Blind instrument response function identification from fluorescence decays.

Time-resolved fluorescence spectroscopy plays a crucial role when studying dynamic properties of complex photochemical systems. Nevertheless, the analysis of measured time decays and the extraction of exponential lifetimes often requires either the experimental assessment or the modeling of the instrument response function (IRF). However, the intrinsic nature of the IRF in the measurement process, which may vary across measurements due to chemical and instrumental factors, jeopardizes the results obtained by reconvolution approaches. In this paper, we introduce a novel methodology, called blind instrument response function identification (BIRFI), which enables the direct estimation of the IRF from the collected data. It capitalizes on the properties of single exponential signals to transform a deconvolution problem into a well-posed system identification problem. To delve into the specifics, we provide a step-by-step description of the BIRFI method and a protocol for its application to fluorescence decays. The performance of BIRFI is evaluated using simulated and time-correlated single-photon counting data. Our results demonstrate that the BIRFI methodology allows an accurate recovery of the IRF, yielding comparable or even superior results compared with those obtained with experimental IRFs when they are used for reconvolution by parametric model fitting.

Cloning and structural basis of fluorescent protein color variants from identical species of sea anemone, Diadumene lineata.

Diadumene lineata is a colorful sea anemone with orange stripe tissue of the body column and plain tentacles with red lines. We subjected Diadumene lineata to expression cloning and obtained genes encoding orange (OFP: DiLiFP561) and red fluorescent proteins (RFPs: DiLiFP570 and DiLiFP571). These proteins formed obligatory tetramers. All three proteins showed bright fluorescence with the brightness of 58.3 mM-1·cm-1 (DiLiFP561), 43.9 mM-1·cm-1 (DiLiFP570), and 31.2 mM-1·cm-1 (DiLiFP571), which were equivalent to that of commonly used red fluorescent proteins. Amplitude-weighted average fluorescence lifetimes of DiLiFP561, DiLiFP570 and DiLiFP571 were determined as 3.7, 3.6 and 3.0 ns. We determined a crystal structure of DiLiFP570 at 1.63 Å resolution. The crystal structure of DiLiFP570 revealed that the chromophore has an extended π-conjugated structure similar to that of DsRed. Most of the amino acid residues surrounding the chromophore were common between DiLiFP570 and DiLiFP561, except M159 of DiLiFP570 (Lysine in DiLiFP561), which is located close to the chromophore hydroxyl group. Interestingly, a similar K-to-M substitution has been reported in a red-shifted variant of DsRed (mRFP1). It is a striking observation that the naturally evolved color-change variants are consistent with the mutation induced via protein engineering processes. The newly cloned proteins are promising as orange and red fluorescent markers for imaging with long fluorescence lifetime.

Dynamics of the energy transfer involved in a diarylethene-perylenebisimide dyad: comparison between the molecule and the nanoparticle level.

Photochromic materials are widely used to achieve fluorescence photoswitching. Understanding the energy transfer processes occurring in these systems would be an advantage for their use and better optimization of their properties. In this scope, we studied a diarylethene-perylenebisimide (DAE-PBI) dyad that presents a bright red emission and a large ON-OFF contrast, both in solution and in an aqueous suspension of nanoparticles (NPs). Using ultrafast transient absorption spectroscopy, the excited state dynamics was characterized for this dyad in THF solution and compared to its behavior in NPs state. An efficient energy transfer process between the PBI fluorophore and the DAE photochromic unit in its closed form was demonstrated, occurring in a few hundreds of femtoseconds.

Is orange carotenoid protein photoactivation a single-photon process?

In all published photoactivation mechanisms of orange carotenoid protein (OCP), absorption of a single photon by the orange dark state starts a cascade of red-shifted OCP ground-state intermediates that subsequently decay within hundreds of milliseconds, resulting in the formation of the final red form OCPR, which is the biologically active form that plays a key role in cyanobacteria photoprotection. A major challenge in deducing the photoactivation mechanism is to create a uniform description explaining both single-pulse excitation experiments, involving single-photon absorption, and continuous light irradiation experiments, where the red-shifted OCP intermediate species may undergo re-excitation. We thus investigated photoactivation of Synechocystis OCP using stationary irradiation light with a biologically relevant photon flux density coupled with nanosecond laser pulse excitation. The kinetics of photoactivation upon continuous and nanosecond pulse irradiation light show that the OCPR formation quantum yield increases with photon flux density; thus, a simple single-photon model cannot describe the data recorded for OCP in vitro. The results strongly suggest a consecutive absorption of two photons involving a red intermediate with ≈100 millisecond lifetime. This intermediate is required in the photoactivation mechanism and formation of the red active form OCPR.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

Oligomerization processes limit photoactivation and recovery of the orange carotenoid protein.

