Fluorescence On-off Switching Research Articles

A General Strategy for the Construction of NIR-emitting Si-rhodamines and Their Application for Mitochondrial Temperature Visualization.

Si-rhodamine (SiR) is an ideal fluorophore because it possesses bright emission in the NIR region and can be implemented flexibly in living cells. Currently, several promising approaches for synthesizing SiR are being developed. However, challenges remain in the construction of SiR containing functional groups for bioimaging application. Herein, we introduce a general and simple approach by a condensation reaction of diarylsilylether and arylaldehyde in o-dichlorobenzene to synthesize a series of SiRs bearing various functional substituents. These SiRs have moderate to high quantum efficiency, tolerance to photobleaching, and high water solubility as well as NIR emitting, and their NIR fluorescence properties can be controlled through the photoinduced electron transfer (PET) mechanism. Fluorescence OFF-ON switching effect is observed for SiR 9 in the presence of acid, which is rationalized by DFT/TDDFT calculations. Moreover, reversible stimuli response toward temperature is achieved. Since positive charge enables mitochondrial targeting ability and chloromethyl unit can covalently immobilize the dyes onto the mitochondrial via click reaction between the benzyl choride and protein sulfhydryls, SiR 8 is identified as a valuable fluorescent marker to visualize the morphology and monitor the temperature change of mitochondria with high photostability.

(Invited) Carbon Nano Onions for Biomedical Applications

Carbon-based nanomaterials have emerged as platforms for biomedical applications because of their low toxicity and ability to be internalized by cells [1]. The development of imaging probes and drug delivery devices based on carbon nanomaterials for biomedical studies requires the understanding of their biological response as well as the efficient and safety exposition of the nanomaterial to the cell compartment where it is designed to operate.Multi-shell fullerenes, also known as carbon nano-onions (CNOs) are structured by concentric shells of carbon atoms and display several unique properties, such as a large surface area to volume ratio, low density and a graphitic multilayer morphology [2]. In my research group we have developed a versatile and robust approach for the functionalisation of CNOs, involving the facile introduction of a number of simple functionalities onto their surface. Our results have shown that chemical functionalization of the CNOs dramatically enhance their solubility and reduce their inflammatory properties in vitro and in vivo [3]. We reported the absence of adverse effects induced by CNOs on short and long term toxicity in Hydra [4] and in zebrafish [5] suggesting a reasonable degree of biosafety of this new class of nanomaterials. We have developed intracellular imaging systems based on CNOs functionalised with BODIPY derivatives [6], with special attention to the biologically important near-infra red (NIR) region [7] and pH dependent fluorescence on-off switching [8]. The on/off emission of the fluorescent CNOs is fast and reversible both in solution and in vitro, making this nanomaterial suitable as pH-dependent probes for diagnostic applications [8].We have also developed a synthetic multi-functionalisation strategy for the introduction of different functionalities (receptor targeting unit and imaging unit) onto the surface of the CNOs The modified CNOs display high brightness and photostability in aqueous solutions and their selective and rapid uptake in two different cancer cell lines without significant cytotoxicity is demonstrated. The localization of the functionalized CNOs in late-endosomes cell compartments is revealed by a correlative approach with confocal and transmission electron microscopy [9].To probe the possible applications of CNOs as a platform for therapeutic and diagnostic interventions on CNS diseases, we injected fluorescent CNOs in vivo in mice hippocampus. We analyzed ex vivo their diffusion within brain tissues and their cellular localization by confocal and electron microscopy. The subsequent fluorescent staining of hippocampal cells populations indicates they efficiently internalize the nanoparticles. Furthermore, the inflammatory potential of the CNOs injection was found comparable to sterile vehicle infusion, and it did not result in manifest neurophysiological and behavioral alterations of hippocampal-mediated functions [10]. These results encourage further development as brain disease-targeted diagnostics or therapeutics nanocarriers.

Understanding Structural Changes through Excited-State Intramolecular Proton Transfer in 4'-N,N-Diethylamino-3-hydroxyflavone (DEAHF) in Solution Based on Quantum Chemical Calculations.

