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ISRN Physical Chemistry
Volume 2012 (2012), Article ID 619251, 15 pages
Laboratory for Molecular Photonics, Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146-0431, USA
Received 25 July 2012; Accepted 16 August 2012
Academic Editors: H. Pal and M. Sliwa
Copyright © 2012 Françisco M. Raymo. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Photoactivatable fluorophores switch from a nonemissive to an emissive state upon illumination at an activating wavelength and then emit after irradiation at an exciting wavelength. The interplay of such activation and excitation events can be exploited to switch fluorescence on in a defined region of space at a given interval of time. In turn, the spatiotemporal control of fluorescence translates into the opportunity to implement imaging and spectroscopic schemes that are not possible with conventional fluorophores. Specifically, photoactivatable fluorophores permit the monitoring of dynamic processes in real time as well as the reconstruction of images with subdiffraction resolution. These promising applications can have a significant impact on the characterization of the structures and functions of biomolecular systems. As a result, strategies to implement mechanisms for fluorescence photoactivation with synthetic fluorophores are particularly valuable. In fact, a number of versatile operating principles have already been identified to activate the fluorescence of numerous members of the main families of synthetic dyes. These methods are based on either the irreversible cleavage of covalent bonds or the reversible opening and closing of rings. This paper overviews the fundamental mechanisms that govern the behavior of these photoresponsive systems, illustrates structural designs for fluorescence photoactivation, and provides representative examples of photoactivatable fluorophores in actions.
Certain organic molecules emit light in the form of fluorescence upon excitation at an appropriate wavelength . This behavior offers the opportunity to probe biological structures and processes with optical methods . Indeed, labeling strategies to introduce fluorescent probes within biological samples, in combination with microscopic techniques to excite the labels and collect their fluorescence, permit the noninvasive visualization of a diversity of specimens in real time . In particular, the basic operating principles of a fluorescence microscope (Figure 1) involve the illumination of the labeled specimen with an excitation source through an objective lens. The radiations focused on the sample excite the many fluorescent labels from their ground state ( in Figure 2) to one of their excited singlet states (e.g., ). The excited species relax thermally to the first singlet excited state () and then radiatively to S0. The emitted light is collected on the detector, through the very same objective lens, to reconstruct an image of the labeled sample with micrometer resolution on a millisecond timescale. In fact, the fluorescence microscope has become an indispensable analytical tool in the biomedical laboratory for the routinary investigation of cells and tissues.
The power of fluorescence microscopy in permitting the visual inspection of biological samples revolves around the ability of individual fluorescent probes to absorb exciting radiations and emit as a result . Thus, the identification of viable strategies to manipulate the photophysical properties of organic molecules is central to the further development of this discipline. In particular, mechanisms to switch molecules from a nonemissive to an emissive state under the influence of optical stimulations translate into the opportunity to photoactivate fluorescence. Interest in such photoactivatable fluorophores was sparked initially by the idea of monitoring the course of photochemical reactions with fluorescence measurements  and then by the possibility of using such compounds for photographic applications . It became eventually apparent, however, that photoactivatable fluorophores can be a valuable complement to conventional ones in a diversity of imaging applications [6–12]. Indeed, fluorescence photoactivation permits the monitoring of dynamic processes in real time [13–24] as well as the acquisition of images with spatial resolution at the nanometer level [25–42]. As a result, significant research efforts are presently directed to the development of strategies for fluorescence photoactivation with fluorescent proteins and synthetic dyes together with their implementation in imaging applications. In fact, the former can conveniently be encoded genetically in a diversity of biological samples and permit the visualization of living specimens [17–22]. The latter can be engineered with superior photochemical and photophysical properties, relying on the aid of chemical synthesis [6–12], and their small dimensions, relative to their natural counterparts, ensure minimal steric perturbations to labeled samples . In this paper, we overview the basic principles governing the photophysical properties of organic molecules, highlight the main mechanisms developed so far to photoactivate fluorescence with synthetic chromophores, illustrate representative examples of photoactivatable synthetic probes, and discuss their promising applications.
2. Photophysical Properties of Organic Molecules
An organic molecule can adopt a finite number of electronic configurations with distinct energies . These electronic states differ in the distribution mode of the many electrons among the available molecular orbitals, and their energy separations approach the energies associated with ultraviolet and visible radiations. As a result, a molecule can gain a sufficient amount of energy to change its electronic configuration by absorbing an ultraviolet or a visible photon.
The electronic configuration with the lowest possible energy (ground state) of a neutral organic molecule distributes pairs of electrons with opposite spin in the half of available molecular orbitals with lowest energy. Therefore the sum () of the individual electronic spins is equal to 0 in the ground state. This value corresponds to a multiplicity () of 1 and, hence, this particular electronic state is a singlet and is indicated with the notation (Figure 2).
Upon absorption of a photon with appropriate energy, an electron in one of the occupied molecular orbitals moves to one of the unoccupied molecular orbitals. During this transition, the electron retains its spin and the multiplicity of the overall system does not change. Thus, the resulting electronic state (excited state) is also a singlet, even though it has two unpaired electrons.
Distinct singlet excited states can be accessed from the very same ground electronic configuration. Indeed, a molecule in the initial state S0 has multiple pairs of occupied and unoccupied molecular orbitals available to permit electronic transitions with different energies. Therefore, excitation from S0 can populate any one of the accessible singlet excited states, depending on the energy of the absorbed photon. For example, the diagram in Figure 2 shows a transition from to the second singlet excited state (S2) upon excitation.
The electronic transition that accompanies the absorption of a photon from occurs on a femtosecond timescale . During this extremely fast process, the nuclear configuration of the molecule does not change, and the geometry adopted in is retained in the final electronic state (e.g., in Figure 2). After the transition, however, the nuclei respond to the change in electronic configuration by adjusting their relative positions. As a result, the excited molecule alters its geometry to release a fraction of the energy gained with excitation and, eventually, changes its electronic configuration to decay nonradiatively to . This relaxation process, called internal conversion (Figure 2), occurs generally on a timescale of picoseconds. Once is populated, the excited molecule can return back to S0 by either releasing thermal energy or emitting light in the form of fluorescence. The decay from to tends to occur over times ranging from hundreds of picoseconds to a few nanoseconds.
The two unpaired electrons of a molecule in S1 have opposite spin. One of them can change its spin to produce an electronic state with a total spin () of 1 and a multiplicity () of 3. Therefore, this particular state is a triplet and is indicated with the notation . The process responsible for the transformation of into that is called intersystem crossing (Figure 2) occurs generally over several nanoseconds and is followed by either the release of thermal energy or the emission of light in the form of phosphorescence to populate . The transformation of into , however, requires one of the two unpaired electrons to change its spin and happens over times ranging from few nanoseconds to several microseconds.
Photochemical reactions also offer the opportunity to release the excitation energy of a molecule . Specifically a compound in can undergo a structural transformation to produce a different species, which can then decay to its ground electronic configuration. Thus four distinct relaxation pathways are available to deactivate , but only one of them is responsible for fluorescence. Indeed can (1) decay to thermally, (2) decay to radiatively, (3) intersystemly cross to , or (4) undergo a photochemical reaction. As a result, the quantum efficiency of fluorescence is ultimately dictated by the rates of these competitive processes . In particular the fluorescence quantum yield () is the ratio between the number of emitted photons () and the number of the absorbed photons () and is related to the rate constants of fluorescence (), nonradiative decay (), intersystem crossing (), and photochemical conversion (), according to (1). Therefore the ability of a fluorophore to emit light can, in principle, be controlled by manipulating these four rate constants with appropriate structural modifications:
3. Mechanisms and Structural Designs for Irreversible Fluorescence Photoactivation
Photoactivatable fluorophores switch from a nonemissive to an emissive state upon illumination at an activating wavelength ( in Figure 3). The photogenerated species is then irradiated at an exciting wavelength ( in Figure 3) to emit light in the form of fluorescence. This behavior can be implemented with two general mechanisms [6–12]. One of them acts on the ability of to absorb the exciting radiations and the other controls the nonradiative deactivation of . In the first instance, the switchable system is designed to absorb at only after the transformation induced by . Under these conditions, activation enables the population of the excited state responsible for fluorescence. In the second instance, the initial species can also absorb at , but nonradiative decay dominates its relaxation. After the transformation induced by is designed to decrease significantly, relative to , in order to enhance , according to (1).
