Photoactivatable Synthetic Dyes for Fluorescence ... - ACS Publications

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Photoactivatable Synthetic Dyes for Fluorescence Imaging at the Nanoscale Françisco M. Raymo* Laboratory for Molecular Photonics, Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146-0431, United States ABSTRACT: The transition from conventional to photoactivatable fluorophores can bring the resolution of fluorescence images from the micrometer to the nanometer level. Indeed, fluorescence photoactivation can overcome the limitations that diffraction imposes on the resolution of optical microscopes. Specifically, distinct fluorophores positioned within the same subdiffraction volume can be resolved only if their emissions are activated independently at different intervals of time. Under these conditions, the sequential localization of multiple probes permits the reconstruction of images with a spatial resolution that is otherwise impossible to achieve with conventional fluorophores. The irreversible photolysis of protecting groups or the reversible transformations of photochromic compounds can be employed to control the emission of appropriate fluorescent chromophores and allow the implementation of these ingenious operating principles for superresolution imaging. Such molecular constructs enable the spatiotemporal control that is required to avoid diffraction and can become invaluable analytical tools for the optical visualization of biological specimens and nanostructured materials with unprecedented resolution.

O

compounds can conveniently be probed on the basis of activation and excitation events. In addition, the photochemical and photophysical properties of photoactivatable fluorophores can be exploited to overcome diffraction and reconstruct fluorescence images with resolution at the nanometer level. Conventional fluorescence microscopes collect the emission of individual fluorescent labels, introduced within a sample of interest, with an objective lens in order to record an image of the overall specimen.8 The radiations propagating through the lens, however, are diffracted. As a result, a light source as small as a single fluorophore appears on the focal plane of the lens in the form of an Airy pattern (Figure 2). The central disk of the pattern concentrates most of the focused radiation, and its radius (r) is related to the wavelength (λ) of the emitted light, the refractive index (n) of the medium interposed between the lens and its focal plane, as well as the semiaperture angle (θ) of the lens, according to eq 1. It follows that a fluorescent molecule, emitting visible radiation in air, generates an Airy pattern of hundreds of nanometers in lateral dimensions. Under these conditions, two fluorophores can be resolved in space only if their separation approaches this length scale. Thus, the phenomenon of diffraction that is inherent to focusing is, ultimately, dictating the spatial resolution of a fluorescence image and prevents the visualization of structural factors at the molecular level.

rganic chromophores can be designed to switch from a nonemissive state to an emissive one (Figure 1) under

Figure 1. Photoactivatable fluorophores switch from a nonemissive to an emissive state upon illumination at an activating wavelength (λAc) and then emit light in the form of fluorescence under irradiation at an exciting wavelength (λEx).

optical control with the aid of chemical synthesis.1−7 Such photoresponsive molecular systems permit the activation of fluorescence within a defined region of space at a given interval of time. Indeed, significant emission occurs only after illumination of the initial species at an activating wavelength (λAc) and irradiation of the photogenerated one at an exciting wavelength (λEx). In turn, the interplay of activating and exciting beams translates into a spatiotemporal control of fluorescence that is otherwise impossible with conventional fluorophores. This unique behavior offers the opportunity to monitor dynamic processes in real time. In fact, the diffusion of biomolecular systems labeled with photoactivatable fluorophores as well as the flow of liquids containing these functional © 2012 American Chemical Society

r=

0.61λ n sin θ

(1)

