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Excited-state lifetime measurements of fluorescent proteins a Excited-state lifetime of rsEGFP as a function of the maximum transmission wavelength (linearly proportional to the cavity length) of the nanocavity. Solid and open circles are data that were measured at maximum fluorescence signal and at 50% of maximum signal, respectively. The fit parameters are the fluorescence quantum yield (Φ) and the free lifetime in the absence of the cavity (τ). See Tables 1 and 2 for more details. b Fluorescence decay curves of rsFastLime at maximum fluorescence signal (blue curve) and at its 10% (red curve).
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One of the key photophysical properties of fluorescent proteins that is most difficult to measure is the quantum yield. It describes how efficiently a fluorophore converts absorbed light into fluorescence. Its measurement using conventional methods become particularly problematic when it is unknown how many of the proposedly fluorescent molecules o...
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Plasmonic nanoparticles (e.g gold, silver) have attracted extensive attentions in biological sensing and imaging as promising nanoprobes. Practical biomedical applications demand small gold nanoparticles with comparable size to quantum dots and fluorescent proteins. While too small nanoparticles with size below Rayleigh limit (usually <30-40 nm) we...
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... The quantum yield is one of the essential properties of a fluorescent compound [31], [32]. It is usually made sense by the relationship between the number of photons absorbed and the number of photons emitted [32], [33]. ...
Thanks to their chemical, thermal, photophysical, and photochemical properties, phenanthroimidazole derivative compounds attract attention in optoelectronic device technology, especially in OLEDs. For this reason, phenanthroimidazole derivatives are the most promising compounds for these applications in the future. In this study, the-diCl bearing phenanthroimidazole compound was synthesized, characterized, and investigated in detail the photophysical and aggregation properties. Spectroscopic studies for its characterization used 1 H-NMR, ATR-IR, and detailed LCMS-MS (mass and product ion scan). The absorption and emission spectra of diCl-PHN have been recorded with solvents of different polarities at room temperature. diCl-PHN exhibit high fluorescence quantum yield in DMSO with 0.78. CIE chromaticity diagram results of diCl-PHN were obtained and displayed good intensity. Additionally, aggregation properties were recorded at seven different water concentrations (water/DMSO %). With the results obtained from the study, it was concluded that this phenanthroimidazole derivative compound is a suitable molecule for fluorescent applications. Keywords: diCl bearing phenanthroimidazole, blue fluorescence, quantum yield, solvent effect-chromaticity, aggregation effects.
... The above results show that the nanocavity-based method allows for an easy sample preparation and assembly, accurate control of the distance between the mirrors, and precise modeling of the fluorophores radiative rate modulation. The method can be applied not only to various complex systems where conventional methods fail, 28,29,47,48 but can be used at the single molecule level. 49 As the excitation intensity-tuned lifetime has been recently used for enhancing spatial resolution in fluorescence imaging, 50 the nanocavity-based method could potentially help to measure single particle quantum yield directly within the biological sample. ...
One of the key phenomena that determine the fluorescence of nanocrystals is the nonradiative Auger-Meitner recombination of excitons. This nonradiative rate affects the nanocrystals' fluorescence intensity, excited state lifetime, and quantum yield. Whereas most of the above properties can be directly measured, the quantum yield is the most difficult to assess. Here we place semiconductor nanocrystals inside a tunable plasmonic nanocavity with subwavelength spacing and modulate their radiative de-excitation rate by changing the cavity size. This allows us to determine absolute values of their fluorescence quantum yield under specific excitation conditions. Moreover, as expected considering the enhanced Auger-Meitner rate for higher multiple excited states, increasing the excitation rate reduces the quantum yield of the nanocrystals.
... The other method relies on comparing the bright state QY (QY bright ) of the VFP to the ensembleaveraged QY obtained in conventional approaches. 20,21 Both methods show that all four VFPs contain a considerable fraction of dark proteins. For SYFP2 in the tandem construct, we find a larger f dark than for the method looking at the nontandem SYFP2. ...
... The obtained fluorescence lifetimes agree well with the data found in the literature. 15,20,28,37 To determine f dark , we start with TCCD to directly observe the fluorescence of individual tandems of VFPs. In a tandem of VFP A and VFP B, both A and B can be either emitting (bright) or non-emitting (dark). ...
Genetically encoded visible fluorescent proteins (VFPs) are a key tool used to visualize cellular processes. However, compared to synthetic fluorophores, VFPs are photophysically complex. This photophysical complexity includes the presence of non-emitting, dark proteins within the ensemble of VFPs. Quantitative fluorescence microcopy approaches that rely on VFPs to obtain molecular insights are hampered by the presence of these dark proteins. To account for the presence of dark proteins, it is necessary to know the fraction of dark proteins (fdark) in the ensemble. To date, fdark has rarely been quantified, and different methods to determine fdark have not been compared. Here, we use and compare two different methods to determine the fdark of four commonly used VFPs: EGFP, SYFP2, mStrawberry, and mRFP1. In the first, direct method, we make use of VFP tandems and single-molecule two-color coincidence detection (TCCD). The second method relies on comparing the bright state fluorescence quantum yield obtained by photonic manipulation to the ensemble-averaged fluorescence quantum yield of the VFP. Our results show that, although very different in nature, both methods are suitable to obtain fdark. Both methods show that all four VFPs contain a considerable fraction of dark proteins. We determine fdark values between 30 and 60% for the different VFPs. The high values for fdark of these commonly used VFPs highlight that fdark has to be accounted for in quantitative microscopy and spectroscopy.