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection by quenching of the excess of light-harvested energy. The photoactivation mechanism remains elusive, in part due to absence of data pertaining to the timescales over which protein structural changes take place. It also remains unclear whether or not oligomerization of the dark-adapted and light-adapted OCP could play a role in the regulation of its energy-quenching activity. Here, we probed photoinduced structural changes in OCP by a combination of static and time-resolved X-ray scattering and steady-state and transient optical spectroscopy in the visible range. Our results suggest that oligomerization partakes in regulation of the OCP photocycle, with different oligomers slowing down the overall thermal recovery of the dark-adapted state of OCP. They furthermore reveal that upon non-photoproductive excitation a numbed state forms, which remains in a non-photoexcitable structural state for at least ≈0.5 µs after absorption of a first photon.

Unifying Perspective of the Ultrafast Photodynamics of Orange Carotenoid Proteins from Synechocystis: Peril of High-Power Excitation, Existence of Different S* States, and Influence of Tagging.

A substantial number of Orange Carotenoid Protein (OCP) studies have aimed to describe the evolution of singlet excited states leading to the formation of a photoactivated form, OCPR. The most recent one suggests that 3 ps-lived excited states are formed after the sub-100 fs decay of the initial S2 state. The S* state, which has the longest reported lifetime of a few to tens of picoseconds, is considered to be the precursor of the first red photoproduct P1. Here, we report the ultrafast photodynamics of the OCP from Synechocystis PCC 6803 carried out using visible-near infrared femtosecond time-resolved absorption spectroscopy as a function of the excitation pulse power and wavelength. We found that a carotenoid radical cation can form even at relatively low excitation power, obscuring the determination of photoactivation yields for P1. Moreover, the comparison of green (540 nm) and blue (470 nm) excitations revealed the existence of an hitherto uncharacterized excited state, denoted as S∼, living a few tens of picoseconds and formed only upon 470 nm excitation. Because neither the P1 quantum yield nor the photoactivation speed over hundreds of seconds vary under green and blue continuous irradiation, this S∼ species is unlikely to be involved in the photoactivation mechanism leading to OCPR. We also addressed the effect of His-tagging at the N- or C-termini on the excited-state photophysical properties. Differences in spectral signatures and lifetimes of the different excited states were observed at a variance with the usual assumption that His-tagging hardly influences protein dynamics and function. Altogether our results advocate for the careful consideration of the excitation power and His-tag position when comparing the photoactivation of different OCP variants and beg to revisit the notion that S* is the precursor of photoactivated OCPR.

Structure-function-dynamics relationships in the peculiar Planktothrix PCC7805 OCP1: Impact of his-tagging and carotenoid type.

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection. Here, we report on the functional, spectral and structural characteristics of the peculiar Planktothrix PCC7805 OCP (Plankto-OCP). We show that this OCP variant is characterized by higher photoactivation and recovery rates, and a stronger energy-quenching activity, compared to other OCP studied thus far. We characterize the effect of the functionalizing carotenoid and of his-tagging on these reactions, and identify the time scales on which these modifications affect photoactivation. The presence of a his-tag at the C-terminus has a large influence on photoactivation, thermal recovery and PBS-fluorescence quenching, and likewise for the nature of the carotenoid that additionally affects the yield and characteristics of excited states and the ns-s dynamics of photoactivated OCP. By solving the structures of Plankto-OCP in the ECN- and CAN-functionalized states, each in two closely-related crystal forms, we further unveil the molecular breathing motions that animate Plankto-OCP at the monomer and dimer levels. We finally discuss the structural changes that could explain the peculiar properties of Plankto-OCP.

Selective population of triplet excited states in heavy-atom-free BODIPY-C60 based molecular assemblies.