To design novel dyes with a controllable fluorescence on-off switching mechanism, understanding the dark state at the atomic level should be a key focus. In this study, we focused on the radiative and nonradiative mechanisms of 4'-N,N-diethylamino-3-hydroxyflavone (DEAHF) based on theoretical and experimental viewpoints. In the excited state, an excited-state intramolecular proton transfer (ESIPT) reaction of DEAHF occurs, and both the normal (N*) and tautomer (T*) forms exist in solution. To discuss the electronic structure changes through ESIPT, we mainly focused on two structural changes: the rotation of the diethylamino group and the bending motion of DEAHF in the excited state. The potential energy surfaces (PESs) passing through the rotation of the diethylamino group indicated that rotation may occur by thermal fluctuation during each phase. When the diethylamino group is rotated by 90° in the N* form, the oscillator strength becomes zero, which may be critical in nonradiative decay pathways. For the bending motion, we found a conical intersection, which could be a key pathway of nonradiative decay. By employing molecular orbital analysis, we concluded that the electronic structure changes induced by ESIPT play a key role in determining the decay pathways. Additionally, we compared the fluorescence quantum yield in acetonitrile with that in cyclohexane and showed that the solvent polarity also affects the radiative and nonradiative mechanisms of DEAHF.

Fluorescence ON-OFF switching, Boolean logic gates like behavior of carbon quantum dots and highly sensitive bovine serum albumin sensing

We demonstrate fluorescence “ON-OFF” switching and photonic logic gates based on fluorescence response of carbon quantum dots (CQDs) in the presence of graphene oxide (GO), reduced graphene oxide (RGO), and bovine serum albumin (BSA). We study the excited state electron and energy transfer interactions among the carbon based materials in detail through steady state fluorescence (SSF) and time resolved fluorescence (TRF) spectroscopy. CQDs function as donor; GO and RGO function as acceptors. SSF results show the fluorescence “turn-OFF” behavior of CQDs in the presence of GO and RGO, and the reason is explained through Stern-Volmer plots. TRF results reveal a decrease in the decay time components of CQDs in the presence of GO and RGO. The gradual recovery of quenched fluorescence of CQDs is observed by the addition of BSA at nanomolar concentrations which shows the highly sensitive “turn-ON” BSA sensing. Single input and two inputs photonic logic gates are implemented based on the fluorescence response of CQDs in the presence of GO, RGO, and BSA in different input combinations. The not, pass1, nor, and implication gates like behavior of CQDs is demonstrated.

Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems

The theoretically unlimited spatial resolution of fluorescence nanoscopy often comes at the expense of time, contrast and increased dose of energy for recording. Here, we developed MoNaLISA, for Molecular Nanoscale Live Imaging with Sectioning Ability, a nanoscope capable of imaging structures at a scale of 45–65 nm within the entire cell volume at low light intensities (W-kW cm−2). Our approach, based on reversibly switchable fluorescent proteins, features three distinctly modulated illumination patterns crafted and combined to gain fluorescence ON–OFF switching cycles and image contrast. By maximizing the detected photon flux, MoNaLISA enables prolonged (40–50 frames) and large (50 × 50 µm2) recordings at 0.3–1.3 Hz with enhanced optical sectioning ability. We demonstrate the general use of our approach by 4D imaging of organelles and fine structures in epithelial human cells, colonies of mouse embryonic stem cells, brain cells, and organotypic tissues.

Sensitive and Selective Fluorescent Probe for Selenol in Living Cells Designed via a p Ka Shift Strategy.