Received: July 23, 2012 Accepted: August 13, 2012 Published: August 13, 2012 2379

dx.doi.org/10.1021/jz301021e | J. Phys. Chem. Lett. 2012, 3, 2379−2385

The Journal of Physical Chemistry Letters

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the many probes between emissive and nonemissive states. In particular, binding events in the ground state, electron transfer in the excited state, as well as the photoinduced cleavage or formation of covalent bonds have all been used successfully to ensure fluorescence switching and permit the implementation of such super-resolution imaging schemes.9−16 Photoactivation localization microscopy (PALM) is one of the many strategies for super-resolution imaging based on fluorescence switching.17,19 This method relies on photoactivatable fluorophores to ensure switching and overcome diffraction. Generally, a sample of interest is labeled with probes in their nonemissive state and then illuminated at λAc with low intensities to switch only a few of them to the emissive state (Figure 3). These irradiation conditions translate into large physical separations between the activated probes and permit their subsequent spatial resolution. Indeed, illumination at λEx excites the activated probes and enables the visualization of their resolved Airy patterns. The spatial coordinates of the center of each one of them can be approximated to the position of the emissive species. Further irradiation at λEx bleaches the activated probes and turns their emission off irreversibly. At this point, another subset of probes can be activated, localized, and bleached. This sequence of events can be reiterated multiple times, storing the coordinates of a new subset of activated probes each time. The collected coordinates can finally be compiled into a single map to reconstruct an image of the sample with spatial resolution that is no longer controlled by diffraction. In fact, the lateral resolution (Δx) of the resulting image is mostly controlled by the number (N) of emitted photons collected per probe and the background noise (b), according to eq 2, together with the pixel size (a) and the standard deviation (s) of the point-spread function.20 In turn, N is related to the ability of the activated probes to absorb the exciting radiations and emit as a result (brightness), while b is mostly a consequence of the ratio (contrast) between the emission intensity of the emissive state and that of the nonemissive one. It follows that probes with large brightness and contrast offer the opportunity to bring Δx down to the nanoscale.

Figure 2. The objective lens of a fluorescence microscope projects the light emitted by a fluorophore on the focal plane in the form of an Airy pattern with most of the focused light concentrated in the central disk.

The stringent limitations imposed by diffraction on the resolution of conventional fluorescence microscopes can be overcome on the basis of switching events. The stringent limitations imposed by diffraction on the resolution of conventional fluorescence microscopes can be overcome on the basis of switching events. Specifically, fluorophores positioned within the same subdiffraction volume can be separated temporally if their emissions are designed to turn on at different intervals of time.9−16 Under these conditions, distinct fluorescent species can be localized sequentially, and images with subdiffraction resolution can eventually be reconstructed after the identification of an appropriate number of fluorophores. Indeed, these ingenious operating principles have already been applied successfully to image a diverse set of specimens in the wake of three seminal reports.17−19 The common requirements of these imaging protocols are the need to (1) maintain only a sparse population of probes in an emissive state at a given time and (2) ensure that distinct subsets of probes are in the emissive state at different intervals of time. Both conditions demand the identifications of viable mechanisms to switch independently

Δx =

s2 a2 4 πs 3b2 + + N 12N aN 2

(2)

Certain photoactivatable fluorescent proteins have sufficient brightness and contrast for the implementation of PALM, and a diverse group of biological samples have already been imaged with subdiffraction resolution on the basis of these genetically encoded labels.21−24 The power of chemical synthesis in manipulating the stereoelectronic signature of organic

Figure 3. Illumination of a sample, labeled with photoactivatable probes, at an activating wavelength (λAc) switches a subset of the labels from a nonemissive to an emissive state. Subsequent irradiation at an exciting wavelength (λEx) permits the localization of the activated probes and, eventually, bleaches the localized labels. 2380



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chromophores, however, can deliver photoactivatable fluorophores with superior photophysical properties. In addition, the small physical dimensions of synthetic dyes, relative to those of fluorescent proteins, can translate into minimal steric perturbations of labeled samples.25 Therefore, photoactivatable synthetic fluorophores can be a valuable complement to their protein counterparts in a diverse number of super-resolution imaging schemes. These considerations are encouraging the application of available photoactivatable dyes in PALM together with the development of synthetic chromophores specifically designed for this imaging strategy. Indeed, photoactivatable coumarin,26−28 dihydrofuran,29−33 fluorescein, 34,35 rhodamine,36−44 and spiropyran45,46 derivatives have already been employed successfully to acquire subdiffraction images.