Photophysical studies on a BODIPY-fullerene-distyryl BODIPY triad (BDP-C60-DSBDP) and its reference dyads (BODIPY-fullerene; BDP-C60 and distyryl BODIPY-fullerene; DSBDP-C60) are presented herein. In the triad, the association of the two chromophore units linked by a fullerene moiety leads to strong near UV-Visible light absorption from 300 to 700 nm. The triplet-excited state was observed upon visible excitation in all these assemblies, and shown to be localized on the C60 or BODIPY moieties. Using quantitative nanosecond transient absorption, we provide a complete investigation on the lifetime and formation quantum yield of the triplet-excited state. In the BDP-C60 dyad, the triplet excited state of C60 (τ = 7 ± 1 µs) was obtained with a quantum yield of 40 ± 8%. For the DSBDP-C60 dyad and BDP-C60-DSBDP triad, a longer-lived triplet excited state with a lifetime of around 250 ± 20 µs centered on the DSBDP moiety was formed, with respective quantum yields of 37 ± 8 and 20 ± 4%. Triplet-triplet annihilation up-conversion is characterized in the BDP-C60 dyad and the bichromophoric triad in the presence of perylene and DSBDP-monomer as respective annihilators. The photo-induced formation of a long-lived 3DSBDP* in the triad coupled with panchromatic light absorption offers potential applications as a heavy-atom-free organic triplet photosensitizer.

Structural Information about the trans-to-cis Isomerization Mechanism of the Photoswitchable Fluorescent Protein rsEGFP2 Revealed by Multiscale Infrared Transient Absorption.

RsEGFP2 is a reversibly photoswitchable fluorescent protein used in super-resolved optical microscopies, which can be toggled between a fluorescent On state and a nonfluorescent Off state. Previous time-resolved ultraviolet-visible spectroscopic studies have shown that the Off-to-On photoactivation extends over the femto- to millisecond time scale and involves two picosecond lifetime excited states and four ground state intermediates, reflecting a trans-to-cis excited state isomerization, a millisecond deprotonation, and protein structural reorganizations. Femto- to millisecond time-resolved multiple-probe infrared spectroscopy (TRMPS-IR) can reveal structural aspects of intermediate species. Here we apply TRMPS-IR to rsEGFP2 and implement a Savitzky-Golay derivative analysis to correct for baseline drift. The results reveal that a subpicosecond twisted excited state precursor controls the trans-to-cis isomerization and the chromophore reaches its final position in the protein pocket within 100 ps. A new step with a time constant of 42 ns is reported and assigned to structural relaxation of the protein that occurs prior to the deprotonation of the chromophore on the millisecond time scale.

Multivariate Curve Resolution Slicing of Multiexponential Time-Resolved Spectroscopy Fluorescence Data.

Time-resolved fluorescence spectroscopy (TRFS), i.e., measurement of fluorescence decay curves for different excitation and/or emission wavelengths, provides specific and sensitive local information on molecules and on their environment. However, TRFS relies on multiexponential data fitting to derive fluorescence lifetimes from the measured decay curves and the time resolution of the technique is limited by the instrumental response function (IRF). We propose here a multivariate curve resolution (MCR) approach based on data slicing to perform tailored and fit-free analysis of multiexponential fluorescence decay curves. MCR slicing, taking as a basic framework the multivariate curve resolution-alternating least-squares (MCR-ALS) soft-modeling algorithm, relies on a hybrid bilinear/trilinear data decomposition. A key feature of the method is that it enables the recovery of individual components characterized by decay profiles that are only partially describable by monoexponential functions. For TRFS data, not only pure multiexponential tail information but also shorter time delay information can be decomposed, where the signal deviates from the ideal exponential behavior due to the limited time resolution. The accuracy of the proposed approach is validated by analyzing mixtures of three commercial dyes and characterizing the mixture composition, lifetimes, and associated contributions, even in situations where only ternary mixture samples are available. MCR slicing is also applied to the analysis of TRFS data obtained on a photoswitchable fluorescent protein (rsEGFP2). Three fluorescence lifetimes are extracted, along with the profile of the IRF, highlighting that decomposition of complex systems, for which individual isomers are characterized by different exponential decays, can also be achieved.

Photoactive Organic/Inorganic Hybrid Materials with Nanosegregated Donor-Acceptor Arrays.

The synthesis of the first mesogenic donor-acceptor polyoxometalate (POM)-based hybrid is herein described. The structural and electronic properties of the hybrid compound were evaluated through combination of small- and wide-angle X-ray scattering, optical microscopy, electrochemistry and photoluminescence. In the solid state, the compound behaves as a birefringent solid, displaying a lamellar organization in which double-layers of POMs and bis(thiophene)thienothiophene organic donors alternate regularly. Noticeably, the sub-unit organizations in the composite are similar to that observed for the individual POM and organic donor precursors. Photophysical studies show that in the hybrid, the fluorescence of the organic donor unit is considerably quenched both in solution and in the solid state, which is attributed to occurrence of intramolecular charge-separated state.