Selenocysteine (Sec) is a primary kind of reactive selenium species in cells, and its vital roles in physiological processes have been featured. Thus, the development of highly sensitive and selective methods for the sensing of Sec is of great significance. This work reports a turn-on fluorescent probe for selenol based on the unique fluorescence OFF-ON switching between the Schiff base (SB) and its complementary protonated Schiff base (PSB) form of merocyanine dyes. The probe consists of a merocyanine Schiff base fluorophore and a 2,4-dinitrobenzenesulfonamide moiety that reacts especially with selenol. The fluorescence turn-on response of MC-Sec is realized via the selective removal of the strongly electron withdrawing 2,4-dinitrobenzenesulfonyl group by Sec, leading to a shift in the p Ka of the imine nitrogen of the probe from 6.40 to 9.04 and thus significantly increasing the population of the fluorescent PSB form of the dye at physiological pH. MC-Sec shows good selectivity and sensitivity for Sec and has been applied in the imaging of exogenous and endogenous selenol in living cells by confocal fluorescence microscopy. The proposed mechanism should be useful for developing future probes directed to other target molecules by employing this simple but effective p Ka shift strategy.

Spectrally Resolved and Functional Super-resolution Microscopy via Ultrahigh-Throughput Single-Molecule Spectroscopy.

As an elegant integration of the spatial and temporal dimensions of single-molecule fluorescence, single-molecule localization microscopy (SMLM) overcomes the diffraction-limited resolution barrier of optical microscopy by localizing single molecules that stochastically switch between fluorescent and dark states over time. While this type of super-resolution microscopy (SRM) technique readily achieves remarkable spatial resolutions of ∼10 nm, it typically provides no spectral information. Meanwhile, current scanning-based single-location approaches for mapping the positions and spectra of single molecules are limited by low throughput and are difficult to apply to densely labeled (bio)samples. In this Account, we summarize the rationale, design, and results of our recent efforts toward the integration of the spectral dimension of single-molecule fluorescence with SMLM to achieve spectrally resolved SMLM (SR-SMLM) and functional SRM ( f-SRM). By developing a wide-field scheme for spectral measurement and implementing single-molecule fluorescence on-off switching typical of SMLM, we first showed that in densely labeled (bio)samples it is possible to record the fluorescence spectra and positions of millions of single molecules synchronously within minutes, giving rise to ultrahigh-throughput single-molecule spectroscopy and SR-SMLM. This allowed us to first show statistically that for many dyes, single molecules of the same species exhibit near identical emission in fixed cells. This narrow distribution of emission wavelengths, which contrasts markedly with previous results at solid surfaces, allowed us to unambiguously identify single molecules of spectrally similar dyes. Crosstalk-free, multiplexed SRM was thus achieved for four dyes that were merely 10 nm apart in emission spectrum, with the three-dimensional SRM images of all four dyes being automatically aligned within one image channel. The ability to incorporate single-molecule fluorescence measurement with SMLM was next utilized to achieve f-SRM. By encoding functional information into the spectral responses of environment-sensing fluorescent probes, f-SRM transcends the structural information provided by typical SRM techniques and reveals the spatiotemporal distribution of physicochemical parameters with single-molecule sensitivity and nanoscale spatial resolution. As one example, by employing the solvatochromic dye Nile Red to sense local chemical polarity, we revealed nanoscale heterogeneity in the membranes of live mammalian cells. This enabled us to unveil substantial polarity differences between the plasma membrane and the membranes of nanoscale intracellular organelles, a result we determined to be due to differences in local cholesterol levels. With the addition of cholesterol or cholera toxin, we further observed the formation of low-polarity, raftlike nanodomains in the plasma membrane. In another study, we generalized SR-SMLM to fluorogenic single-molecule reactions. As a wide-field technique, SR-SMLM readily captures the emission spectra of individual product fluorescent molecules that are stochastically produced from nonfluorescent reactants at random locations over large sample areas, and therefore, it provides the unique possibility to spectrally identify and characterize single product molecules in a high-throughput fashion. Using the ring-opening reaction of a photochromic spiropyran as an example, we demonstrated that the capability to resolve the emission spectra of single product molecules could unveil rich, multipath reaction pathways. In summary, by integrating the spatial, temporal, and spectral dimensions of single-molecule fluorescence, SR-SMLM and f-SRM add rich spectral and functional dimensions to SRM and thus open up new ways of probing biological and chemical systems at the single-molecule and nanoscale levels.

Superior Fluorogen-Activating Protein Probes Based on 3-Indole-Malachite Green.

