Ground- and First-Excited-Singlet-State Electric Dipole Moments of Some Photochromic Spirobenzopyrans in Their Spiropyran and Merocyanine Form

Bletz, Michael; Pfeifer-Fukumura, Ursula; Kolb, Ute; Baumann, Wolfram

doi:10.1021/jp012562q

Cited by 97 publications

(88 citation statements)

References 14 publications

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“…OLID using optical switch probes described herein affords key advantages for high-contrast imaging: (i) the mechanisms of optical switching of nitroBIPS (17)(18)(20)(21) and related photochromes (22) and Dronpa (19,23) are known, and do not require chemical additives; (ii) optical switching between the two states (Ͻ2 s) is much faster than probes used in PALM and STORM; (iii) optically driven transitions occur in the entire population of probe molecules, providing large signals and rapid imaging; and (iv) kinetic signatures and quantum yields for excited-state transitions can easily be measured in the sample and are used for lock-in detection. Thus, the properties of optically switched organic dyes and fluorescent proteins in OLID are compatible with rapid imaging of specific proteins and structures in live cells and within live animals and can be readily used in most laboratories on existing microscopes.…”

mentioning

confidence: 99%

Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells

Marriott

Mao²,

Sakata³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

206

268

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One of the limitations on imaging fluorescent proteins within living cells is that they are usually present in small numbers and need to be detected over a large background. We have developed the means to isolate specific fluorescence signals from background by using lock-in detection of the modulated fluorescence of a class of optical probe termed ''optical switches.'' This optical lock-in detection (OLID) approach involves modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and nonfluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from nonmodulated background signals. Cross-correlation analysis provides a measure of correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching and a reference waveform detected within the same image over the same switching cycles. This approach to imaging provides a means to selectively detect the emission from optical switch probes among a larger population of conventional fluorescent probes and is compatible with conventional microscopes. OLID using nitrospirobenzopyran-based probes and the genetically encoded Dronpa fluorescent protein are shown to generate high-contrast images of specific structures and proteins in labeled cells in cultured and explanted neurons and in live Xenopus embryos and zebrafish larvae.high-contrast ͉ optical switches ͉ "ac"-imaging ͉ fluorescence microscopy U nderstanding the molecular basis for the regulation of complex biological processes such as cell motility and proliferation requires analysis of the distribution and dynamics of protein interactions within living cells in culture and in intact tissue (1). Tremendous advances have been made toward the development of new optical probes (2, 3) and imaging techniques that are capable of detecting proteins down to the level of single molecules (4-11). However, in living cells, such detection is compromised by autofluorescence, which can amount to several thousand equivalents of fluorescein per cell (12), as well as by light scattering (13). A major challenge in live-cell imaging, therefore, is to develop classes of probes and imaging techniques that are capable of resolving fluorescence signals from synthetic probes or genetically encoded fluorescent proteins in living cells and tissue against large background signals that may vary in time and space.A simple and highly-effective approach for isolating a specific fluorescence signal from a large background is to reversibly modulate the fluorescence intensity of only a probe of interest that is bound to a specific protein by applying a uniform, rapid and specific perturbation (e.g., a change in temperature (14), pressure (15), or voltage (16) to which that probe is uniquely attuned. The modulated fluorescence can be isolated from other steady sources of background fluorescence by lock-in detection, making it possible to specifically extract the probe fluoresc...

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mentioning

confidence: 99%

Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells

Marriott

Mao²,

Sakata³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

206

268

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“…The largest difference in energy for the five differentially linked SP states on G-actin is 504 Ϯ 85 cm Ϫ1 (Ϯ1 nm at 345 nm) between compounds 3 and 6 and 12. This value represents only 27% of the equivalent difference found for the most divergent MC states within G-actin, and, interestingly, this value scales with the strength of the dipole moments of SP and MC of 5 and 20 D, respectively (22). We note that the difference in energy between the most divergent MC conjugates (1,868 cm Ϫ1 ) and the most divergent SP conjugates (504 cm Ϫ1 ) is 1,363 cm Ϫ1 , which is comparable to the binding energy of G-actin with regulatory proteins and ligands.…”

Section: Optical Spectroscopy Of Spirobenzopyran In Solventsmentioning

confidence: 81%

“…1 A). The permanent charge on the nitrogen and the highly polarized nitro group probably account for the fact that MC has a very high ground-state dipole moment (20D) (22).…”

Section: Optical Spectroscopy Of Spirobenzopyran In Solventsmentioning

confidence: 99%

“…The anomalous polar solvent-induced blue shifts in the MC absorption spectrum and the insensitivity of MC fluorescence to polar solvents can be explained by considering the unusual property of the MC dipole moment, which is lower in the excited state (14 D) compared with the ground state (20 D) (22). The inversion of the strength of the dipole moment in the excited state will lead to interactions between MC and solvent or protein dipoles that are stronger in the ground state (Figs.…”

Section: Optical Spectroscopy Of Spirobenzopyran In Solventsmentioning

confidence: 99%

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Optical switching of dipolar interactions on proteins

Sakata

Yao

Marriott

2005

Proc. Natl. Acad. Sci. U.S.A.