Both mechanisms for fluorescence activation can be implemented by integrating switchable and fluorescent components within the same covalent skeleton [6–12]. The local excitation of the former can then be achieved with an activating beam operating at to induce the cleavage of one or more covalent bonds. This photolytic process separates irreversibly the two components and, in some instances, is also followed by a rearrangement or a supramolecular event. Ultimately, the photoinduced structural transformation either enables the absorption of the fluorescent fragment at or suppresses the nonradiative decay of its excited state. This general design logic is extremely versatile and can easily be adapted to most of the main families of fluorescent probes with the aid of chemical synthesis. Indeed, photoactivatable acridinone , anthracene , benzofurazan , borondipyrromethene , chromone , coumarin [51–56], dihydrofuran [57–59], fluorescein [60–67], naphthalene , pyranine , pyrazolines , resorufin , rhodamine [66, 72–76], thioxanthone  and xanthone  derivatives have been developed on the basis of these operating principles. Furthermore, this general strategy has been adapted to activate also the luminescence of conjugated oligomers  and semiconductor quantum dots [80, 81].
The behaviors of compounds 1a  and 2a  (Figure 4) are representative examples of the two main mechanisms for fluorescence photoactivation. The former has a photocleavable azide group attached to a dihydrofuran fluorophore. The latter has a photocleavable nitrobenzyl group attached to a fluorescein fluorophore. The absorption spectrum of 1a in ethanol shows a band centered at 424 nm . Upon illumination at a of 407 nm, the azide group cleaves to generate irreversibly 1b. This structural transformation shifts the absorption band to 570 nm. Thus, irradiation at a (594 nm) positioned within this band can excite exclusively the photogenerated species 1b. It follows that the fluorescence of this molecule is observed only after activation at and excitation at .
The absorption spectrum of 2a in neutral buffer reveals an intense band in the visible region for the heterocyclic chromophore . Upon illumination at of 490 nm, 2a changes its electronic configuration to populate S1. However, the transfer of one electron from the phenoxy appendage to the heterocyclic fragment encourages the nonradiative deactivation of . As a result, the fluorescence quantum yield of 2a is negligible. Upon irradiation at a of 350 nm, the nitrobenzyl group attached to the heterocyclic chromophore cleaves to generate 2b. The anionic character of the photogenerated species prevents intramolecular electron transfer in and favors, instead, the radiative decay of this state. In fact, illumination of 2b at is accompanied by significant emission. Thus, also for this particular system, fluorescence is observed only after activation at and excitation at .
The irreversible photolysis of the azide group of 1a and nitrobenzyl appendage of 2a releases an intact fluorescent fragment that can then emit upon excitation at an appropriate wavelength [57, 64]. In some instances, however, the released fragment must associate with a supramolecular partner in order to discourage nonradiative decay and enable emission [67, 68]. In other instances, the photochemical reaction does not release the intact fluorophore and, instead, generates an intermediate species that spontaneously rearranges into the final fluorescent fragment [50, 52, 54, 56]. Alternatively, such photolytic events can be coupled with energy transfer to activate fluorescence [65, 66]. For example, compound 3a (Figure 5) pairs a coumarin donor to a fluorescein acceptor within its covalent skeleton . The nitrobenzyl appendage attached to the donor cleaves upon illumination at a of 365 nm in neutral buffer. This structural transformation shifts bathochromically the absorption of the coumarin fragment and enables its excitation at a of 410 nm. The excited donor can then transfer energy to the fluorescein acceptor, which then emits at 520 nm. Thus, the fluorescence of the acceptor is eventually activated through the manipulation of the absorption properties of the donor.
4. Mechanisms and Structural Designs for Reversible Fluorescence Photoactivation
The operating principles for fluorescence activation associated with 1a–3a are centered around the photolysis of a covalent bond [57, 64, 65]. This photoinduced process is irreversible and, therefore, the fluorescence of the photochemical product cannot be switched off after activation. Reversible fluorescence activation can, instead, be achieved relying on the inherent reversibility of photochromic transformations [82–87]. Indeed, photochromic compounds switch from a colorless to a colored state under illumination at an appropriate wavelength and return to the original form either thermally or upon irradiation at another wavelength. In fact, the photoinduced coloration of a photochromic compound is, essentially, imposing a bathochromic shift on the absorption spectrum similar to that associated with the interconversion of 1a into 1b. It follows that the photogenerated state of the photochromic component can, eventually, be excited selectively at an appropriate . If this species is fluorescent, then its emission is observed after activation at a designed to trigger the photochromic transformation and illumination at to excite the photogenerated state [87–94]. This behavior is identical to that associated with the photoinduced conversion of 1a into 1b except for the fact that the photochromic process is reversible. Therefore, the original nonemissive state can be regenerated and the system can undergo multiple fluorescence activation/deactivation cycles. Nonetheless, the fluorescence quantum yields of most photochromic compounds are fairly modest, relative to those of the main families of fluorescent probes, with the exception of certain diarylethenes [95–97], rhodamines [98–102], and spiropyrans [103, 104].
The behaviors of compounds 4a  and 5a  (Figure 6) are representative examples of reversible fluorescence activation on the basis of photochromic transformations. The former has a lactam ring connected to a xanthene heterocycle through a spirocenter. Upon illumination at a of 366 nm in a poly(vinyl alcohol) matrix, the [C–N] bond at the spirocenter cleaves to generate the rhodamine 4b. This transformation is accompanied by a significant bathochromic shift in absorption. In fact, the photogenerated isomer can be excited selectively with a of 530 nm. The diarylethene 5b has three adjacent [C=C] bonds at its core. Upon irradiation at a of 313 nm in dioxane, they undergo an electrocyclic rearrangement to close a six-membered ring in the form of compound of 5b. Once again, this structural transformation imposes a significant bathochromic shift in absorption and the photogenerated isomer can be excited selectively at a of 488 nm. Thus, both systems offer the opportunity to photoactivate fluorescence with a mechanism that is essentially identical to that governing the behavior of 1a. In these instances, however, the photogenerated species 4b and 5b can switch back to the original nonemissive states 4a and 5a, respectively, offering the opportunity to perform multiple activation/deactivation cycles. Nonetheless, 4b is thermally unstable, while 5b is thermally stable. The former reverts spontaneously to 4a on a millisecond timescale and the latter converts to 5a photochemically under visible illumination.
The photochromic transformations of 4a and 5a result in the formation of a fluorescent chromophore that was not originally present in the initial structure [97, 98]. Alternatively a preformed fluorophore can be connected to an appropriate photochromic component and the photoinduced interconversion of the latter can be exploited to control the emission of the former [87, 89–94]. Indeed, numerous examples of fluorophore-photochrome dyads have already been developed successfully, relying on this general design logic [105–127]. In most of these systems, however, the photogenerated state of the photochromic component is engineered to quench the emission of the fluorescent partner on the basis of either electron or energy transfer. As a result, the behavior of these functional compounds translates into fluorescence deactivation. A remarkable exception is a family of dyads specifically designed for fluorescence activation [128–130]. In these compounds, the photochromic component regulates reversibly the ability of the fluorescent fragment to absorb at and permits the implementation of an activation mechanism similar to that governing the behavior of 1a. For example, compound 6a (Figure 7) pairs a coumarin fluorophore to an oxazine photochrome within its molecular skeleton. Upon illumination at a of 355 nm in acetonitrile, the oxazine ring opens to generate the zwitterionic isomer 6b. This structural transformation brings the coumarin appendage in conjugation with the cationic fragment of the photogenerated isomer and shifts its absorption band bathochromically by ca. 160 nm. As a result, irradiation at a of 532 nm excites selectively 6b. Therefore, the fluorescence of this species is observed only after activation at and excitation at . The photogenerated species, however, reverts spontaneously to the original one on a microsecond timescale and, in fact, the fluorescence of this system can be switched on and off for hundreds of cycles, relying on the reversible interconversion of the two isomers.