Under similar illumination conditions, the nonemissive compound 3a releases nitrogen to generate a carbene that rearranges and, if the reaction is performed in methanol, is eventually trapped by a solvent molecule to generate the emissive species 3b.39 These photochemical transformations alter the structure of the main chromophoric fragment of these systems and impose a significant bathochromic shift on the absorption spectrum. Irradiation at a λEx positioned within the shifted absorption excites selectively the photochemical product and induces its emission. As a result, significant fluorescence occurs only after illumination at λAc to activate the nonemissive species and irradiation at λEx to excite the emissive one. The photoinduced conversions of 1a−3a into 1b−3b involve the cleavage of covalent bonds.29,34,39 These photochemical processes are irreversible, and as a result, the original nonemissive species cannot be regenerated. Once fluorescence is activated, it can be switched off only by bleaching the emissive species. Nonetheless, the photoinduced bathochromic shifts responsible for fluorescence activation can be replicated with photochromic transformations.47 In turn, the inherent reversibility of such photochemical processes translates into the opportunity to perform multiple activation/deactivation cycles. For example, the lactame ring of 4a and the pyran heterocycle of 5a open reversibly upon illumination at an appropriate λAc.36,45 These structural transformations generate rhodamine and merocyanine chromophores in the form of 4b and 5b, respectively, with a concomitant bathochromic shift in absorption. Illumination at a λEx positioned within the shifted absorption excites selectively the photogenerated isomers, encouraging their emission. Once again, significant fluorescence is observed only after illumination at λAc to activate the

Photoactivatable synthetic fluorophores can be a valuable complement to their protein counterparts in a diverse number of super-resolution imaging schemes. Representative examples of photoactivatable fluorophores employed in PALM are illustrated in Figures 4 and 5, and the main photochemical and photophysical parameters for some of these compounds are listed in Table 1. Illumination of the nonemissive species 1a and 2a at an appropriate λAc induces the cleavage of nitrobenzyl and azide groups, respectively, with the formation of the corresponding emissive species 1b and 2b.29,34

Figure 4. Illumination of the nonemissive species 1a−3a at an activating wavelength (λAc) generates the emissive species 1b−3b. Subsequent irradiation at an exciting wavelength (λEx) excites selectively the photochemical products to encourage their emission in the form of fluorescence. 2381



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Figure 5. Illumination of the nonemissive species 4a−6a at an activating wavelength (λAc) generates the emissive species 4b−6b. Subsequent irradiation at an exciting wavelength (λEx) excites selectively the photochemical products to encourage their emission in the form of fluorescence. The emissive isomers revert spontaneously back to the original nonemissive species on time scales ranging from few microseconds to several minutes.

Table 1. Photochemical and Photophysical Parametersa Associated with Representative Examples of Photoactivatable Fluorophores 2a/b 3a/b 4a/b 6a/b

λAc (nm)

εAc (mM−1 cm−1)

ϕP

λEx (nm)

εEx (mM−1 cm−1)

ϕF

ref

407 312 366 355

29 17

0.0059

594 559 530 532

54 66

0.025 0.37

83

0.09

29 39 36 26

9

0.02

a

The reported parameters were measured in EtOH for 2a/b, in MeCN for 3a, in MeOH for 3b, in poly(vinyl alcohol) for 4a/b, and in MeCN for 6a/b. εAc and εEx were determined at the wavelengths corresponding to the maxima of the corresponding absorption bands, rather than at the actual λAc and λEx. The missing data were not reported in the original articles.

