Ultrafast Dynamics and Photoresponse of a Fungi-Derived Pigment Xylindein from Solution to Thin Films.

Organic semiconductor materials have recently gained momentum due to their non-toxicity, low cost, and sustainability. Xylindein is a remarkably photostable pigment secreted by fungi that grow on decaying wood, and its relatively strong electronic performance is enabled by π-π stacking and hydrogen-bonding network that promote charge transport. Herein, femtosecond transient absorption spectroscopy with a near-IR probe was used to unveil a rapid excited-state intramolecular proton transfer reaction. Conformational motions potentially lead to a conical intersection that quenches fluorescence in the monomeric state. In concentrated solutions, nascent aggregates exhibit a faster excited state lifetime due to excimer formation, confirmed by the excimer→charge-transfer excited-state absorption band of the xylindein thin film, thus limiting its optoelectronic performance. Therefore, extending the xylindein sidechains with branched alkyl groups may hinder the excimer formation and improve optoelectronic properties of naturally derived materials.

Shedding light on ultrafast ring-twisting pathways of halogenated GFP chromophores from the excited to ground state.

Since green fluorescent protein (GFP) has revolutionized molecular and cellular biology for about three decades, there has been a keen interest in understanding, designing, and controlling the fluorescence properties of GFP chromophore (i.e., HBDI) derivatives from the protein matrix to solution. Amongst these cross-disciplinary efforts, the elucidation of excited-state dynamics of HBDI derivatives holds the key to correlating the light-induced processes and fluorescence quantum yield (FQY). Herein, we implement steady-state electronic spectroscopy, femtosecond transient absorption (fs-TA), femtosecond stimulated Raman spectroscopy (FSRS), and quantum calculations to study a series of mono- and dihalogenated HBDI derivatives (X = F, Cl, Br, 2F, 2Cl, and 2Br) in basic aqueous solution, gaining new insights into the photophysical reaction coordinates. In the excited state, the halogenated "floppy" chromophores exhibit an anti-heavy atom effect, reflected by strong correlations between FQY vs. Franck-Condon energy (EFC) or Stokes shift, and knrvs. EFC, as well as a swift bifurcation into the I-ring (major) and P-ring (minor) twisting motions. In the ground state, both ring-twisting motions become more susceptible to sterics and exhibit spectral signatures from the halogen-dependent hot ground-state absorption band decay in TA data. We envision this type of systematic analysis of the halogenated HBDI derivatives to provide guiding principles for the site-specific modification of GFP chromophores, and expand design space for brighter and potentially photoswitchable organic chemical probes in aqueous solution with discernible spectral signatures throughout the photocycle.

Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca2+ Biosensor.

Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca2+ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca2+-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca2+ switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor's functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca2+ imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (ΔR/R0) of ~300%, outperforming many FRET- and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths.

An Engineered Biliverdin-Compatible Cyanobacteriochrome Enables a Unique Ultrafast Reversible Photoswitching Pathway.

Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward Pfr → Po transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse Po → Pfr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm-1. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances.

Time-Resolved Changes in Dielectric Constant of Metal Halide Perovskites under Illumination.

Despite their impressive performance as a solar absorber, much remains unknown on the fundamental properties of metal halide perovskites (MHPs). Their polar nature in particular is an intense area of study, and the relative permittivity (εr) is a parameter widely used to quantify polarization over a range of different time scales. In this report, we have exploited frequency-dependent time-resolved microwave conductivity (TRMC) to study how εr values of a range of MHPs change as a function of time, upon optical illumination. Further characterization of charge carriers and polarizability are conducted by femtosecond transient absorption and stimulated Raman spectroscopy. We find that changes in εr are roughly proportional to photogenerated carrier density but decay with a shorter time constant than conductance, suggesting that the presence of charge carriers alone does not determine polarization.

Ultrafast excited-state proton transfer dynamics in dihalogenated non-fluorescent and fluorescent GFP chromophores.