5. Photochemical and Photophysical Parameters Associated with Fluorescence Activation
The operating principles behind most photoactivatable fluorescent probes revolve around the coupling of a photochemical reaction (activation) with a photophysical process (fluorescence). The first of these two steps requires the nonemissive species to absorb photons at and switch to the emissive state. As a result, the molar extinction coefficient () of the former at and the quantum yield () for this photochemical transformation should be as large as possible. The second step demands the emissive state to absorb photons at and emit light in the form of fluorescence. Thus, the molar extinction coefficient () of this species at and should also be as large as possible. As representative examples, the values of these parameters for 1a–6a are listed in Table 1.
Compounds 2a–6a have acceptable and values to switch to the respective emissive counterparts at moderate activation intensities [64, 65, 97, 98, 128]. However, they require a in the ultraviolet region. In fact, most of the photoactivatable fluorophores developed so far have essentially the very same limitation. Indeed, biological samples have plenty of intrinsic chromophores able to absorb in the ultraviolet region and, therefore, can coabsorb activating radiations in this wavelength range with harmful consequences. This problem can be overcome with structural modifications of the photocleavable group aimed at shifting its absorption from the ultraviolet to the visible region. For example, the introduction of two methoxy substituents on a nitrobenzyl group can be exploited to enable activation with wavelengths longer than 400 nm [8, 9]. Alternatively, two-photon absorption processes can be invoked to induce activation [46, 51–53, 69, 98, 99]. Under these conditions, the simultaneous absorption of two near-infrared photons can encourage the transformation of the nonemissive species into the emissive one. Such protocols offer also the excellent penetration depths and spatial resolutions inherent to two-photon excitation schemes. Nonetheless, they are limited to photocleavable groups with sufficiently large two-photon absorption cross sections.
Compounds 1b–6b can all be excited with a in the visible region and have all a relatively large [57, 64, 65, 97, 98, 128]. Both conditions generally apply to most of the photoactivatable fluorophores developed so far for biological applications. Instead, tends to vary over a broad range of values. The of 1b, for example, is only 0.025 , while that of 5b is up to 0.87 . Nonetheless, a modest is often compensated by a large and the product of the two (brightness) for 1b is sufficiently large to permit the detection of this fluorophore at the single-molecule level .
In addition to the wavelengths, molar extinction coefficients, and quantum yields listed in Table 1, two additional parameters contribute to define the performance of a photoactivatable fluorophore. One of them is the ratio (contrast) between the emission intensity of the emissive state and that of the nonemissive one. Ideally, the nonemissive state should not fluoresce upon illumination at . In a photoactivatable system operating on the basis of changes in the rate of nonradiative decay, however, the nonemissive state can also absorb at and the partial radiative deactivation of its excited state might contribute some fluorescence. For example, 2a absorbs at and emits with a close to 0.001 . After activation and the formation of 2b, increases to 0.86 with a significant enhancement in detected fluorescence. Nonetheless, the minimal fluorescence contribution of the nonemissive state in such systems can have detrimental effects on contrast and might limit the use these probes in imaging applications requiring negligible background signal.
The other parameter of significance is the photobleaching resistance of the emissive state. The population of the S1 of this species, upon excitation at , can be followed by intersystem crossing to . Both and can undergo photochemical transformations that result in the bleaching of the emissive species. These processes are generally not particularly efficient but, over the course of multiple excitation cycles, degradation can become significant. It follows that photoactivatable fluorophores with limited ability to resist photobleaching might not be particularly useful in imaging applications requiring prolonged excitation.
6. Applications of Photoactivatable Fluorophores
The unique combination of photochemical and photophysical properties of photoactivatable fluorophores translates into the opportunity to control the spatial and temporal distribution of fluorescence. As a result, these functional fluorescent probes permit the implementation of imaging and spectroscopic protocols that are otherwise inaccessible with conventional fluorophores. Indeed, photoactivatable fluorophores are routinely used as calibration standards in photolysis experiments [131–133], to probe the activity and dynamics of biomolecular systems [48, 49, 55, 61, 62, 68, 71, 134–142], to monitor the flow dynamics of fluids [143–152], and to reconstruct subdiffraction images [57–59, 74–76, 98, 99, 103, 104, 129, 153–160]. The common theme of all these strategies is the interplay of activating and exciting beams to switch fluorescence on within a defined region of space at a given interval of time.
The ability to activate fluorescence under optical control can be exploited to monitor the diffusion of molecules in a diversity of media [13–24]. In fact, the dynamics of a photoactivatable fluorophore within a substrate of interest can be probed by irradiating at , a portion of the sample and at , the entire substrate, while measuring the intensity of the emitted light. This illumination protocol permits the monitoring of the temporal change in the spatial distribution of the activated fluorescence. For example, the photoinduced transformation of the nonemissive species 7a (Figure 8) into the emissive one 7b offers the opportunity to follow the diffusion of the latter in glycerol on a millisecond timescale . Specifically, the tip of an optical fiber can be positioned on a glycerol drop containing 7a to illuminate a relatively small area of the sample at a of 365 nm. This irradiation conditions cleave the two nitrobenzyl groups to generate 7b within the illuminated area. The simultaneous irradiation of the whole substrate at a of 490 nm reveals fluorescence mostly within the activated area (Figure 8(a)). However, images recorded with the same illumination protocol after a delay of 400 and 900 ms (Figures 8(b) and 8(c)) show clearly a significant widening of the fluorescent area. Thus, the photogenerated species 7b is diffusing away from the activated area over the course of this temporal scale.
The illumination protocol to monitor diffusion with photoactivatable fluorophores is conceptually related to fluorescence recovery after photobleaching (FRAP) [162–164]. However, FRAP demands high-irradiation intensities to bleach fluorophores and switch emission off in the illuminated area. By contrast, moderate illumination intensities are generally sufficient to switch photoactivatable fluorophores and, therefore, they can conveniently be adapted to monitor diffusion in biological specimens [13–24]. In a seminal study, for example, the dynamics of actin microfilaments could be visualized on a timescale of seconds with the aid of a photoactivatable resorufin . Specifically, one of cystein residues of actin can be connected covalently to a photoactivatable label in the form of the conjugate 8a (Figure 9). Illumination of 9a at a of 360 nm cleaves the nitrobenzyl group to generate 8b, which can be excited at a of 575 nm to emit light in the form of fluorescence. The injection of 8a into goldfish epithelial keratocytes results in its incorporation within the native actin microfilaments. Illumination at of a defined area within a labeled keratocyte activates fluorescence. Sequences of images (Figures 9(a)–9(c)), recorded upon irradiation at over the course of several seconds, reveal clearly a change in the spatial distribution of the activated fluorescence that is indicative of microfilament dynamics. In fact, similar imaging protocols have been extended to the investigation of the dynamics of a diversity of biomolecular systems [134, 135, 138–142].
In addition to the diffusion of individual molecules within a media of interest, similar configurations can be adapted to the investigation of fluid dynamics [143–152]. In particular, a solution of a photoactivatable fluorophore flowing through the channels of a microfluidic device can be probed, once again, on the basis of activation and excitation events. For example, a dilute solution of 7a in basic buffer can be introduced within a rectangular channel of micrometer dimensions fabricated within a poly(dimethyl siloxane) matrix . Illumination at a of 337 nm of a fixed position within the channel cleaves the two nitrobenzyl groups of 7a to generate its emissive counterpart 7b. The activated species travels along the channel to reach, eventually, an area that is illuminated at a of 488 nm. The fluorescence emitted from the excited area can be measured simultaneously to build a temporal profile of the number of detected photons. The resulting plots show that photons are detected after a delay from activation that is directly related to the flow rate. Indeed, an increase in flow rate from 11 cm s−1 to 16 m s−1 results in a decrease in delay from milliseconds to microseconds (Figures 10(a) and 10(b)).