nonemissive species and irradiation at λEx to excite the emissive one. In both instances, however, the photogenerated isomers 4b and 5b revert spontaneously back to the original ones 4a and 5a on time scales of milliseconds and minutes, respectively. Furthermore, the transformation of 5b back to 5a can be accelerated with visible illumination to facilitate the trans → cis reisomerization of its central [CC] bond. Thus, the fluorescence of these systems can be activated and then deactivated multiple times, relying on the reversible interconversion of their two isomers. The unique behavior of the photoactivatable fluorophores 1a−5a permits the implementation of the sequence of events outlined in Figure 3 and, eventually, the reconstruction of images with subdiffraction resolution.29,34,36,39,45 For example, the tubulin structure of PtK2 cells was labeled with 4a.36 The

Synthetic fluorophores with photoactivatable fluorescence allow the visualization of biological specimens and nanostructured materials with a spatial resolution that is otherwise impossible to achieve with conventional fluorescent probes. resulting specimen was illuminated with a λAc of 375 nm to activate 4a and with a λEx of 532 nm to excite the photogenerated 4b. After the acquisition of 10 000 frames 2382



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over the course of ∼100 s, the coordinates of the localized probes were compiled into a single map. The resulting reconstructed image (a in Figure 6) clearly reveals single

the interconversion of the two isomers.27 Specifically, the doped polymer nanoparticles were deposited on glass and illuminated with a λAc of 355 nm to activate 6a and a λEx of 532 nm to excite 6b. After the sequential acquisition of 90 000 frames over the course of ∼900 s, the coordinates of the localized probes were compiled into a single map. The resulting reconstructed image (a in Figure 7) reveals individual

Figure 6. Reconstructed (a) and diffraction-limited (b) images (scale bar = 2 μm) of the tubulin network of PtK2 cells immunolabeled with the photoactivatable fluorophore 4a (Reproduced from ref 36 with permission). Figure 7. Reconstructed (a) and diffraction-limited (b) images (scale bar = 0.2 μm) of polymer nanoparticles doped with the photoactivatable fluorophore 6a (Reproduced from ref 27 with permission).

tubulin filaments with a thickness ranging from 55 to 70 nm. By contrast, these nanoscaled structures cannot be discerned in the conventional diffraction-limited image (b in Figure 6) of the very same sample, which instead reveals features with a thickness approaching 500 nm at best. Thus, the ability to activate the fluorescence of the labels under optical control offers the opportunity to overcome diffraction and appreciate structural features that would otherwise be impossible to visualize with conventional fluorophores. The photoinduced conversions of 4a and 5a into 4b and 5b are designed to alter the structure of the initial species in order to generate a fluorescent fragment within the final one. Indeed, the rhodamine and merocyanine chromophores responsible for the fluorescence of 4b and 5b are only formed after the photoinduced opening of the lactame and pyran rings of 4a and 5a, respectively. An alternative approach to activate fluorescence reversibly involves the attachment of a switchable auxochrome to a pre-existing fluorescent chromophore. The photoinduced transformation of the former can be exploited to control the ability of the latter to absorb at an appropriate λEx and, therefore, also to emit. Compound 6a is a representative example of a photoactivatable fluorophore based on these operating principles.26,27 Its molecular skeleton combines a coumarin fluorophore and an oxazine photochrome. The chiral center at the junction of the two heterocyclic fragments of the latter isolates the two main components electronically. Upon irradiation at λAc, however, the oxazine ring of 6a opens to bring the coumarin appendage in conjugation with the indolium cation of 6b. This structural transformation imposes a bathochromic shift of 160 nm on the main absorption band of the coumarin fluorophore. As a result, illumination at a λEx positioned within the shifted band excites selectively the zwitterionic isomer 6b with concomitant fluorescence. The photogenerated species, however, reverts spontaneously back to the original one on a microsecond time scale in acetonitrile. It follows that the fluorescence of this particular system can be turned on and off for hundreds of switching cycles, relying on the interplay of activation and excitation events. Furthermore, 6a can be trapped within the hydrophobic interior of micellar assemblies of an amphiphilic polymer.28 The resulting supramolecular constructs have nanoscaled dimensions and can be dispersed in aqueous environments. Within these nanostructured assemblies, the nonemissive species 6a retains its photochemical behavior and can be switched to the emissive isomer 6b. Indeed, images with subdiffraction resolution of such nanoscaled objects can be reconstructed on the basis of

nanoparticles with a diameter of 30 nm and separations of only 110 nm. By contrast, these nanoscaled objects cannot be distinguished in the diffraction-limited image (b in Figure 7) of the very same sample, which reveals instead a single and wide fluorescent area.

