A Single Fluorescent Protein-Based Indicator with a Time-Resolved Fluorescence Readout for Precise pH Measurements in the Alkaline Range.

The real-time monitoring of the intracellular pH in live cells with high precision represents an important methodological challenge. Although genetically encoded fluorescent indicators can be considered as a probe of choice for such measurements, they are hindered mostly by the inability to determine an absolute pH value and/or a narrow dynamic range of the signal, making them inefficient for recording the small pH changes that typically occur within cellular organelles. Here, we study the pH sensitivity of a green-fluorescence-protein (GFP)-based emitter (EGFP-Y145L/S205V) with the alkaline-shifted chromophore's pKa and demonstrate that, in the pH range of 7.5-9.0, its fluorescence lifetime changes by a factor of ~3.5 in a quasi-linear manner in mammalian cells. Considering the relatively strong lifetime response in a narrow pH range, we proposed the mitochondria, which are known to have a weakly alkaline milieu, as a target for live-cell pH measurements. Using fluorescence lifetime imaging microscopy (FLIM) to visualize the HEK293T cells expressing mitochondrially targeted EGFP-Y145L/S205V, we succeeded in determining the absolute pH value of the mitochondria and recorded the ETC-uncoupler-stimulated pH shift with a precision of 0.1 unit. We thus show that a single GFP with alkaline-shifted pKa can act as a high-precision indicator that can be used in a specific pH range.

Protein Mobility Measurements through Oxidative Green-to-Red Photoconversion of EGFP.

Protein dynamics plays a key role in live cell functioning, stimulating the development of new experimental techniques for studying protein transport phenomena. Here, we introduce a relaxation method that is based on the rapid formation of a nonequilibrium concentration profile of the enhanced green fluorescent protein (EGFP) across a sample by its oxidative green-to-red photoconversion. Following the blue-light irradiation of a part of a sample containing EGFP and an oxidant, the diffusion-controlled response of a system is monitored. Changes in the concentration of the initial green-emitting and oxidized red-emitting forms are simultaneously tracked by fluorescence lifetime measurements using the time-correlated single photon counting. We show that the diffusion coefficient of EGFP in water, determined by this method, is in good agreement with previously published data. This approach opens a way for the studies of intracellular viscosity changes combined with sensing of elevated levels of reactive oxygen species.

Nitrogen-Doped Carbon Nanodots Produced by Femtosecond Laser Synthesis for Effective Fluorophores.

Understanding the effect of heteroatom doping is crucial for the design of carbon nanodots (CNDs) with enhanced luminescent properties for fluorescence imaging and light-emitting devices. Here, we study the effect and mechanisms of luminescence enhancement through nitrogen doping in nanodots synthesized by the bottom-up route in an intense femtosecond laser field using the comparative analysis of CNDs obtained from benzene and pyridine. We demonstrate that laser irradiation of aromatic compounds produces hybrid nanoparticles consisting of a nanocrystalline core with a shell of surface-bonded aromatic rings. These nanoparticles exhibit excitation-dependent visible photoluminescence typical for CNDs. Incorporation of nitrogen into pyridine-derived CNDs enhances their luminescence characteristics through the formation of small pyridine-based fluorophores peripherally bonded to the nanoparticles. We identify oxidation of surface pyridine rings as a mechanism of formation of several distinct blue- and green-emitting fluorophores in nanodots, containing pyridine moieties. These findings shed additional light on the nature and formation mechanism of effective fluorophores in nitrogen-doped carbon nanodots produced by the bottom-up route.

Mechanochemistry for the synthesis of non-classical N-metalated palladium(II) pincer complexes.

The peculiarities of cyclopalladation of a series of non-classical pincer-type ligands based on monothiooxalyl amides bearing ancillary N- or S-donor groups in the amide units have been scrutinized both under conditions of conventional solution-based synthesis and in the absence of a solvent according to a solid-phase methodology including mechanochemical activation. Grinding the functionalized monothiooxamides with PdCl2(NCPh)2 in a mortar or vibration ball mill is shown to serve as an efficient and green alternative to the synthesis of these complex metal-organic systems in solution that can offer such advantages as the absence of any auxiliary and significant rate and yield enhancement, especially for the challenging ligands. The realization of S,N,N- or S,N,S-monoanionic tridentate coordination in the resulting pincer complexes has been confirmed by multinuclear NMR (including 2D NMR) and IR spectroscopy and, in some cases, X-ray diffraction. The course and outcome of the solid-phase reactions have been studied by a combination of different spectroscopic methods as well as SEM/EDS analysis. The preliminary evaluation of cytotoxic activity against several human cancer cell lines has revealed the high potency of some of the cyclopalladated derivatives obtained, rendering further development of solvent-free synthetic routes to this type of complexes very urgent.

Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants.