The phenomenon of diffraction limits the lateral resolution of the fluorescence microscope to hundreds of nanometers . As a result, individual fluorophores can be resolved in space only if their distance is significantly greater than their physical dimensions. It follows that this convenient imaging technique cannot appreciate the subtle factors that govern biological processes and define biological structures at the molecular level. These stringent limitations can be overcome with the aid of photoactivatable fluorophores [26–42]. Indeed, the ability to switch fluorescence on under optical control permits the separation of distinct probes in time and the sequential reconstruction of images with subdiffraction resolution. Specifically, a biological sample can be labeled with photoactivatable probes in their nonemissive state and illuminated at with low intensities. Under these conditions, only a small fraction of probes switches to their emissive state (Figure 11), ensuring a large physical separations between them. At this point, the activated sample can be illuminated at , and the few emissive species can be localized at the single-molecule level with nanoscaled precision, if their brightness and contrast are designed to be sufficiently large. The coordinates of the localized probes can be recorded, and the emissive species can be irradiated further at until they bleach. Then, the sequence of activation, excitation, localization, and bleaching events can be reiterated multiple times, recording for every single sequence the coordinates of a new subset of probes. Eventually this procedure permits the accumulation of a sufficient number of coordinates to compile a single image of the sample with a lateral resolution that is no longer controlled by diffraction. For example, the tubulin structure of PtK2 cells can be immunolabeled with the photochromic rhodamine 4a . Reiterative sequences of activation, excitation, localization, and bleaching events can then be exploited to map the spatial distribution of the fluorescent probes with subdiffraction resolution. Comparison of the resulting image with its diffraction-limited counterpart (in Figures 11(a) and 11(b)) clearly reveals a drastic enhancement in resolution. Individual tubulin filaments can be distinguished in the former, while they appear as blurred objects in the latter. In fact, this powerful imaging protocol has been extended already to other numerous photoactivatable fluorophores [57–59, 74–76, 98, 99, 103, 104, 129, 153–160] to permit the visualization of a diversity of biological specimens with a resolution that is otherwise impossible to achieve with conventional probes.
Synthetic fluorophores can be designed to emit light in the form of fluorescence upon excitation at one wavelength only after activation at another wavelength. Such photoactivatable fluorophores can be constructed by connecting a photocleavable group to a fluorescent chromophore. The photoinduced cleavage of the former can be exploited to control the ability of the latter to absorb at the exciting wavelength. Alternatively, this photochemical process can alter the rate of nonradiative decay of the fluorescent component. Both mechanisms translate into irreversible fluorescence activation. Similarly, photochromic transformations can be invoked to control the ability of a fluorescent component to absorb at the exciting wavelength. The inherent reversibility of photochromic processes, however, offers also the opportunity to deactivate the activated fluorophore. Indeed, multiple activation/deactivation cycles can be performed under optical control with these switchable systems.
The photochemical and photophysical behavior of photoactivatable fluorophores permits the control of the spatial and temporal distribution of fluorescence. These unique characteristics translate into the possibility of implementing imaging and spectroscopic protocols that are otherwise impossible with conventional fluorophores. Specifically, the diffusion of chemicals labeled with photoactivatable fluorophores and the dynamics of fluids containing such switchable compounds can both be probed in a diversity of media, relying on the interplay of activation and excitation events. Furthermore, the ability to activate the emission of individual fluorophores at different intervals of time offers the opportunity to resolve temporally probes that are separated by relatively short distances. This protocol permits to overcome the limitations imposed by diffraction on the resolution of conventional fluorescence images and, hence, enables the visualization of biological specimens at the nanometer level. Thus, photoactivatable fluorophores are versatile molecular probes for the noninvasive investigation of the dynamics and structures of a diversity of samples.
The National Science Foundation (CAREER Award CHE-0237578, CHE-0749840, and CHE-1049860) is acknowledged for supporting our research program.
- J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer, New York, NY, USA, 2006.
- D. B. Murphy, Fundamentals of Light Microscopy and Electronic Imaging, Wiley-Liss, New York, NY, USA, 2001.
- J. B. Pawley, Ed., Handbook of Biological Confocal Microscopy, Springer, New York, NY, USA, 2006.
- T. Förster, “Diabatic and adiabatic processes in photochemistry,” Pure and Applied Chemistry, vol. 24, no. 3, pp. 443–449, 1970.
- A. Zweig, “Photochemical generation of stable fluorescent compounds (photofluorescence),” Pure and Applied Chemistry, vol. 33, no. 2-3, pp. 389–410, 1973.
- G. A. Krafft, R. T. Cummings, and J. P. Dizio, “Fluorescence photoactivation and dissipation (FPD),” in Nucleocytoplasmic Transport, R. Peters and M. Trendelenburg, Eds., pp. 35–52, Springer, Berlin, Germany, 1986.
- T. J. Mitchison, K. E. Sawin, J. A. Theriot, K. Gee, and A. Mallavarapu, “Caged fluorescent probes,” Methods in Enzymology, vol. 291, pp. 63–78, 1998.
- A. P. Pelliccioli and J. Wirz, “Photoremovable protecting groups: reaction mechanisms and applications,” Photochemical and Photobiological Sciences, vol. 1, no. 7, pp. 441–458, 2002.
- M. Goeldner and and R. Givens, Eds., Dynamic Studies in Biology: Phototriggers, Photoswitches and Caged Biomolecules, Wiley-VCH, Weinheim, Germany, 2005.
- L. M. Wysocki and and L. D. Davis, “Advances in the chemistry of small molecule fluorescent probes,” Current Opinion in Chemical Biology, vol. 15, no. 6, pp. 752–759, 2011.
- D. Puliti, D. Warther, C. Orange, A. Specht, and M. Goeldner, “Small photoactivatable molecules for controlled fluorescence activation in living cells,” Bioorganic and Medicinal Chemistry, vol. 19, no. 3, pp. 1023–1029, 2011.
- W.-H. Li and and G. Zheng, “Photoactivatable fluorophores and techniques for biological imaging applications,” Photochemical and Photobiological Sciences, vol. 11, no. 3, pp. 460–471, 2012.
- S. R. Adams and R. Y. Tsien, “Controlling cell chemistry with caged compounds,” Annual Review of Physiology, vol. 55, pp. 755–784, 1993.
- J. P. Y. Kao and S. R. Adams, “Photosensitive caged compounds: design, properties and biological applications,” in Optical Microscopy: Emerging Methods and Applications, B. Herman and and J. J. Lemasters, Eds., pp. 27–85, Academic Press, San Diego, Calif, USA, 1993.
- J. C. Politz, “Use of caged fluorochromes to track macromolecular movement in living cells,” Trends in Cell Biology, vol. 9, no. 7, pp. 284–287, 1999.
- R. W. Dirks, C. Molenaar, and H. J. Tanke, “Methods for visualizing RNA processing and transport pathways in living cells,” Histochemistry and Cell Biology, vol. 115, no. 1, pp. 3–11, 2001.
- J. Lippincott-Schwartz, N. Altan-Bonnet, and G. H. Patterson, “Photobleaching and photoactivation: following protein dynamics in living cells,” Nature Reviews Molecular Cell Biology, vol. 4, pp. S7–S14, 2003.
- J. Lippincott-Schwartz and G. H. Patterson, “Development and use of fluorescent protein markers in living cells,” Science, vol. 300, no. 5616, pp. 87–91, 2003.
- V. V. Verkhusha and K. A. Lukyanov, “The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins,” Nature Biotechnology, vol. 22, no. 3, pp. 289–296, 2004.
- N. C. Shaner, G. H. Patterson, and M. W. Davidson, “Advances in fluorescent protein technology,” Journal of Cell Science, vol. 120, no. 24, pp. 4247–4260, 2007.
- G. C. R. Ellis-Davies, “Caged compounds: photorelease technology for control of cellular chemistry and physiology,” Nature Methods, vol. 4, no. 8, pp. 619–628, 2007.
- S. Manley, J. M. Gillette, and J. Lippincott-Schwartz, “Single-particle tracking photoactivated localization microscopy for mapping single-molecule dynamics,” Methods in Enzymology, vol. 475, no. C, pp. 109–120, 2010.
- I. Johnson and M. T. Z. Spence, The Molecular Probes Handbook—A Guide to Fluorescent Probes and Labeling Technologies, Life Technologies, Carlsbad, Calif, USA, 11th edition, 2010.
- Y. Xu, T. J. Melia, and D. T. Toomre, “Using light to see and control membrane traffic,” Current Opinion in Chemical Biology, vol. 15, pp. 822–830, 2011.