Significant synthetic efforts together with detailed spectroscopic studies are very much needed to unravel the interrelation of structural design and excitation dynamics in photoactivatable fluorophores and permit their operation in water. Synthetic fluorophores with photoactivatable fluorescence allow the visualization of biological specimens and nanostructured materials with a spatial resolution that is otherwise impossible to achieve with conventional fluorescent probes. The further development of these functional compounds can therefore have significant implications in biomedical research and materials science. Their overall behavior, however, relies on the concatenation of a photochemical process (activation) with a photophysical event (fluorescence). As a result, the fundamental understanding of the basic factors governing both processes is essential for the rational design of activatable systems with improved performance. Indeed, the nonemissive species should ideally absorb as many photons as possible at λAc and switch efficiently to the emissive species. Similarly, the photogenerated product should also absorb as many photons as possible at λEx and emit efficiently as a consequence. In addition to learning how to control the molar extinction coefficients of the two species at the two wavelengths as well as the quantum yields for activation and fluorescence, it is also crucial to understand how to regulate the photobleaching resistances and, for reversible systems, the reisomerization kinetics. In fact, the emissive species should survive for a sufficiently long time to permit its localization at the single-molecule level with nanoscaled precision but also lose its ability to emit within a relatively short time to allow the subsequent localization of 2383