Green fluorescent protein (GFP) has enabled a myriad of bioimaging advances due to its photophysical and photochemical properties. To deepen the mechanistic understanding of such light-induced processes, novel derivatives of GFP chromophore p-HBDI were engineered by fluorination or bromination of the phenolic moiety into superphotoacids, which efficiently undergo excited-state proton transfer (ESPT) in aqueous solution within the short lifetime of the excited state, as opposed to p-HBDI where efficient ESPT is not observed. In addition, we tuned the excited-state lifetime from picoseconds to nanoseconds by conformational locking of the p-HBDI backbone, essentially transforming the nonfluorescent chromophores into highly fluorescent ones. The unlocked superphotoacids undergo a barrierless ESPT without much solvent activity, whereas the locked counterparts exhibit two distinct solvent-involved ESPT pathways. Comparative analysis of femtosecond transient absorption spectra of these unlocked and locked superphotoacids reveals that the ESPT rates adopt an "inverted" kinetic behavior as the thermodynamic driving force increases upon locking the backbone. Further experimental and theoretical investigations are expected to shed more light on the interplay between the modified electronic structure (mainly by dihalogenation) and nuclear motions (by conformational locking) of the functionalized GFP derivatives (e.g., fluorescence on and off).

Dual Illumination Enhances Transformation of an Engineered Green-to-Red Photoconvertible Fluorescent Protein.

The molecular mechanisms for the photoconversion of fluorescent proteins remain elusive owing to the challenges of monitoring chromophore structural dynamics during the light-induced processes. We implemented time-resolved electronic and stimulated Raman spectroscopies to reveal two hidden species of an engineered ancestral GFP-like protein LEA, involving semi-trapped protonated and trapped deprotonated chromophores en route to photoconversion in pH 7.9 buffer. A new dual-illumination approach was examined, using 400 and 505 nm light simultaneously to achieve faster conversion and higher color contrast. Substitution of UV irradiation with visible light benefits bioimaging, while the spectral benchmark of a trapped chromophore with characteristic ring twisting and bridge-H bending motions enables rational design of functional proteins. With the improved H-bonding network and structural motions, the photoexcited chromophore could increase the photoswitching-aided photoconversion while reducing trapped species.

Reversible Insertion of Mg-Cl Superhalides in Graphite as a Cathode for Aqueous Dual-Ion Batteries.

Oxidative anion insertion into graphite in an aqueous environment represents a significant challenge in the construction of aqueous dual-ion batteries. In dilute aqueous electrolytes, the oxygen evolution reaction (OER) dominates the anodic current before anions can be inserted into the graphite gallery. Herein, we report that the reversible insertion of Mg-Cl superhalides in graphite delivers a record-high reversible capacity of 150 mAh g-1 from an aqueous deep eutectic solvent comprising magnesium chloride and choline chloride. The insertion of Mg-Cl superhalides in graphite does not form staged graphite intercalation compounds; instead, the insertion of Mg-Cl superhalides makes the graphite partially turbostratic.

Delayed vibrational modulation of the solvated GFP chromophore into a conical intersection.

Green fluorescent protein (GFP) has revolutionized bioimaging and life sciences. Its successes have inspired modification of the chromophore structure and environment to tune emission properties, but outside the protein cage, the chromophore is essentially non-fluorescent. In this study, we employ the tunable femtosecond stimulated Raman spectroscopy (FSRS) and transient absorption (TA) to map the energy dissipation pathways of GFP model chromophore (HBDI) in basic aqueous solution. Strategic tuning of the Raman pump to 550 nm exploits the stimulated emission band to enhance excited state vibrational motions as HBDI navigates the non-equilibrium potential energy landscape to pass through a conical intersection. The time-resolved FSRS uncovers prominent anharmonic couplings between a global out-of-plane bending mode of ∼227 cm-1 and two modes at ∼866 and 1572 cm-1 before HBDI reaches the twisted intramolecular charge transfer (TICT) state on the ∼3 ps time scale. Remarkably, the wavelet transform analysis reveals a ∼500 fs delayed onset of the coupling peaks, in correlation with the emergence of an intermediate charge-separated state en route to the TICT state. This mechanism is corroborated by the altered coupling matrix for the HBDI Raman modes in the 50% (v/v) water-glycerol mixture, and a notable lengthening of the picosecond time constant. The real-time molecular "movie" of the general rotor-like HBDI isomerization reaction following photoexcitation represents a significant advance in comprehending the photochemical reaction pathways of the solvated GFP chromophore, therefore providing a crucial foundation to enable rational design of diverse nanomachines from efficient molecular rotors to bright fluorescent probes.

