. Biochem. 2009, 78, 993–1016. Ji, N.; Shroff, H.; Zhong, H.; Betzig, E. Advances in the Speed and Resolution of Light Microscopy. Curr. Opin. Neurobiol. 2008, 18, 605–616. Gustafsson, M. G. L. Super-Resolution Light Microscopy Goes Live. Nat. Methods 2008, 5, 385–387. Fernandez-Su arez, M.; Ting, A. Y. Fluorescent Probes for Super-Resolution Imaging in Living Cells. Nat. Rev. Mol. Cell. Biol. 2008, 9, 929–943. Heilemann, M.; Dedecker, P.; Hofkens, J.; Sauer, M. Photoswitches: Key Molecules for Subdiffraction-Resolution Fluorescence. Laser Photon Rev. 2009, 3, 180–202. Lippincott-Schwartz, J.; Manley, S. Putting Super-Resolution Fluorescence Microscopy to Work. Nat. Methods 2009, 6, 21–23.
r 2010 American Chemical Society
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
1692
Lippincott-Schwartz, J.; Patterson, G. H. Photoactivatable Fluorescent Proteins for Diffraction-Limited and Super-Resolution Imaging. Trends Cell Biol. 2009, 19, 555–565. Hess, S. T. Red Lights, Camera, Photoactivation!. Nat. Methods 2009, 6, 124–125. Kuz'min, M. G.; Koz'menko, M. V. Luminescence of Photochromic Compounds. In Organic Photochromes; El'tsov, A. V., Ed.; Consultants Bureau: New York, 1990; pp 245-265. Irie, M., Ed. Photochromism Memories and Switches. Chem. Rev. 2000, 100, 1683-1890. Raymo, F. M.; Tomasulo, M. Electron and Energy Transfer Modulation with Photochromic Switches. Chem. Soc. Rev. 2005, 34, 327–336. Raymo, F. M.; Tomasulo, M. Fluorescence Modulation with Photochromic Switches. J. Phys. Chem. A 2005, 109, 7343– 7352. Yildiz, I.; Deniz, E.; Raymo, F. M. Fluorescence Modulation with Photochromic Switches in Nanostructured Constructs. Chem. Soc. Rev. 2009, 38, 1859–1867. Cusido, J.; Deniz, E.; Raymo, F. M. Fluorescent Switches Based on Photochromic Compounds. Eur. J. Org. Chem. 2009, 2031–2045. Hofmann, M.; Eggeling, C.; Jakobs, S.; Hell, S. W. Breaking the Diffraction Barrier in Fluorescence Microscopy at Low Light Intensities by Using Reversibly Photoswitchable Proteins. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17565– 17569. Bossi, M.; F€ olling, J.; Dyba, M.; Westphal, V.; Hell, S. W. STED Microscopy Resolves Nanoparticle Assemblies. New J. Phys. 2006, 8, 275–284. Schwentker, M. A.; Bock, H.; Hofmann, M.; Jakobs, S.; Bewersdorf, J.; Eggeling, C.; Hell, S. W. Wide-Field Subdiffraction RESOLFT Microscopy Using Fluorescent Protein Photoswitching. Microsc. Res. Tech. 2007, 70, 269–280. F€ olling, J.; Polyakova, S.; Belov, V.; van Blaaderen, A.; Bossi, M. L.; Hell, S. W. Synthesis and Characterization of Photoswitchable Fluorescent Silica Nanoparticles. Small 2008, 4, 134–142. Tomasulo, M.; Deniz, E.; Alvarado, R. J.; Raymo, F. M. Photoswitchable Fluorescent Assemblies Based on Hydrophilic BODIPY-Spiropyran Conjugates. J. Phys. Chem. C 2008, 112, 8038–8045. Tomasulo, M.; Sortino, S.; White, A. J. P.; Raymo, F. M. Fast and Stable Photochromic Oxazines. J. Org. Chem. 2005, 70, 8180–8189. Tomasulo, M.; Sortino, S.; Raymo, F. M. A Fast and Stable Photochromic Switch Based on the Opening and Closing of an Oxazine Ring. Org. Lett. 2005, 7, 1109–1112.
DOI: 10.1021/jz100510u |J. Phys. Chem. Lett. 2010, 1, 1690–1693
pubs.acs.org/JPCL
(30)
(31)
(32)
(33)
(34)
(35)
Tomasulo, M.; Sortino, S.; Raymo, F. M. Amplification of the Coloration Efficiency of Photochromic Oxazines. Adv. Mater. 2008, 20, 832–835. Tomasulo, M.; Sortino, S.; Raymo, F. M. Bichromophoric Photochromes Based on the Opening and Closing of a Single Oxazine Ring. J. Org. Chem. 2008, 73, 118–126. Deniz, E.; Tomasulo, M.; Sortino, S.; Raymo, F. M. Substituent Effects on the Photochromism of Bichromophoric Oxazines. J. Phys. Chem. C 2009, 113, 8491–8497. Åxman Petersen, M.; Deniz, E.; Brøndsted Nielsen, M.; Sortino, S.; Raymo, F. M. Photochromic Oxazines with Extended Conjugation. Eur. J. Org. Chem. 2009, 4333–4339. Tomasulo, M.; Deniz, E.; Benelli, T.; Sortino, S.; Raymo, F. M. Photochromic Polymers Based on the Photoinduced Opening and Thermal Closing of [1,3]Oxazine Rings. Adv. Funct. Mater. 2009, 19, 3956–3961. Tomasulo, M.; Deniz, E.; Sortino, S.; Raymo, F. M. Hydrophilic and Photochromic Switches Based on the Opening and Closing of [1,3]Oxazine Rings. Photochem. Photobiol. Sci. 2010, 9, 136–140.
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