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(11) Hell, S. W. Microscopy and Its Focal Switch. Nat. Methods 2009, 6, 24−32. (12) Toomre, D.; Bewersdorf, J. A New Wave of Cellular Imaging. Ann. Rev. Cell Develop. Biol. 2010, 26, 285−314. (13) Cusido, J.; Impellizzeri, S.; Raymo, F. M. Molecular Strategies to Read and Write at the Nanoscale with Far-Field Optics. Nanoscale 2011, 3, 59−70. (14) van de Linde, S.; Heilemann, M.; Sauer, M. Live-Cell SuperResolution Imaging with Synthetic Fluorophores. Annu. Rev. Phys. Chem. 2012, 63, 519−540. (15) Ha, T.; Tinnefeld, P. Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging. Annu. Rev. Phys. Chem. 2012, 63, 595−617. (16) Moerner, W. E. Microscopy Beyond the Diffraction Limit Using Actively Controlled Single Molecules. J. Microsc. 2012, 246, 213−220. (17) Hess, S. T.; Girirajan, T. P. K.; Mason, M. D. Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy. Biophys. J. 2006, 91, 4258−4272. (18) Rust, M. J.; Bates, M.; Zhuang, X. Sub-Diffraction-Limit Imaging by Stochastic Optical Reconstruction Microscopy (STORM). Nat. Methods 2006, 3, 793−795. (19) Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.; Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; Lippincott-Schwartz, J.; Hess, H. F. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 2006, 313, 1642−1645. (20) Thompson, R. E.; Larson, D. R.; Webb, W. W. Precise Nanometer Localization Analysis for Individual Fluorescent Probes. Biophys. J. 2002, 82, 2775−2783. (21) Stepanenko, O. V.; Stepanenko, O. V.; Shcherbakova, D. M.; Kutznetsova, I. M.; Turoverov, K. K.; Verkhusha, V. V. Modern Fluorescent Proteins: from Chromophore Formation to Novel Intracellular Applications. Biotechniques 2011, 51, 313−327. (22) Lippincott-Schwartz, J. Bridging Structure and Process in Developmental Biology Through New Imaging Technologies. Develop. Cell 2011, 21, 5−10. (23) Patterson, G. H. Highlights of the Optical Highlighter Fluorescent Proteins. J. Microsc. 2011, 243, 1−7. (24) Wiedenmann, J.; Gayda, S.; Adam, V.; Oswald, F.; Nienhaus, K.; Bourgeois, D.; Nienhaus, G. U. From EosFP to mIrisFP: StructureBased Development of Advanced Photoactivatable Marker Proteins of the GFP-Family. J. Biophotonics 2011, 4, 377−390. (25) Prescher, J. A.; Bertozzi, C. R. Chemistry in Living Systems. Nat. Chem. Biol. 2005, 1, 13−21. (26) Deniz, E.; Sortino, S.; Raymo, F. M. Fluorescence Switching with a Photochromic Auxochrome. J. Phys. Chem. Lett. 2010, 1, 3506− 3509. (27) Deniz, E.; Tomasulo, M.; Cusido, J.; Yildiz, I.; Petriella, M.; Bossi, M. L.; Sortino, S.; Raymo, F. M. Photoactivatable Fluorophores for Super-Resolution Imaging Based on Oxazine Auxochromes. J. Phys. Chem. C 2012, 116, 6058−6068. (28) Cusido, J.; Battal, M.; Deniz, E.; Yildiz, I.; Sortino, S.; Raymo, F. M. Fast Fluorescence Switching within Hydrophilic Supramolecular Assemblies. Chem.Eur. J. 2012, 18, 10399−10407. (29) Lord, S. J.; Conley, N. R.; Lee, H. D.; Samuel, R.; Liu, N.; Twieg, R. J.; Moerner, W. E. A Photoactivatable Push−Pull Fluorophore for Single-Molecule Imaging in Live Cells. J. Am. Chem. Soc. 2008, 130, 9204−9205. (30) Lord, S. J.; Conley, N. R.; Lee, H. D.; Nishimura, S. Y.; Pomerantz, A. K.; Willets, K. A.; Lu, Z.; Wang, H.; Liu, N.; Samuel, R.; et al. DCDHF Fluorophores for Single-Molecule Imaging in Cells. ChemPhysChem 2009, 10, 55−65. (31) Pavani, S. R. P.; Thompson, M. A.; Biteen, J. S.; Lord, S. J.; Liu, N.; Twieg, R. J.; Piestum, R.; Moerner, E. W. Three-Dimensional, Single-Molecule Fluorescence Imaging Beyond the Diffraction Limit by Using a Double-Helix Point Spread Function. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 2995−2999. (32) Lee, H. D.; Lord, S. J.; Iwanaga, S.; Zhan, K.; Xie, H.; Williams, J. C.; Wang, H.; Bowman, G. R.; Goley, E. D.; Shapiro, L.; et al. Superresolution Imaging of Targeted Proteins in Fixed and Living

other probes. Finally, it is also essential to learn how to introduce these switchable probes within samples of interest without compromising their performance. In this context, either structural modifications of the basic chromophoric units or their encapsulation within appropriate supramolecular containers can be exploited to impose hydrophilic character and ensure compatibility with biological specimens. Thus, significant synthetic efforts together with detailed spectroscopic studies are very much needed to unravel the interrelation of structural design and excitation dynamics in photoactivatable fluorophores and permit their operation in water. The synergism of both will contribute to develop this promising area of research even further and, hopefully, will ultimately deliver optimal photoactivatable probes for a diverse number of imaging schemes with spatial resolution at the nanometer level.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biography Françisco M. Raymo is Professor of Chemistry at the University of Miami, where he directs the Laboratory for Molecular Photonics. His research interests combine the design, synthesis, and analysis of switchable molecular constructs for imaging applications. He is the author of more than 190 publications. http://www.as.miami.edu/ chemistry/Raymo

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ACKNOWLEDGMENTS The author thanks the National Science Foundation (CAREER Awards CHE-0237578, CHE-0749840, and CHE-1049860) for supporting his research program.

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REFERENCES

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