- S. W. Hell, “Toward fluorescence nanoscopy,” Nature Biotechnology, vol. 21, no. 11, pp. 1347–1355, 2003.
- M. Sauer, “Reversible molecular photoswitches: a key technology for nanoscience and fluorescence imaging,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 27, pp. 9433–9434, 2005.
- S. W. Hell, “Far-field optical nanoscopy,” Science, vol. 316, no. 5828, pp. 1153–1158, 2007.
- M. Bates, B. Huang, and X. Zhuang, “Super-resolution microscopy by nanoscale localization of photo-switchable fluorescent probes,” Current Opinion in Chemical Biology, vol. 12, no. 5, pp. 505–514, 2008.
- M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nature Reviews Molecular Cell Biology, vol. 9, no. 12, pp. 929–943, 2008.
- B. Huang, M. Bates, and X. Zhuang, “Super-resolution fluorescence microscopy,” Annual Review of Biochemistry, vol. 78, pp. 993–1016, 2009.
- M. Heilemann, P. Dedecker, J. Hofkens, and M. Sauer, “Photoswitches: key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification,” Laser and Photonics Reviews, vol. 3, no. 1-2, pp. 180–202, 2009.
- S. T. Hess, T. J. Gould, M. Gunewardene, J. Bewersdorf, and M. D. Mason, “Ultrahigh resolution imaging of biomolecules by fluorescence photoactivation localization microscopy,” Methods in molecular biology (Clifton, N.J.), vol. 544, pp. 483–522, 2009.
- S. W. Hell, “Microscopy and its focal switch,” Nature Methods, vol. 6, no. 1, pp. 24–32, 2009.
- D. Toomre and J. Bewersdorf, “A new wave of cellular imaging,” Annual Review of Cell and Developmental Biology, vol. 26, pp. 285–314, 2010.
- J. Vogelsang, C. Steinhauer, C. Forthmann et al., “Make them blink: probes for super-resolution microscopy,” ChemPhysChem, vol. 11, no. 12, pp. 2475–2490, 2010.
- M. A. Thompson, J. S. Biteen, S. J. Lord, N. R. Conley, and W. E. Moerner, “Molecules and methods for super-resolution imaging,” Methods in Enzymology, vol. 475, no. C, pp. 27–59, 2010.
- T. Fukaminato, “Single-molecule fluorescence photoswitching: design and synthesis of photoswitchable fluorescent molecules,” Journal of Photochemistry and Photobiology C, vol. 12, no. 3, pp. 177–208, 2011.
- J. Cusido, S. Impellizzeri, and F. M. Raymo, “Molecular strategies to read and write at the nanoscale with far-field optics,” Nanoscale, vol. 3, no. 1, pp. 59–70, 2011.
- S. Van De Linde, S. Wolter, and M. Sauer, “Single-molecule photoswitching and localization,” Australian Journal of Chemistry, vol. 64, no. 5, pp. 503–511, 2011.
- J. C. Vaughan and X. Zhuang, “New fluorescent probes for super-resolution imaging,” Nature Biotechnology, vol. 29, no. 10, pp. 880–881, 2011.
- S. van de Linde, M. Heilemann, and M. Sauer, “Live-cell Super-resolution imaging with synthetic fluorophores,” Annual Reviews of Physical Chemistry, vol. 63, pp. 519–540, 2012.
- T. Ha and P. Tinnefeld, “Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging,” Annual Reviews of Physical Chemistry, vol. 63, pp. 595–617, 2012.
- J. A. Prescher and C. R. Bertozzi, “Chemistry in living systems,” Nature Chemical Biology, vol. 1, no. 1, pp. 13–21, 2005.
- I. N. Levine, Quantum Chemistry, Prentice Hall, Englewoods Cliffs, NJ, USA, 1991.
- N. J. Turro, V. Ramamurthy, and J. C. Scaiano, Principles of Molecular Photochemistry: An Introduction, University Science Book, Herndon, Va, USA, 2009.
- D. Warther, F. Bolze, J. Léonard et al., “Live-cell one- And two-photon uncaging of a far-red emitting acridinone fluorophore,” Journal of the American Chemical Society, vol. 132, no. 8, pp. 2585–2590, 2010.
- R. T. Cummings and G. A. Krafft, “Photoactivable fluorophores. 1. Synthesis and photoactivation of o-nitrobenzyl-quenched fluorescent carbamates,” Tetrahedron Letters, vol. 29, no. 1, pp. 65–68, 1988.
- W. F. Veldhuyzen, Q. Nguyen, G. McMaster, and D. S. Lawrence, “A light-activated probe of intracellular protein kinase activity,” Journal of the American Chemical Society, vol. 125, no. 44, pp. 13358–13359, 2003.
- T. Kobayashi, T. Komatsu, M. Kamiya, et al., “Highly activatable and environment-insensitive optical highlighters for selective spatiotemporal imaging of target proteins,” Journal of the American Chemical Society, vol. 134, no. 27, pp. 11153–11160, 2012.
- R. T. Cummings, J. P. DiZio, and G. A. Krafft, “Photoactivable fluorophores. 2. Synthesis and photoactivation of functionalized 3-aroyl-2-(2-furyl)-chromones,” Tetrahedron Letters, vol. 29, no. 1, pp. 69–72, 1988.
- Y. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez, and W. H. Li, “New caged Coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications,” Journal of the American Chemical Society, vol. 126, no. 14, pp. 4653–4663, 2004.
- N. Gagey, P. Neveu, and L. Jullien, “Two-photon uncaging with the efficient 3,5-dibromo-2,4-dihydroxycinnamic caging group,” Angewandte Chemie, vol. 46, no. 14, pp. 2467–2469, 2007.
- C. Orange, A. Specht, D. Puliti et al., “Synthesis and photochemical properties of a light-activated fluorophore to label His-tagged proteins,” Chemical Communications, no. 10, pp. 1217–1219, 2008.
- N. Gagey, M. Emond, P. Neveu et al., “Alcohol uncaging with fluorescence reporting: evaluation of o-acetoxyphenyl methyloxazolone precursors,” Organic Letters, vol. 10, no. 12, pp. 2341–2344, 2008.
- G. Zheng, L. Cochella, J. Liu, O. Hobert, and W. H. Li, “Temporal and spatial regulation of microRNA activity with photoactivatable cantimirs,” American Chemical Society Chemical Biology, vol. 6, no. 12, pp. 1332–1338, 2011.
- X.-Y. Duan, B.-C. Zhai, and Q.-H. Song, “Water-s o luble o-hydroxycinnamate as an efficient photoremovable protecting group of alcohols with fluorescence reporting,” Photochemical and Photobiological Sciences, vol. 11, no. 3, pp. 593–598, 2012.
- S. J. Lord, N. R. Conley, H. L. D. Lee et al., “A photoactivatable push-pull fluorophore for single-molecule imaging in live cells,” Journal of the American Chemical Society, vol. 130, no. 29, pp. 9204–9205, 2008.
- S. J. Lord, N. R. Conley, H. L. D. Lee et al., “DCDHF fluorophores for single-molecule imaging in cells,” ChemPhysChem, vol. 10, no. 1, pp. 55–65, 2009.
- S. J. Lord, H. L. D. Lee, R. Samuel et al., “Azido push-pull fluorogens photoactivate to produce bright fluorescent labels,” Journal of Physical Chemistry B, vol. 114, no. 45, pp. 14157–14167, 2010.
- G. A. Krafft, W. R. Sutton, and R. T. Cummings, “Photoactivable fluorophores. 3. Synthesis and photoactivation of fluorogenic difunctionalized fluoresceins,” Journal of the American Chemical Society, vol. 110, no. 1, pp. 301–303, 1988.
- T. J. Mitchison, “Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence,” Journal of Cell Biology, vol. 109, no. 2, pp. 637–652, 1989.
- J. P. Vincent and P. H. O'Farrell, “The state of engrailed expression is not clonally transmitted during early Drosophila development,” Cell, vol. 68, no. 5, pp. 923–931, 1992.
- J. P. Pellois, M. E. Hahn, and T. W. Muir, “Simultaneous triggering of protein activity and fluorescence,” Journal of the American Chemical Society, vol. 126, no. 23, pp. 7170–7171, 2004.