A Dual Plating Battery with the Iodine/[ZnIx (OH2 )4-x ]2-x Cathode.

Plating battery electrodes typically deliver higher specific capacity values than insertion or conversion electrodes because the ion charge carriers represent the sole electrode active mass, and a host electrode is unnecessary. However, reversible plating electrodes are rare for electronically insulating nonmetals. Now, a highly reversible iodine plating cathode is presented that operates on the redox couples of I2 /[ZnIx (OH2 )4-x ]2-x in a water-in-salt electrolyte. The iodine plating cathode with the theoretical capacity of 211 mAh g-1 plates on carbon fiber paper as the current collector, delivering a large areal capacity of 4 mAh cm-2 . Tunable femtosecond stimulated Raman spectroscopy coupled with DFT calculations elucidate a series of [ZnIx (OH2 )4-x ]2-x superhalide ions serving as iodide vehicles in the electrolyte, which eliminates most free iodide ions, thus preventing the consequent dissolution of the cathode-plated iodine as triiodides.

Photoinduced proton transfer inside an engineered green fluorescent protein: a stepwise-concerted-hybrid reaction.

Photoactivated proton transfer (PT) wire is responsible for the glow of green fluorescent protein (GFP), which is crucial for bioimaging and biomedicine. In this work, a new GFP-S65T/S205V double mutant is developed from wild-type GFP in which the PT wire is significantly modified. We implement femtosecond transient absorption (fs-TA) and femtosecond stimulated Raman spectroscopy (FSRS) to delineate the PT process in action. The excited state proton transfer proceeds on the ∼110 ps timescale, which infers that the distance of one key link (water to T203) in the PT wire of GFP-S205V is shortened by the extra S65T mutation. The rise of an imidazolinone ring deformation mode at ∼871 cm-1 in FSRS further suggests that this PT reaction is in a concerted manner. A ∼4 ps component prior to large-scale proton dissociation through the PT wire is also retrieved, indicative of some small-scale proton motions and heavy-atom rearrangement in the vicinity of the chromophore. Our work provides deep insights into the novel hybrid PT mechanism in engineered GFP and demonstrates the power of tunable FSRS methodology in tracking ultrafast photoreactions with the desirable structural specificity in physiological environments.

Excited State Structural Evolution of a GFP Single-Site Mutant Tracked by Tunable Femtosecond-Stimulated Raman Spectroscopy.

Tracking vibrational motions during a photochemical or photophysical process has gained momentum, due to its sensitivity to the progression of reaction and change of environment. In this work, we implemented an advanced ultrafast vibrational technique, femtosecond-stimulated Raman spectroscopy (FSRS), to monitor the excited state structural evolution of an engineered green fluorescent protein (GFP) single-site mutant S205V. This mutation alters the original excited state proton transfer (ESPT) chain. By strategically tuning the Raman pump to different wavelengths (i.e., 801, 539, and 504 nm) to achieve pre-resonance with transient excited state electronic bands, the characteristic Raman modes of the excited protonated (A*) chromophore species and intermediate deprotonated (I*) species can be selectively monitored. The inhomogeneous distribution/population of A* species go through ESPT with a similar ~300 ps time constant, confirming that bridging a water molecule to protein residue T203 in the ESPT chain is the rate-limiting step. Some A* species undergo vibrational cooling through high-frequency motions on the ~190 ps time scale. At early times, a portion of the largely protonated A* species could also undergo vibrational cooling or return to the ground state with a ~80 ps time constant. On the photoproduct side, a ~1330 cm-1 delocalized motion is observed, with dispersive line shapes in both the Stokes and anti-Stokes FSRS with a pre-resonance Raman pump, which indicates strong vibronic coupling, as the mode could facilitate the I* species to reach a relatively stable state (e.g., the main fluorescent state) after conversion from A*. Our findings disentangle the contributions of various vibrational motions active during the ESPT reaction, and offer new structural dynamics insights into the fluorescence mechanisms of engineered GFPs and other analogous autofluorescent proteins.