Control of the Photo-Isomerization Mechanism in 3H-Naphthopyrans to Prevent Formation of Unwanted Long-Lived Photoproducts.

In the photochromic reactions of 3H-naphthopyrans, two colored isomers TC (transoid-cis) and TT (transoid-trans) are formed. In terms of optimized photo-switchable materials, synthetic efforts are nowadays evolving toward developing 3H-naphthopyran derivatives that would not be able to photoproduce the long-living transoid-trans, TT, photoproduct. The substitution with a methoxy group at position 10 results in significant reduction of the TT isomer formation yield. The TC photophysics responsible for TT suppression were revealed here using a combination of multi-scale time resolved absorption UV-vis spectroscopy and ab initio calculations. The substitution changes the TC excited-state potential energy landscape, the bicycle-pedal isomerization path is favored over the rotation around a single double bond. The bicycle-pedal path is aborted in halfway to TT formation due to S1→S0 internal conversion populating back the TC species in the ground electronic state. This is validated by a shorter TC S1 state lifetime for methoxy derivative in comparison to that of the parent-unsubstituted compound (0.47 ± 0.05 ps vs. 0.87 ± 0.09 ps) in cyclohexane.

All Visible Light Switch Based on the Dimethyldihydropyrene Photochromic Core.

Two photoswitchable compounds that can operate under visible light irradiation are prepared and investigated using spectroscopic and computational studies. These all-visible systems are based on the dimethyldihydropyrene (DHP)/cyclophanediene (CPD) photochromic couple connected either to a bipyridine (bpy) unit or to a (tris(bpy)ruthenium(II)) complex through a pyridinium bridge. In these compounds, the DHP to CPD isomerization and the reverse CPD to DHP conversion can be triggered by illumination with red (>630 nm) and blue (460 nm) lights, respectively. The unambiguous and reversible response of these systems triggered by visible light make them potential candidates for biological purposes and electronic devices.

Mechanistic Insights into the Triplet Sensitized Photochromism of Diarylethenes.

Operating photoswitchable molecules repetitively and reliably is crucial for most of their applications, in particular in (opto)electronic devices, and related to reversibility and fatigue resistance, which both critically depend on the photoisomerization mechanism defined by the substitution pattern. Two diarylethene photoswitches bearing biacetyl triplet sensitizers either at the periphery or at the core were investigated using both stationary as well as transient UV/Vis absorption spectroscopy ranging from the femtosecond to the microsecond time scale. The diarylethene with two biacetyl moieties at the periphery is switching predominantly from the triplet excited state, giving rise to an enhanced fatigue resistance. In contrast, the diarylethene bearing one diketone at the photoreactive inner carbon atom cyclizes from the singlet excited state and shows significantly higher quantum yields for both cyclization and cycloreversion.

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.

Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the µs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.

Restriction of the conrotatory motion in photo-induced 6π electrocyclic reaction: formation of the excited state of the closed-ring isomer in the cyclization.

The electrocyclic reaction dynamics of a photochromic dithiazolylarylene derivative, 2,3-dithiazolylbenzothiophene (DTA) was investigated by using time-resolved transient absorption and fluorescence spectroscopies. The closed-ring isomer of DTA undergoes cycloreversion through the conical intersection mediating the potential energy surfaces of the excited and ground states, which is in agreement with the Woodward-Hoffmann rules for the electrocyclic reactions of 6π electron systems. On the other hand, a large portion of the open-ring isomer undergoes cyclization along the distinct reaction scheme, in which the cyclization takes place in the excited state manifold leading to the formation of the excited state of the closed-ring isomer. The suppression of the geometrical motion of DTA due to the intramolecular interaction could open a new efficient reaction pathway resulting in the formation of the electronically excited state of the product.

Photochromic Reaction by Red Light via Triplet Fusion Upconversion.

Red or near-infrared (NIR) light responsive molecules have received much attention for biological and material applications because potentially harmful UV light for materials and cells is not required for the photochemical reactions. Although some molecular designs for photochromic molecules to increase the photosensitivity to red or NIR light have been reported, the strategies are limited to the extension of π-conjugation length and the utilization of charge transfer transition or energy and electron transfers. Triplet fusion is an attractive tool to cause chemical reactions by converting low-energy excitation light to high-energy upconversion light. However, the efficient use of the high energy of upconversion light is difficult because almost all reported triplet fusion systems rely on reabsorption of upconversion light. Here, we demonstrated red-light-driven photochromism via the triplet fusion of a phenoxyl-imidazolyl radical complex, Pery-RPIC, that has a covalently bonded perylene as an annihilator unit. The femtosecond time-resolved absorption and fluorescence spectroscopy revealed that this photochromic reaction proceeds by the highly efficient singlet energy transfer from the annihilator unit to the photochromic unit. This strategy can be applied not only to the development of visible and NIR light responsive photochromic system but also to various photochemical reactions.