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This work shows that optical switching between the spiro (SP) and merocyanine (MC) states of different photochromes specifically labeled to G-actin can be used to rapidly and reversibly modulate specific dipolar interactions within the conjugate. Members of a common spirobenzopyran photochrome and a related spironaphthoxazine that differ only in the locations of their alkylating groups were selectively labeled to Cys-374 on G-actin. The nature of MC and SP interactions within G-actin was investigated by using optical spectroscopy. The average absorption energy of the highly polarized MC is sensitive to interactions with polar groups on solvents and G-actin; the average absorption energy of the corresponding SP state was found to be relatively constant, consistent with its lower dipole moment compared with MC (5 and 20 D, respectively). Alternate excitation of spirobenzopyran G-actin conjugates with 365 and 546 nm leads to rapid transitions from the SP to MC states and MC to SP states, respectively; optical switching within spirobenzopyran-G-actin occurs with high fidelity and the recovery of specific dipolar interactions between the protein and the MC and SP states. The difference in the free energy for specific dipolar interactions between different MC states within G-actin (6 kcal͞mol) is similar to that found for complexes of G-actin and its regulatory proteins. We propose, therefore, that optical switching between SP and MC within an appropriately labeled conjugate could be used to inhibit a functional interaction with a ligand in the MC, but not the SP, state. protein engineeringT he increasing need to understand biomolecular and cellular processes in terms of the mechanisms of protein-mediated reactions is driving the development of new optical probes capable of specifically manipulating and mapping protein interactions and activities in complex environments (1-6). We have previously shown that protein interactions and activities can be manipulated in vitro and in vivo by using light-directed activation of caged proteins (3,6). This perturbation technique can yield important kinetic and functional information on the protein with high temporal and spatial resolution (6). However, the photoisomerization of the 2-nitrophenyl group limits the usefulness of this technique because the reaction is irreversible, slow, and generates toxic photoproducts.We propose a previously undescribed approach for optical switching of protein interactions (7) that is based on optical control of specific dipolar interactions within proteins specifically labeled with a single photochromic probe. The spirobenzopyran probe is well suited for this task because it undergoes rapid, and reversible, light-directed transitions between a colorless spiro (SP) state and a colorful merocyanine (MC) state without the release of photoproducts (7-13). Optical spectroscopy is used to characterize the interactions of the two switch states within the conjugates and to provide information on their dipolar interactions with G-actin. We argue that if ...

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“…This is consistent with conversions achievable in chloroform solution. Generally, we can state that the quantum yield for coloration of this particular type of spiropyran is very low 28,36,37 and this explains the high fluences required to achieve this conversion. This is opposite, yet analogous, to the case of spirooxazine ring closure where Bohne et al used laser fluences up to 600 mJ per pulse to photobleach merocyanine isomers due to the very low quantum yield of this process.…”

Section: Photochemical and Photobiological Sciencesmentioning

confidence: 99%

Transient absorption spectroscopy on spiropyran monolayers using nanosecond pump—probe Brewster angle reflectometry

Siebenhofer

Gorelik

Lear

et al. 2013

Photochem Photobiol Sci

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Self-assembled monolayers of 11-(3',3'-dimethyl-6,8-dinitrospiro[chromene-2,2'-indoline]-1'-yl) undecanoic acid (amphiphilic spiropyran) at the air-water interface are studied using Brewster angle reflectometry.Transient kinetics of the spiropyran to merocyanine conversion are recorded in a UV-pump, VIS-probe configuration. By varying the probe wavelength using an optical parametric oscillator, we are able to reconstruct absorption spectra of intermediate states with a time-resolution of 10 nanoseconds, limited by the temporal convolution of the two laser pulses. After UV irradiation, spiropyran converts to merocyanine in two stages. The first occurs within a timescale of several tens of nanoseconds and is heavily convoluted with the system response time, whereas the second stage occurs over a few hundred nanoseconds. During the rise time there is a small red shift in the transient absorption spectrum of ∼20 nm. We assign the red shift and the slower kinetics to the isomerization of a merocyanine isomer cis about the central methine bond to those that are trans about the same bond.

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Ground- and First-Excited-Singlet-State Electric Dipole Moments of Some Photochromic Spirobenzopyrans in Their Spiropyran and Merocyanine Form

Cited by 97 publications

References 14 publications

Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells

Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells

Optical switching of dipolar interactions on proteins

Transient absorption spectroscopy on spiropyran monolayers using nanosecond pump—probe Brewster angle reflectometry

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