Tuning calcium biosensors with a single-site mutation: structural dynamics insights from femtosecond Raman spectroscopy.

Fluorescent protein biosensors are popular reporters for biological processes and life sciences, but their fundamental working mechanisms remain unclear. To characterize the functional fluorescence events on their native timescales, we implemented wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) to shed light on a blue-green emission-ratiometric fluorescent protein based Ca2+ biosensor with a single Pro377Arg mutation. The transient Raman modes of the embedded chromophore from ca. 1000-1650 cm-1 exhibit characteristic intensity and frequency dynamics which infer the underlying atomic motions and photochemical reaction stages. Our experimental study reveals the hidden structural inhomogeneity of the protein local environment upon Ca2+ binding with the mutated arginine residue trapping multiple chromophore subpopulations, which manifest distinct time constants of ∼16 and 90 ps for excited state proton transfer (ESPT) following 400 nm photoexcitation. The altered ESPT reaction pathways and emission properties of the Ca2+ biosensor represent the foundational step of rationally designing advanced fluorescent protein biosensors to tune their functionalities by site-specifically altering the local environment (e.g., the active site) of the embedded chromophore.

Dynamic Raman Line Shapes on an Evolving Excited-State Landscape: Insights from Tunable Femtosecond Stimulated Raman Spectroscopy.

Tracking molecular motions in real time remains a formidable challenge in science and engineering fields because the experimental methodology requires simultaneously high spatial and temporal resolutions. Building on early successes and future potential of femtosecond stimulated Raman spectroscopy (FSRS) as a structural dynamics technique, we present a comprehensive study of stimulated Raman line shapes of a photosensitive molecule in solution with tunable Raman pump and probe pulses. Following femtosecond 400 nm electronic excitation, the model photoacid pyranine exhibits dynamic and mode-dependent Raman line shapes when the Raman pump is tuned from the red side toward and across the excited-state absorption (ESA) band (e.g., from S1) with varying resonance conditions. On the anti-Stokes FSRS side, low-frequency modes below ∼1000 cm-1 exhibit a line shape change from gain to dispersive to loss, whereas the dispersive intermediate is much less notable for high-frequency modes. The characteristic mode frequency blue shift involving vibrationally hot states in S1 with time constants of ∼9.6 and 58.6 ps reveals the sensitivity of anti-Stokes FSRS to vibrational cooling and solvation. This work lays the foundation for expanding tunable FSRS technology on both the Stokes and anti-Stokes sides to investigate a variety of photoinduced processes in solution with sufficient resolution to expose functional motions and increased sensitivity to monitor vibrational cooling.

Cascaded four-wave mixing for broadband tunable laser sideband generation.

We demonstrate the versatile broadband wavelength tunability of frequency upconverted multicolor cascaded four-wave-mixing (CFWM) signals spanning the continuous wavelength range from UV to near IR in a thin type-I BBO crystal using 35 fs, 800 nm fundamental and chirped IR supercontinuum white light pulses. Two sets of spatially dispersed CFWM laser sidebands are concomitantly generated from two incident pulses as well as their second-harmonic-generation and sum-frequency-generation pulses in a crossing geometry. The tunable cascaded signals with ultrabroad bandwidth can be readily achieved via spatially rotating the BBO crystal to different phase-matching conditions and temporally varying the time delay between the two incident near-IR pulses.