- T. Kobayashi, Y. Urano, M. Kamiya, T. Ueno, H. Kojima, and T. Nagano, “Highly activatable and rapidly releasable caged fluorescein derivatives,” Journal of the American Chemical Society, vol. 129, no. 21, pp. 6696–6697, 2007.
- G. Zheng, Y. M. Guo, and W. H. Li, “Photoactivatable and water soluble FRET dyes with high uncaging cross section,” Journal of the American Chemical Society, vol. 129, no. 35, pp. 10616–10617, 2007.
- D. Maurel, S. Banala, T. Laroche, and K. Johnsson, “Photoactivatable and photoconvertible fluorescent probes for protein labeling,” ACS Chemical Biology, vol. 5, no. 5, pp. 507–516, 2010.
- L. Yuan, W. Lin, Z. Cao, L. Long, and J. Song, “Photocontrollable analyte-responsive fluorescent probes: a photocaged copper-responsive fluorescence turn-on probe,” Chemistry, vol. 17, no. 2, pp. 689–696, 2011.
- M. E. Vázquez, M. Nitz, J. Stehn, M. B. Yaffe, and B. Imperiali, “Fluorescent caged phosphoserine peptides as probes to investigate phosphorylation-dependent protein associations,” Journal of the American Chemical Society, vol. 125, no. 34, pp. 10150–10151, 2003.
- N. I. Kiskin, R. Chillingworth, J. A. McCray, D. Piston, and D. Ogden, “The efficiency of two-photon photolysis of a “caged” fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic terminals,” European Biophysics Journal, vol. 30, no. 8, pp. 588–604, 2002.
- Z. Yu, L. Y. Ho, and Q. Lin, “Rapid, photoactivatable turn-on fluorescent probes based on an intramolecular photoclick reaction,” Journal of the American Chemical Society, vol. 133, no. 31, pp. 11912–11915, 2011.
- J. A. Theriot and T. J. Mitchison, “Actin microfilament dynamics in locomoting cells,” Nature, vol. 352, no. 6331, pp. 126–131, 1991.
- J. Ottl, D. Gabriel, and G. Marriott, “Preparation and photoactivation of caged fluorophores and caged proteins using a new class of heterobifunctional, photocleavable cross-linking reagents,” Bioconjugate Chemistry, vol. 9, no. 2, pp. 143–151, 1998.
- K. R. Gee, E. S. Weinberg, and D. J. Kozlowski, “Caged Q-rhodamine dextran: a new photoactivated fluorescent tracer,” Bioorganic and Medicinal Chemistry Letters, vol. 11, no. 16, pp. 2181–2183, 2001.
- V. N. Belov, C. A. Wurm, V. P. Boyarskiy, S. Jakobs, and S. W. Hell, “Rhodamines NN: a novel class of caged fluorescent dyes,” Angewandte Chemie, vol. 49, no. 20, pp. 3520–3523, 2010.
- L. M. Wysocki, J. B. Grimm, A. N. Tkachuk, T. A. Brown, E. Betzig, and L. D. Lavis, “Facile and general synthesis of photoactivatable xanthene dyes,” Angewandte Chemie, vol. 50, no. 47, pp. 11206–11209, 2011.
- K. Kolmakov, C. Wurm, M. V. Sednev, M. L. Bossi, V. N. Belov, and S. W. Hell, “Masked red-emitting carbopyronine dyes with photosensitive 2-diazo-1-indanone caging group,” Photochemical and Photobiological Sciences, vol. 11, no. 3, pp. 522–532, 2012.
- J. R. R. Majjigapu, A. N. Kurchan, R. Kottani, T. P. Gustafson, and A. G. Kutateladze, “Release and report: a new photolabile caging system with a two-photon fluorescence reporting function,” Journal of the American Chemical Society, vol. 127, no. 36, pp. 12458–12459, 2005.
- J. A. Blake, M. Lukeman, and J. C. Scaiano, “Photolabile protecting groups based on the singlet state photodecarboxylation of xanthone acetic acid,” Journal of the American Chemical Society, vol. 131, no. 11, pp. 4127–4135, 2009.
- R. H. Pawle, V. Eastman, and S. W. Thomas III, “UV-induced fluorescence recovery and solubility modulation of photocaged conjugated oligomers,” Journal of Materials Chemistry, vol. 21, no. 36, pp. 14041–14047, 2011.
- G. Han, T. Mokari, C. Ajo-Franklin, and B. E. Cohen, “Caged quantum dots,” Journal of the American Chemical Society, vol. 130, no. 47, pp. 15811–15813, 2008.
- S. Impellizzeri, B. McCaughan, J. F. Callan, and F. M. Raymo, “Photoinduced enhancement in the luminescence of hydrophilic quantum dots coated with photocleavable ligands,” Journal of the American Chemical Society, vol. 134, no. 4, pp. 2276–2283, 2012.
- G. H. Dorion and A. F. Wiebe, Photochromism, Focal Press, New York, NY, USA, 1970.
- G. H. Brown, Ed., Photochromism, Wiley, New York, NY, USA, 1971.
- A. V. El'tsov, Organic Photochromes, Consultants Bureau, New York, NY, USA, 1990.
- H. Bouas-Laurent and H. Dürr, Eds., Photochromism: Molecules and Systems, Elsevier, Amsterdam, The Netherlands, 1990.
- J. C. Crano and R. Guglielmetti, Eds., Organic Photochromic and Thermochromic Compounds, Plenum Press, New York, NY, USA, 1999.
- M. Irie, Ed., “Photochromism: memories and switches—introduction,” Chemical Reviews, vol. 100, no. 5, pp. 1683–1890, 2000.
- M. G. Kuz'min and M. V. Koz'menko, “Luminescence of Photochromic Compounds,” in Organic Photochromes, A. V. El'tsov, Ed., pp. 245–265, Consultants Bureau, New York, NY, USA, 1990.
- F. M. Raymo and M. Tomasulo, “Electron and energy transfer modulation with photochromic switches,” Chemical Society Reviews, vol. 34, no. 4, pp. 327–336, 2005.
- F. M. Raymo and M. Tomasulo, “Fluorescence modulation with photochromic switches,” Journal of Physical Chemistry A, vol. 109, no. 33, pp. 7343–7352, 2005.
- J. Cusido, E. Deniz, and F. M. Raymo, “Fluorescent switches based on photochromic compounds,” European Journal of Organic Chemistry, no. 13, pp. 2031–2045, 2009.
- I. Yildiz, E. Deniz, and F. M. Raymo, “Fluorescence modulation with photochromic switches in nanostructured constructs,” Chemical Society Reviews, vol. 38, no. 7, pp. 1859–1867, 2009.
- J. Cusido, E. Deniz, and F. M. Raymo, “Photochromic compounds for fluorescence nanoscopy,” Current Physical Chemistry, no. 3, pp. 232–241, 2009.
- C. Yun, J. You, J. Kim, J. Huh, and E. Kim, “Photochromic fluorescence switching from diarylethenes and its applications,” Journal of Photochemistry and Photobiology C, vol. 10, no. 3, pp. 111–129, 2009.
- Y. C. Jeong, S. I. Yang, K. H. Ahn, and E. Kim, “Highly fluorescent photochromic diarylethene in the closed-ring form,” Chemical Communications, no. 19, pp. 2503–2505, 2005.
- Y. C. Jeong, S. I. Yang, E. Kim, and K. H. Ahn, “Development of highly fluorescent photochromic material with high fatigue resistance,” Tetrahedron, vol. 62, no. 25, pp. 5855–5861, 2006.
- K. Uno, H. Niikura, M. Morimoto, Y. Ishibashi, H. Miyasaka, and M. Irie, “In situ preparation of highly fluorescent dyes upon photoirradiation,” Journal of the American Chemical Society, vol. 133, no. 34, pp. 13558–13564, 2011.
- J. Fölling, V. Belov, R. Kunetsky et al., “Photochromic rhodamines provide nanoscopy with optical sectioning,” Angewandte Chemie, vol. 46, no. 33, pp. 6266–6270, 2007.