A series of novel fluorogenic dyes based on 3-indole-Malachite Green, MGs 5-7, have been developed that are dark in solution but highly fluorescent when bound to the cognate reporter, fluorogen-activating protein (FAP). Significantly, the new MGs 5-7 probes are superior to the traditional MG 1 with high fluorescent efficiency and low toxicity to cells while maintaining the large "pseudo-Stokes" shifts (Δλ = λex - λem) and the malachite green (MG)-like fluorescence OFF-ON switching mechanism in both live mammalian cells and bacterial cells when bound to FAP.

Cascade OFF–ON–OFF fluorescent probe: Dual detection of Fe3+ ions and thiols

Abstract A new novel Rhodamine B-based fluorescent probe was synthesized for the selective cascade signaling of Fe 3+ and thiols (glutathione, homo-cysteine, cysteine). Non-fluorescent rhodamine derivatives can selectively detect Fe 3+ over some other metal ions in ethanol-water (4/1, v/v, pH 7.0) media, leading to prominent fluorescence OFF–ON switching. The obtained LDF-Fe 3+ ensemble can subsequently serve as a sensitive and selective chemosensor for thiols, exhibiting complete signal quenching (fluorescence ON–OFF switching). The practical use of its Fe 3+ ions ensemble was demonstrated by its application to the detection of thiols in the living cells.

A cyanine-based colorimetric and fluorescent probe for highly selective sensing and bioimaging of phosphate ions

This paper reports a near-infrared chemosensor for phosphate ions (Pi) based on Pi-controlled fluorescence off-on switching mechanism. The Pi sensor displayed colorimetric responses towards Pi with a distinct color change from green to yellow in aqueous media. Apyrase, a hydrolytic enzyme, was used to accelerate the endogenous Pi production for evaluating in vivo fluorescent sensing ability of the new cyanine based probe (1). In addition, the present study demonstrated potential of the newly developed chemosensor in bioimaging by performing experiments in Chinese hamster ovary (CHO) cells both in vitro and in vivo.

An efficient quinoline-based fluorescence sensor for zinc(II) and its application in live-cell imaging

A novel and efficient quinoline-based fluorescent chemosensor (QLSA) for zinc(II) ion (Zn2+) detection was reported. This sensor was facilely synthesized via a simple linker NH2–NH2 connected quinoline-2-carboxylic acid and salicylaldehyde. This non-fluorescent quinoline derivative can detect Zn2+ with high sensitivity and favorable specificity and lead to significant fluorescence OFF–ON switching. The detection limit for Zn2+ was found to be 2.1×10−8M. The mechanism of the interaction between the sensor and Zn2+ has been investigated in detail through NMR and HRMS analysis. The sensor exhibited low cytotoxicity and larger Stokes shifts (171nm) which are important and desired features for a fluorophore in live-cell imaging. Moreover, further application of the sensor in fluorescence imaging was studied in Hela cell by confocal fluorescence microscopy, suggesting that the sensor was proved to be a useful tool for tracking Zn2+ levels in vivo.

Fluorescence ON–OFF switching using micelle of stimuli-responsive double hydrophilic block copolymers: Nile Red fluorescence in micelles of poly(acrylic acid-b-N-isopropylacrylamide)

The dual-mode fluorescence ON–OFF switching of Nile Red (NR) by using stimuli-responsive polymeric micelle of poly(acrylic acid-b-N-isopropylacrylamide) (PAA-b-PNIPAM) has been studied. PAA-b-PNIPAM, one of double hydrophilic block copolymers, is known to form PNIPAM-core/PAA-corona micelles in aqueous solutions when the temperature of the solution is elevated up to the lower critical solution temperature (LCST) of PNIPAM block. It also forms PAA-core/PNIPAM-corona micelles when the anionic PAA block is charge-neutralized with cationic cetyltrimethylammonium ion. Fluorescence properties of NR in the micelles are elucidated by observing various fluorescence parameters such as intensity, polarization, and quantum yield. It is found that the fluorescence intensity is negligibly low (OFF-state) when PAA-b-PNIPAM exists as a form of unimer, whereas it is remarkably enhanced (ON-state) when the PNIPAM-core or PAA-core micelles are formed. These results demonstrate that a novel fluorescence ON–OFF switching system can be constructed by using PAA-b-PNIPAM micelles and NR.