To twist or not to twist: From chromophore structure to dynamics inside engineered photoconvertible and photoswitchable fluorescent proteins.

Green-to-red photoconvertible fluorescent proteins (FPs) are vital biomimetic tools for powerful techniques such as super-resolution imaging. A unique Kaede-type FP named the least evolved ancestor (LEA) enables delineation of the evolutionary step to acquire photoconversion capability from the ancestral green fluorescent protein (GFP). A key residue, Ala69, was identified through several steady-state and time-resolved spectroscopic techniques that allows LEA to effectively photoswitch and enhance the green-to-red photoconversion. However, the inner workings of this functional protein have remained elusive due to practical challenges of capturing the photoexcited chromophore motions in real time. Here, we implemented femtosecond stimulated Raman spectroscopy and transient absorption on LEA-A69T, aided by relevant crystal structures and control FPs, revealing that Thr69 promotes a stronger π-π stacking interaction between the chromophore phenolate (P-)ring and His193 in FP mutants that cannot photoconvert or photoswitch. Characteristic time constants of ~60-67 ps are attributed to P-ring twist as the onset for photoswitching in LEA (major) and LEA-A69T (minor) with photoconversion capability, different from ~16/29 ps in correlation with the Gln62/His62 side-chain twist in ALL-GFP/ALL-Q62H, indicative of the light-induced conformational relaxation preferences in various local environments. A minor subpopulation of LEA-A69T capable of positive photoswitching was revealed by time-resolved electronic spectroscopies with targeted light irradiation wavelengths. The unveiled chromophore structure and dynamics inside engineered FPs in an aqueous buffer solution can be generalized to improve other green-to-red photoconvertible FPs from the bottom up for deeper biophysics with molecular biology insights and powerful bioimaging advances.

Capturing excited-state structural snapshots of evolutionary green-to-red photochromic fluorescent proteins.

Photochromic fluorescent proteins (FPs) have proved to be indispensable luminous probes for sophisticated and advanced bioimaging techniques. Among them, an interplay between photoswitching and photoconversion has only been observed in a limited subset of Kaede-like FPs that show potential for discovering the key mechanistic steps during green-to-red photoconversion. Various spectroscopic techniques including femtosecond stimulated Raman spectroscopy (FSRS), X-ray crystallography, and femtosecond transient absorption were employed on a set of five related FPs with varying photoconversion and photoswitching efficiencies. A 3-methyl-histidine chromophore derivative, incorporated through amber suppression using orthogonal aminoacyl tRNA synthetase/tRNA pairs, displays more dynamic photoswitching but greatly reduced photoconversion versus the least-evolved ancestor (LEA). Excitation-dependent measurements of the green anionic chromophore reveal that the varying photoswitching efficiencies arise from both the initial transient dynamics of the bright cis state and the final trans-like photoswitched off state, with an exocyclic bridge H-rocking motion playing an active role during the excited-state energy dissipation. This investigation establishes a close-knit feedback loop between spectroscopic characterization and protein engineering, which may be especially beneficial to develop more versatile FPs with targeted mutations and enhanced functionalities, such as photoconvertible FPs that also feature photoswitching properties.

Observation of sum-frequency-generation-induced cascaded four-wave mixing using two crossing femtosecond laser pulses in a 0.1 mm beta-barium-borate crystal.

We demonstrate the simultaneous generation of multicolor femtosecond laser pulses spanning the wavelength range from UV to near IR in a 0.1 mm Type I beta-barium borate crystal from 800 nm fundamental and weak IR super-continuum white light (SCWL) pulses. The multicolor broadband laser pulses observed are attributed to two concomitant cascaded four-wave mixing (CFWM) processes as corroborated by calculation: (1) directly from the two incident laser pulses; (2) by the sum-frequency generation (SFG) induced CFWM process (SFGFWM). The latter signal arises from the interaction between the frequency-doubled fundamental pulse (400 nm) and the SFG pulse generated in between the fundamental and IR-SCWL pulses. The versatility and simplicity of this spatially dispersed multicolor self-compressed laser pulse generation offer compact and attractive methods to conduct femtosecond stimulated Raman spectroscopy and time-resolved multicolor spectroscopy.