Cobalt(II) Pentaaza-Macrocyclic Schiff Base Complex as Catalyst for Light-Driven Hydrogen Evolution in Water: Electrochemical Generation and Theoretical Investigation of the One-Electron Reduced Species.

We previously reported that the tetraazamacrocyclic Schiff base complex [CoIII(CR14)(X)2]n+ (CR14 = 2,12-dimethyl-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),2,11,13,15-pentaene, X = Cl (n = 1) (1-Cl2) or H2O (n = 3) (1-(H2O)2)) is a very efficient H2-evolving catalyst (HEC) in fully aqueous solutions at pH 4.0-4.5 when used in a photocatalytic system including a photosensitizer and ascorbate as sacrificial electron donor. The excellent H2-evolving activity of this complex, compared to other cobalt and rhodium catalysts studied in the same photocatalytic conditions, can be related to the high stability of its two-electron reduced form, the putative "Co(I)" state. These very interesting results led us to investigate the H2-evolving performances of a series of compounds from a close-related family, the pentaaza-macrocyclic cobalt [CoII(CR15)(H2O)2]Cl2 complex (2, CR15 = 2,13-dimethyl-3,6,9,12,18-pentaazabicyclo[12.3.1]octadeca-1(18),2,12,14,16-pentaene), which comprises a larger macrocycle with five nitrogen atoms instead of four. Electrochemical as well as spectroscopic investigations in CH3CN coupled to density functional theory (DFT) calculations point to decoordination of one of the amine upon reduction of Co(II) to the low-valent "Co(I)" form. The resulting unchelated amine could potentially act as a proton relay promoting the H2 formation via proton-coupled-electron transfer (PCET) reactions. Besides, the iron, manganese, and zinc analogues, [FeII(CR15)(X)2]n+ (X = Cl (n = 0) or H2O (n = 2)) (3), [MnII(CR15)(CH3CN)2](PF6)2 (4), and {[ZnII(CR15)Cl](PF6)}n (5) were also synthesized and investigated. The photocatalytic activity of 2-5 toward proton reduction was then evaluated in a tricomponent system containing the [RuII(bpy)3]Cl2 photosensitizer and ascorbate, in fully aqueous solution. The photocatalytic activity of 2 was also compared with that of 1 in the same experimental conditions. It was found that the number of catalytic cycles versus catalyst for 2 are slightly lower than that for 1, suggesting that if the amine released upon reduction of 2 plays a role in promoting the H2-evolving catalytic activity, other factors balance this effect. Finally, photophysical and nanosecond transient absorption spectroscopies were used to investigate the photocatalytic system.

Study of conformational transitions of i-motif DNA using time-resolved fluorescence and multivariate analysis methods.

Recently, the presence of i-motif structures at C-rich sequences in human cells and their regulatory functions have been demonstrated. Despite numerous steady-state studies on i-motif at neutral and slightly acidic pH, the number and nature of conformation of this biological structure are still controversial. In this work, the fluorescence lifetime of labelled molecular beacon i-motif-forming DNA sequences at different pH values is studied. The influence of the nature of bases at the lateral loops and the presence of a Watson-Crick-stabilized hairpin are studied by means of time-correlated single-photon counting technique. This allows characterizing the existence of several conformers for which the fluorophore has lifetimes ranging from picosecond to nanosecond. The information on the existence of different i-motif structures at different pH values has been obtained by the combination of classical global decay fitting of fluorescence traces, which provides lifetimes associated with the events defined by the decay of each sequence and multivariate analysis, such as principal component analysis or multivariate curve resolution based on alternating least squares. Multivariate analysis, which is seldom used for this kind of data, was crucial to explore similarities and differences of behaviour amongst the different DNA sequences and to model the presence and identity of the conformations involved in the pH range of interest. The results point that, for i-motif, the intrachain contact formation and its dissociation show lifetimes ten times faster than for the open form of DNA sequences. They also highlight that the presence of more than one i-motif species for certain DNA sequences according to the length of the sequence and the composition of the bases in the lateral loop.