Easily accessible ferric ion chemosensor based on rhodamine derivative and its reversible OFF–ON fluorescence response

An easily accessible ferric ion (Fe3+) fluorescence chemosensor was designed and synthesized via microwave reaction based on rhodamine derivative. This sensor displayed a favorable selectivity for Fe3+ ion over a range of other common metal cations in acetonitrile/Tris buffer (4:1, v/v, pH 7.0) with a 1:1 stoichiometry, leading to prominent fluorescence OFF–ON switching of the rhodamine fluorophore. The association constant between the sensor and Fe3+ was determined to be 1.51×105, and the corresponding detection limit was calculated to be 3.2×10−7 M according to fluorescence titration analysis. The recognizing behavior of the chemosensor toward Fe3+ has been investigated in detail by FTIR, NMR, and HRMS measurements. Additionally, the recognition process of the chemosensor for Fe3+ is chemically reversible on the addition of phosphate anion (PO43−).

Cascade sensitive and selective fluorescence OFF–ON–OFF sensor for Cr3+ cation and F− anion

Cascade recognition of trivalent chromium cation (Cr3+) and fluoride anion (F−) via rhodamine-urea methacrylate-based fluorescent probe (RhBUA) and its subsequent cationic complex was reported. The non-fluorescent rhodamine derivatives can selectively detect Cr3+ cation over some other metal ions in CH3CN–H2O (90/10, v/v, pH 7.0) solutions with a 1:1 stoichiometry, leading to prominent fluorescence OFF–ON switching. The obtained RhBUA–Cr3+ complex can subsequently serve as a sensitive and selective chemosensor for F− ion. Complete signal quenching (fluorescence ON–OFF switching) can also be observed, ascribing to the extraction of Cr3+ cation and the resulting deformation of RhBUA–Cr3+ complex.

Modification of Fluorescent Photoinduced Electron Transfer (PET) Sensors/Switches To Produce Molecular Photo-Ionic Triode Action

The fluorophore-spacer1-receptor1-spacer2-receptor2 system (where receptor2 alone is photoredox-inactive) shows ionically tunable proton-induced fluorescence off-on switching, which is reminiscent of thermionic triode behavior. This also represents a new extension to modular switch systems based on photoinduced electron transfer (PET) towards the emulation of analogue electronic devices.

A Stimuli‐Responsive Nanogel‐Based Sensitive and Selective Fluorescent Sensor for Cr3+ with Thermo‐Induced Tunable Detection Sensitivity

Stimuli-responsive poly(N-isopropylacrylamide) nanogel with covalently labeled rhodamine B urea derivatives (P(NIPAM-co-RhBUA)) is utilized as a sensitive fluorescent probe for Cr(3+) in aqueous solution, and its thermo-induced tunable detection capacity is investigated. At 20 °C, non-fluorescent nanogel can selectively bind with Cr(3+) over some other metal ions, leading to prominent fluorescence OFF-ON switching due to the recognition of RhBUA with Cr(3+) . Upon heating above the phase transition temperature, enhanced fluorescence intensity is observed (≈61-fold increase at 45 °C) for the nanogel in the presence of Cr(3+) , accompanied with an improved detection sensitivity, which suggest that hydrophobic microenvironment generated in the collapsed nanogel plays an active role for their detection performance.