- J. Fölling, V. Belov, D. Riedel et al., “Fluorescence nanoscopy with optical sectioning by two-photon induced molecular switching using continuous-wave lasers,” ChemPhysChem, vol. 9, no. 2, pp. 321–326, 2008.
- I. Testa, A. Schönle, C. V. Middendorff et al., “Nanoscale separation of molecular species based on their rotational mobility,” Optics Express, vol. 16, no. 25, pp. 21093–21103, 2008.
- V. N. Belov, M. L. Bossi, J. Fölling, V. P. Boyarskiy, and S. W. Hell, “Rhodamine spiroamides for multicolor single-molecule switching fluorescent nanoscopy,” Chemistry, vol. 15, no. 41, pp. 10762–10776, 2009.
- H. Montenegro, M. Di Paolo, D. Capdevila, P. F. Aramendía, and M. L. Bossi, “The mechanism of the photochromic transformation of spirorhodamines,” Photochemical and Photobiological Sciences, vol. 11, no. 6, pp. 1081–1086, 2012.
- D. Hu, Z. Tian, W. Wu, W. Wan, and A. D. Q. Li, “Photoswitchable nanoparticles enable high-resolution cell imaging: PULSAR microscopy,” Journal of the American Chemical Society, vol. 130, no. 46, pp. 15279–15281, 2008.
- Z. Tian, A. D. Q. Li, and D. Hu, “Super-resolution fluorescence nanoscopy applied to imaging core-shell photoswitching nanoparticles and their self-assemblies,” Chemical Communications, vol. 47, no. 4, pp. 1258–1260, 2011.
- T. Inada, S. Uchida, and Y. Yokoyama, “Perfect on/off switching of emission of fluorescence by photochromic reaction of a binaphthol-condensed fulgide derivative,” Chemistry Letters, no. 4, pp. 321–322, 1997.
- A. J. Myles and N. R. Branda, “Controlling photoinduced electron transfer within a hydrogen-bonded porphyrin-phenoxynaphthacenequinone photochromic system,” Journal of the American Chemical Society, vol. 123, no. 1, pp. 177–178, 2001.
- J. L. Bahr, G. Kodis, L. De la Garza et al., “Photoswitched singlet energy transfer in a porphyrin-spiropyran dyad,” Journal of the American Chemical Society, vol. 123, no. 29, pp. 7124–7133, 2001.
- A. Myles and N. R. Branda, “1,2-Dithienylethene photochromes and non-destructive erasable memory,” Advanced Functional Materials, vol. 12, no. 3, pp. 167–173, 2002.
- L. Giordano, T. M. Jovin, M. Irie, and E. A. Jares-Erijman, “Diheteroarylethenes as thermally stable photoswitchable acceptors in photochromic fluorescence resonance energy transfer (pcFRET),” Journal of the American Chemical Society, vol. 124, no. 25, pp. 7481–7489, 2002.
- M. Irie, T. Fukaminato, T. Sasaki, N. Tamai, and T. Kawai, “A digital fluorescent molecular photoswitch,” Nature, vol. 420, no. 6917, pp. 759–760, 2002.
- T. Kawai, M. S. Kim, T. Sasaki, and M. Irie, “Fluorescence switching of photochromic diarylethenes,” Optical Materials, vol. 21, no. 1-3, pp. 275–278, 2003.
- R. T. F. Jukes, V. Adamo, F. Hartl, P. Belser, and L. De Cola, “Photochromic dithienylethene derivatives containing Ru(II) or Os(II) metal units. Sensitized photocyclization from a triplet state,” Inorganic Chemistry, vol. 43, no. 9, pp. 2779–2792, 2004.
- T. Fukaminato, T. Sasaki, T. Kawai, N. Tamai, and M. Irie, “Digital photoswitching of fluorescence based on the photochromism of diarylethene derivatives at a single-molecule level,” Journal of the American Chemical Society, vol. 126, no. 45, pp. 14843–14849, 2004.
- J. Andréasson, G. Kodis, Y. Terazono et al., “Molecule-based photonically switched half-adder,” Journal of the American Chemical Society, vol. 126, no. 49, pp. 15926–15927, 2004.
- Y. Terazono, G. Kodis, J. Andréasson et al., “Photonic control of photoinduced electron transfer via switching of redox potentials in a photochromic moiety,” Journal of Physical Chemistry B, vol. 108, no. 6, pp. 1812–1814, 2004.
- E. A. Jares-Erijman, L. Giordano, C. Spagnuolo, J. Kawior, R. J. Verneij, and T. M. Jovin, “Photochromic fluorescence resonance energy transfer (pcFRET): formalism, implementation, and perspectives,” Proceedings of the Society of the Photo-Optical Instrumentation Engineers, vol. 5323, no. 13, pp. 13–26, 2004.
- S. D. Straight, J. Andréasson, G. Kodis et al., “Molecular AND and INHIBIT gates based on control of porphyrin fluorescence by photochromes,” Journal of the American Chemical Society, vol. 127, no. 26, pp. 9403–9409, 2005.
- M. Bossi, V. Belov, S. Polyakova, and S. W. Hell, “Reversible red fluorescent molecular switches,” Angewandte Chemie, vol. 45, no. 44, pp. 7462–7465, 2006.
- Y. Odo, T. Fukaminato, and M. Irie, “Photoswitching of fluorescence based on intramolecular electron transfer,” Chemistry Letters, vol. 36, no. 2, pp. 240–241, 2007.
- L. H. Liu, K. Nakatani, R. Pansu, J. J. Vachon, P. Tauc, and E. Ishow, “Fluorescence patterning through photoinduced migration of squaraine-functionalized azo derivatives,” Advanced Materials, vol. 19, no. 3, pp. 433–436, 2007.
- A. De Meijere, Z. Ligang, V. N. Belov, M. Bossi, M. Noltemeyer, and S. W. Hell, “1,3-bicyclo[1.1.1]pentanediyl: the shortest rigid linear connector of phenylated photochromic units and a 1,5-dimethoxy-9,10-di(phenylethynyl) anthracene fluorophore,” Chemistry, vol. 13, no. 9, pp. 2503–2516, 2007.
- T. Fukaminato, T. Umemoto, Y. Iwata et al., “Photochromism of diarylethene single molecules in polymer matrices,” Journal of the American Chemical Society, vol. 129, no. 18, pp. 5932–5938, 2007.
- M. Berberich, A. M. Krause, M. Orlandi, F. Scandola, and F. Würthner, “Toward fluorescent memories with nondestructive readout: photoswitching of fluorescence by intramolecular electron transfer in a diaryl ethene-perylene bisimide photochromic system,” Angewandte Chemie, vol. 47, no. 35, pp. 6616–6619, 2008.
- J. Fölling, S. Polyakova, V. Belov, A. Van Blaaderen, M. L. Bossi, and S. W. Hell, “Synthesis and characterization of photoswitchable fluorescent silica nanoparticles,” Small, vol. 4, no. 1, pp. 134–142, 2008.
- T. Fukaminato, T. Doi, M. Tanaka, and M. Irie, “Photocyclization reaction of diarylethene-perylenebisimide dyads upon irradiation with visible (>500 nm) light,” Journal of Physical Chemistry C, vol. 113, no. 27, pp. 11623–11627, 2009.
- T. Fukaminato, M. Tanaka, T. Doi et al., “Fluorescence photoswitching of a diarylethene-perylenebisimide dyad based on intramolecular electron transfer,” Photochemical and Photobiological Sciences, vol. 9, no. 2, pp. 181–187, 2010.
- T. Fukaminato, T. Doi, N. Tamaoki et al., “Single-molecule fluorescence photoswitching of a diarylethene- perylenebisimide dyad: non-destructive fluorescence readout,” Journal of the American Chemical Society, vol. 133, no. 13, pp. 4984–4990, 2011.
- E. Deniz, S. Sortino, and F. M. Raymo, “Fluorescence switching with a photochromic auxochrome,” Journal of Physical Chemistry Letters, vol. 1, no. 24, pp. 3506–3509, 2010.
- E. Deniz, M. Tomasulo, and J. Cusido, “Photoactivatable fluorophores for super-resolution imaging based on oxazine auxochromes,” Journal of Physical Chemistry C, vol. 116, no. 10, pp. 6058–6068, 2012.