Transient electronic and vibrational signatures during reversible photoswitching of a cyanobacteriochrome photoreceptor.

Cyanobacteriochromes (CBCRs) are an emerging class of photoreceptors that are distant relatives of the phytochromes family. Unlike phytochromes, CBCRs have gained popularity in optogenetics due to their highly diverse spectral properties spanning the UV to near-IR region and only needing a single compact binding domain. AnPixJg2 is a CBCR that can reversibly photoswitch between its red-absorbing (15ZPr) and green-absorbing (15EPg) forms of the phycocyanobilin (PCB) cofactor. To reveal primary events of photoconversion, we implemented femtosecond transient absorption spectroscopy with a homemade LED box and a miniature peristaltic pump flow cell to track transient electronic responses of the photoexcited AnPixJg2 on molecular time scales. The 525 nm laser-induced Pg-to-Pr reverse conversion exhibits a ~3 ps excited-state lifetime before reaching the conical intersection (CI) and undergoing further relaxation on the 30 ps time scale to generate a long-lived Lumi-G ground state intermediate en route to Pr. The 650 nm laser-induced Pr-to-Pg forward conversion is less efficient than reverse conversion, showing a longer-lived excited state which requires two steps with ~13 and 217 ps time constants to enter the CI region. Furthermore, using a tunable ps Raman pump with broadband Raman probe on both the Stokes and anti-Stokes sides, we collected the pre-resonance ground-state femtosecond stimulated Raman spectroscopy (GS-FSRS) data with mode assignments aided by quantum calculations. Key vibrational marker bands at ~850, 1050, 1615, and 1649 cm-1 of the Pr conformer exhibit a notable blueshift to those of the Pg conformer inside AnPixJg2, reflecting the PCB chromophore terminal D (major) and A (minor) ring twist along the primary photoswitching reaction coordinate. This integrated ultrafast spectroscopy and computational platform has the potential to elucidate photochemistry and photophysics of more CBCRs and photoactive proteins in general, providing the highly desirable mechanistic insights to facilitate the rational design of functional molecular sensors and devices.

Excitation ratiometric chloride sensing in a standalone yellow fluorescent protein is powered by the interplay between proton transfer and conformational reorganization.

Natural and laboratory-guided evolution has created a rich diversity of fluorescent protein (FP)-based sensors for chloride (Cl-). To date, such sensors have been limited to the Aequorea victoria green fluorescent protein (avGFP) family, and fusions with other FPs have unlocked ratiometric imaging applications. Recently, we identified the yellow fluorescent protein from jellyfish Phialidium sp. (phiYFP) as a fluorescent turn-on, self-ratiometric Cl- sensor. To elucidate its working mechanism as a rare example of a single FP with this capability, we tracked the excited-state dynamics of phiYFP using femtosecond transient absorption (fs-TA) spectroscopy and target analysis. The photoexcited neutral chromophore undergoes bifurcated pathways with the twisting-motion-induced nonradiative decay and barrierless excited-state proton transfer. The latter pathway yields a weakly fluorescent anionic intermediate , followed by the formation of a red-shifted fluorescent state that enables the ratiometric response on the tens of picoseconds timescale. The redshift results from the optimized π-π stacking between chromophore Y66 and nearby Y203, an ultrafast molecular event. The anion binding leads to an increase of the chromophore pK a and ESPT population, and the hindrance of conversion. The interplay between these two effects determines the turn-on fluorescence response to halides such as Cl- but turn-off response to other anions such as nitrate as governed by different binding affinities. These deep mechanistic insights lay the foundation for guiding the targeted engineering of phiYFP and its derivatives for ratiometric imaging of cellular chloride with high selectivity.