Analogs of Changsha near-infrared dyes with large Stokes Shifts for bioimaging

The construction of fluorescent imaging probes has contributed significantly to the recent advances in biology and medicine. Near infrared (NIR) fluorescent probes are favorable to be employed in fluorescence imaging in living animals. Rhodamine dyes have been widely used as a robust platform for development of fluorescent probes for a wide variety of targets. However, the absorption and emission wavelengths of classical rhodamine derivatives are below 600 nm. Thus, it is desirable to construct rhodamine analogs with longer absorption and emission wavelengths, preferably in the NIR region. Toward this end, our group has previously constructed Changsha (CS) NIR dyes while retaining the rhodamine-like fluorescence ON–OFF switching mechanism. However, like classic rhodamines, these rhodamine NIR derivatives still have small Stokes shifts (typically less than 35 nm), which can lead to serious self-quenching and fluorescence detection error due to excitation backscattering effects. This shortcoming may constrain the full potential of their applications. Thus, there is a need to develop rhodamine NIR derivatives with large Stokes shifts. In this work, we have designed and synthesized a class of analogs of CS NIR dyes with large Stokes shifts. Among the new dyes presented herein, the dye 1c displays a high fluorescence quantum yield in biological media and thus promising for in vivo imaging applications. Furthermore, using 1c as a platform, we further constructed the NIR fluorescent turn-on probe 3, which is suitable for imaging endogenously produced HClO in the RAW264.7 macrophage cells, demonstrating the value of our new NIR functional fluorescent dye 1c.

Piezochromism: Unique Piezochromic Fluorescence Behavior of Dicyanodistyrylbenzene Based Donor–Acceptor–Donor Triad: Mechanically Controlled Photo‐Induced Electron Transfer (eT) in Molecular Assemblies (Adv. Mater. 40/2012)

A donor–acceptor–donor triad, which exhibits fluorescence on-off switching with high contrast ratio (ca. 1000) in response to mechanical stimulus in the solid state, is reported by Soo Young Park and co-workers on page 5487. This system provides a very unique example of high contrast fluorescence switching that is driven by a mechanical force controlled photo-induced electron transfer (eT) in molecular assemblies.

Unique Piezochromic Fluorescence Behavior of Dicyanodistyrylbenzene Based Donor–Acceptor–Donor Triad: Mechanically Controlled Photo‐Induced Electron Transfer (eT) in Molecular Assemblies

A novel donor-acceptor-donor triad that exhibits fluorescence on-off switching with high contrast ratio (ca. 10(3) ) in response to a mechanical stimulus in the solid state is reported. This system provides a very unique example of high-contrast fluorescence switching that is driven by a mechanical-force-controlled photo-induced electron transfer (eT) in molecular assemblies.

A Unique Class of Near-Infrared Functional Fluorescent Dyes with Carboxylic-Acid-Modulated Fluorescence ON/OFF Switching: Rational Design, Synthesis, Optical Properties, Theoretical Calculations, and Applications for Fluorescence Imaging in Living Animals

Fluorescence imaging is one of the most powerful techniques for monitoring biomolecules in living systems. Fluorescent sensors with absorption and emission in the near-infrared (NIR) region are favorable for biological imaging applications in living animals, as NIR light leads to minimum photodamage, deep tissue penetration, and minimum background autofluorescence interference. Herein, we have introduced a new strategy to design NIR functional dyes with the carboxylic-acid-controlled fluorescence on-off switching mechanism by the spirocyclization. Based on the design strategy, we have developed a series of Changsha (CS1-6) NIR fluorophores, a unique new class of NIR functional fluorescent dyes, bearing excellent photophysical properties including large absorption extinction coefficients, high fluorescence quantum yields, high brightness, good photostability, and sufficient chemical stability. Significantly, the new CS1-6 NIR dyes are superior to the traditional rhodamine dyes with both absorption and emission in the NIR region while retaining the rhodamine-like fluorescence ON-OFF switching mechanism. In addition, we have performed quantum chemical calculations with the B3LYP exchange functional employing 6-31G* basis sets to shed light on the structure-optical properties of the new CS1-6 NIR dyes. Furthermore, using CS2 as a platform, we further constructed the novel NIR fluorescent TURN-ON sensor 7, which is capable of imaging endogenously produced HClO in the living animals, demonstrating the value of our new CS NIR functional fluorescent dyes. We expect that the design strategy may be extended for development of a wide variety of NIR functional dyes with a suitable fluorescence-controlled mechanism for many useful applications in biological studies.