- J. Cusido, M. Battal, E. Deniz, I. Yildiz, S. Sortino, and F. M. Raymo, “Fast fluorescence switching within hydrophilic supramolecular assemblies,” Chemistry—A European Journal, vol. 18, pp. 10399–10407, 2012.
- G. P. Ahern, S. F. Hsu, and M. B. Jackson, “Direct actions of nitric oxide on rat neurohypophysial K+ channels,” Journal of Physiology, vol. 520, no. 1, pp. 165–176, 1999.
- E. Korkotian, D. Holcman, and M. Segal, “Dynamic regulation of spine-dendrite coupling in cultured hippocampal neurons,” European Journal of Neuroscience, vol. 20, no. 10, pp. 2649–2663, 2004.
- E. Korkotian and M. Segal, “Morphological constraints on calcium dependent glutamate receptor trafficking into individual dendritic spine,” Cell Calcium, vol. 42, no. 1, pp. 41–57, 2007.
- L. Cramer and T. J. Mitchison, “Moving and stationary actin filaments are involved in spreading of postmitotic PtK2 cells,” Journal of Cell Biology, vol. 122, no. 4, pp. 833–843, 1993.
- V. I. Rodionov, S. S. Lim, V. I. Gelfand, and G. G. Borisy, “Microtubule dynamics in fish melanophores,” Journal of Cell Biology, vol. 126, no. 6, pp. 1455–1464, 1994.
- D. J. Kozlowski, T. Murakami, R. K. Ho, and E. S. Weinberg, “Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein,” Biochemistry and Cell Biology, vol. 75, no. 5, pp. 551–562, 1997.
- D. J. Kozlowski and E. S. Weinberg, “Photoactivatable (caged) fluorescein as a cell tracer for fate mapping in the zebrafish embryo,” Methods in Molecular Biology, vol. 135, pp. 349–355, 2000.
- K. Dakin, Y. Zhao, and W. H. Li, “LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling,” Nature Methods, vol. 2, no. 1, pp. 55–62, 2005.
- J. C. R. Politz, R. A. Tuft, K. V. Prasanth et al., “Rapid, diffusional shuttling of poly(A) RNA between nuclear speckles and the nucleoplasm,” Molecular Biology of the Cell, vol. 17, no. 3, pp. 1239–1249, 2006.
- K. Dakin and W. H. Li, “Infrared-LAMP: two-photon uncaging and imaging of gap junctional communication in three dimensions,” Nature Methods, vol. 3, no. 12, p. 959, 2006.
- Y. M. Guo, S. Chen, P. Shetty, G. Zheng, R. Lin, and W. H. Li, “Imaging dynamic cell-cell junctional coupling in vivo using Trojan-LAMP,” Nature Methods, vol. 5, no. 9, pp. 835–841, 2008.
- I. A. Shestopalov, C. L. W. Pitt, and J. K. Chen, “Spatiotemporal resolution of the Ntla transcriptome in axial mesoderm development,” Nature Chemical Biology, vol. 8, no. 3, pp. 270–276, 2012.
- W. R. Lempert, P. Ronney, K. Magee, K. R. Gee, and R. P. Haugland, “Flow tagging velocimetry in incompressible flow using photo-activated nonintrusive tracking of molecular motion (PHANTOMM),” Experiments in Fluids, vol. 18, no. 4, pp. 249–257, 1995.
- J. E. Guilkey, K. R. Gee, P. A. McMurtry, and J. C. Klewicki, “Use of caged fluorescent dyes for the study of turbulent passive scalar mixing,” Experiments in Fluids, vol. 21, no. 4, pp. 237–242, 1996.
- J. E. Guilkey, A. R. Kerstein, P. A. McMurtry, and J. C. Klewicki, “Mixing mechanisms in turbulent pipe flow,” Physics of Fluids, vol. 9, no. 3, pp. 717–723, 1997.
- P. H. Paul, M. G. Garguilo, and D. J. Rakestraw, “Imaging of pressure- and electrokinetically driven flows through open capillaries,” Analytical Chemistry, vol. 70, no. 13, pp. 2459–2467, 1998.
- S. L. R. Barker, D. Ross, M. J. Tarlov, M. Gaitan, and L. E. Locascio, “Control of flow direction in microfluidic devices with polyelectrolyte multilayers,” Analytical Chemistry, vol. 72, no. 24, pp. 5925–5929, 2000.
- T. J. Johnson, D. Ross, M. Gaitan, and L. E. Locascio, “Laser modification of preformed polymer microchannels: application to reduce band broadening around turns subject to electrokinetic flow,” Analytical Chemistry, vol. 73, no. 15, pp. 3656–3661, 2001.
- D. Ross and L. E. Locascio, “Effect of caged fluorescent dye on the electroosmotic mobility in microchannels,” Analytical Chemistry, vol. 75, no. 5, pp. 1218–1220, 2003.
- J. P. Shelby and D. T. Chiu, “Mapping fast flows over micrometer-length scales using flow-tagging velocimetry and single-molecule detection,” Analytical Chemistry, vol. 75, no. 6, pp. 1387–1392, 2003.
- Y. Rondelez, G. Tresset, K. V. Tabata et al., “Microfabricated arrays of femtoliter chambers allow single molecule enzymology,” Nature Biotechnology, vol. 23, no. 3, pp. 361–365, 2005.
- S. Hapuarachchi, S. P. Premeau, and C. A. Aspinwall, “High-speed capillary zone electrophoresis with online photolytic optical injection,” Analytical Chemistry, vol. 78, no. 11, pp. 3674–3680, 2006.
- H. L. D. Lee, S. J. Lord, S. Iwanaga et al., “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” Journal of the American Chemical Society, vol. 132, no. 43, pp. 15099–15101, 2010.
- M. F. Juette, T. J. Gould, M. D. Lessard et al., “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nature Methods, vol. 5, no. 6, pp. 527–529, 2008.
- S. R. P. Pavani, M. A. Thompson, J. S. Biteen et al., “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 9, pp. 2995–2999, 2009.
- F. Cella Zanacchi, Z. Lavagnino, and M. Perrone Donnorso, “Live-cell 3D superresolution imaging in thick biological samples,” Nature Methods, vol. 8, no. 12, pp. 1047–1049, 2011.
- M. Bossi, J. Föiling, V. N. Belov et al., “Multicolor far-field fluorescence nanoscopy through isolated detection of distinct molecular species,” Nano Letters, vol. 8, no. 8, pp. 2463–2468, 2008.
- D. Aquino, A. Schönle, C. Geisler et al., “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nature Methods, vol. 8, no. 4, pp. 353–359, 2011.
- M. J. Roberti, J. Fölling, M. S. Celej, M. L. Bossi, T. M. Jovin, and E. A. Jares-Erijman, “Imaging nanometer-sized b-synuclein aggregates by superresolution fluorescence localization microscopy,” Biophysical Journal, vol. 102, no. 7, pp. 1598–1607, 2012.
- H. Aoki, K. Mori, and S. Ito, “Conformational analysis of single polymer chains in three dimensions by super-resolution fluorescence microscopy,” Soft Matter, vol. 8, pp. 4390–4395, 2012.
- Y. Bernardinelli, C. Haeberli, and J. Y. Chatton, “Flash photolysis using a light emitting diode: an efficient, compact, and affordable solution,” Cell Calcium, vol. 37, no. 6, pp. 565–572, 2005.
- A. Lopez, L. Dupou, A. Altibelli, J. Trotard, and J. F. Tocanne, “Fluorescence recovery after photobleaching (FRAP) experiments under conditions of uniform disk illumination. Critical comparison of analytical solutions, and a new mathematical method for calculation of diffusion coefficient D,” Biophysical Journal, vol. 53, no. 6, pp. 963–970, 1988.
- D. E. Wolf, “Theory of fluorescence recovery after photobleaching measurements on cylindrical surfaces,” Biophysical Journal, vol. 61, no. 2 I, pp. 487–493, 1992.
- K. Braeckmans, L. Peeters, N. N. Sanders, S. C. De Smedt, and J. Demeester, “Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope,” Biophysical Journal, vol. 85, no. 4, pp. 2240–2252, 2003.