The bright ultimately short lifetime enhanced emitter (BrUSLEE) green fluorescent protein, which differs from the enhanced green fluorescent protein (EGFP) in three mutations, exhibits an extremely short fluorescence lifetime at a relatively high brightness. An important contribution to shortening the BrUSLEE fluorescence lifetime compared to EGFP is provided by the T65G substitution of chromophore-forming residue and the Y145M mutation touching the chromophore environment. Although the influence of the T65G mutation was studied previously, the role of the 145th position in determining the GFPs physicochemical characteristics remains unclear. In this work, we show that the Y145M substitution, both alone and in combination with the F165Y mutation, does not shorten the fluorescence lifetime of EGFP-derived mutants. Thus, the unlocking of Y145M as an important determinant of lifetime tuning is possible only cooperatively with mutations at position 65. We also show here that the introduction of a T65G substitution into EGFP causes complex photobehavior of the respective mutants in the lifetime domain, namely, the appearance of two fluorescent states with different lifetimes, preserved in any combination with the Y145M and F165Y substitutions.

Probing Intracellular Dynamics Using Fluorescent Carbon Dots Produced by Femtosecond Laser In Situ.

Fluorescent particle tracking is a powerful technique for studying intracellular transport and microrheological properties within living cells, which in most cases employs exogenous fluorescent tracer particles delivered into cells or fluorescent staining of cell organelles. Herein, we propose an alternative strategy, which is based on the generation of fluorescent species in situ with ultrashort laser pulses. Using mouse germinal vesicle oocytes as a model object, we demonstrate that femtosecond laser irradiation produces compact dense areas in the intracellular material containing fluorescent carbon dots synthesized from biological molecules. These dots have tunable persistent and excitation-dependent emission, which is highly advantageous for fluorescent imaging. We further show that tight focusing and tuning of irradiation parameters allow precise control of the location and size of fluorescently labeled areas and minimization of damage inflicted to cells. Pieces of the intracellular material down to the submicrometer size can be labeled with laser-produced fluorescent dots in real time and then employed as probes for detecting intracellular motion activity via fluorescent tracking. Analyzing their diffusion in the oocyte cytoplasm, we arrive to realistic characteristics of active forces generated within the cell and frequency-dependent shear modulus of the cytoplasm. We also quantitatively characterize the level of metabolic activity and density of the cytoskeleton meshwork. Our findings establish a new technique for probing intracellular mechanical properties and also promise applications in tracking individual cells in population or studies of spatiotemporal cell organization.

Plasmon-Enhanced Fluorescence of EGFP on Short-Range Ordered Ag Nanohole Arrays.

Fluorescence of organic molecules can be enhanced by plasmonic nanostructures through coupling to their locally amplified electromagnetic field, resulting in higher brightness and better photostability of fluorophores, which is particularly important for bioimaging applications involving fluorescent proteins as genetically encoded biomarkers. Here, we show that a hybrid bionanosystem comprised of a monolayer of Enhanced Green Fluorescent Protein (EGFP) covalently linked to optically thin Ag films with short-range ordered nanohole arrays can exhibit up to 6-fold increased brightness. The largest enhancement factor is observed for nanohole arrays with a propagating surface plasmon mode, tuned to overlap with both excitation and emission of EGFP. The fluorescence lifetime measurements in combination with FDTD simulations provide in-depth insight into the origin of the fluorescence enhancement, showing that the effect is due to the local amplification of the optical field near the edges of the nanoholes. Our results pave the way to improving the photophysical properties of hybrid bionanosystems based on fluorescent proteins at the interface with easily fabricated and tunable plasmonic nanostructures.

Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP.

Enhanced green fluorescent protein (EGFP)-one of the most widely applied genetically encoded fluorescent probes-carries the threonine-tyrosine-glycine (TYG) chromophore. EGFP efficiently undergoes green-to-red oxidative photoconversion ("redding") with electron acceptors. Enhanced yellow fluorescent protein (EYFP), a close EGFP homologue (five amino acid substitutions), has a glycine-tyrosine-glycine (GYG) chromophore and is much less susceptible to redding, requiring halide ions in addition to the oxidants. In this contribution we aim to clarify the role of the first chromophore-forming amino acid in photoinduced behavior of these fluorescent proteins. To that end, we compared photobleaching and redding kinetics of EGFP, EYFP, and their mutants with reciprocally substituted chromophore residues, EGFP-T65G and EYFP-G65T. Measurements showed that T65G mutation significantly increases EGFP photostability and inhibits its excited-state oxidation efficiency. Remarkably, while EYFP-G65T demonstrated highly increased spectral sensitivity to chloride, it is also able to undergo redding chloride-independently. Atomistic calculations reveal that the GYG chromophore has an increased flexibility, which facilitates radiationless relaxation leading to the reduced fluorescence quantum yield in the T65G mutant. The GYG chromophore also has larger oscillator strength as compared to TYG, which leads to a shorter radiative lifetime (i.e., a faster rate of fluorescence). The faster fluorescence rate partially compensates for the loss of quantum efficiency due to radiationless relaxation. The shorter excited-state lifetime of the GYG chromophore is responsible for its increased photostability and resistance to redding. In EYFP and EYFP-G65T, the chromophore is stabilized by π-stacking with Tyr203, which suppresses its twisting motions relative to EGFP.

Excited-state locked amino analogues of the green fluorescent protein chromophore with a giant Stokes shift.

We design a novel class of excited-state locked GFP chromophores by introducing an amine group at the double exo-bond and a difluoroboryl bridge. We show that these chromophores intrinsically exhibit a very large Stokes shift of 1 eV. Further tuning through chemical modifications of their aryl substituents makes them environmentally sensitive.