... We consider GFPD as signaling molecules, as GFPD molecules can be reversibly switched and read out by fluorescence, which is required for media modulation according to Section II. The GFPD specific parameter values are taken from [13], [20]- [24]. The parameter values related to the duct are chosen such that they have the same order of magnitude as those of the human cardiovascular system [25,Chap. ...
In conventional molecular communication (MC) systems, the signaling molecules used for information transmission are stored, released, and then replenished by a transmitter (TX). However, the replenishment of signaling molecules at the TX is challenging in practice. Furthermore, in most envisioned MC applications, e.g., in the medical field, it is not desirable to insert the TX into the MC system, as this might impair natural biological processes. In this paper, we propose the concept of media modulation based MC where the TX is placed outside the channel and utilizes signaling molecules already present inside the system. The signaling molecules can assume different states which can be switched by external stimuli. Hence, in media modulation based MC, the TX modulates information into the state of the signaling molecules. In particular, we exploit the group of photochromic molecules, which undergo light-induced reversible state transitions, for media modulation. We study the usage of these molecules for information transmission in a three-dimensional duct system, which contains an eraser, a TX, and a receiver for erasing, writing, and reading of information via external light, respectively. We develop a statistical model for the received signal. We adopt a maximum likelihood detector and show that it can be reduced to a threshold based detector. Furthermore, we derive analytical expressions for the optimal threshold value and the resulting bit error rate (BER), respectively. Finally, we investigate the impact of various system parameters on the BER of media modulation based MC. Both the statistical model and BER results are verified by computer simulations. Our results reveal that media modulation enables reliable information transmission, validating it as a promising alternative to MC based on molecule emitting TXs.
... 16,52 On a practical note, dark state conversion, which is distinct from saturation of the optical transition in a 2level system, can lead to inaccuracies in measurements of fluorescence quantum yield, as shown by Ruhlandt and coworkers for photoswitching FPs. 53 Partially in response to this complication, Prangsma and co-workers developed methods to accurately determine the fluorescence quantum yield by tuning the local photon density of states near a metal surface. 13 Looking beyond the influences on brightness, investigations of dark-state conversion and engineering of fluorescence blinking are valuable for probe development in super-resolution microscopy. ...
Fluorescent proteins (FPs) have transformed cell biology through their use in fluorescence microscopy, enabling precise labeling of proteins via genetic fusion. A key advancement is altering primary sequences to customize their photophysical properties for specific imaging needs. A particularly notable family of engineered mutants is constituted by Reversible Switching Fluorescent Proteins (RSFPs), i.e. variant whose optical properties can be toggled between a bright and a dark state, thereby adding a further dimension to microscopy imaging. RSFPs have strongly contributed to the super-resolution (nanoscopy) revolution of optical imaging that has occurred in the last 20 years and afforded new knowledge of cell biochemistry at the nanoscale. Beyond high-resolution applications, the flexibility of RSFPs has been exploited to apply these proteins to other non-conventional imaging schemes such as photochromic fluorescence resonance energy transfer (FRET). In this work, we explore the origins and development of photochromic behaviors in FPs and examine the intricate relationships between structure and photoswitching ability. We also discuss a simple mathematical model that accounts for the observed photoswitching kinetics. Although we review most RSFPs developed over the past two decades, our main goal is to provide a clear understanding of key switching phenotypes and their molecular bases. Indeed, comprehension of photoswitching phenotypes is crucial for selecting the right protein for specific applications, or to further engineer the existing ones. To complete this picture, we highlight in some detail the exciting applications of RSFPs, particularly in the field of super-resolution microscopy.
Recent years have seen dramatic improvements in the design of organic fluorophores based on limiting non-radiative decay pathways. We sought to extend this understanding to benzothiadiazoles that have been used as turn-on fluorescent substrates for the self-labeling protein HaloTag. When conjugated to HaloTag, the benzothiadiazoles reside in a narrow tunnel that precludes twisted internal charge transfer, which allowed us to explore steric and electronic effects on other non-radiative decay pathways. By minimizing both non-radiative decay and nonspecific interactions with cellular components, we produced improved turn-on dyes with 136-fold increase in fluorescence over background in cells.
Fluorogenic dyes enable highly sensitive, real-time fluorescent readouts in live cells. Recent applications of fluorogenic dyes with self-labeling enzymes has expanded their applications, allowing high spatial and temporal resolution. However, improving fluorogenic self-labeling enzyme systems has largely focused on synthetically modifying the substrate dye and not the self-labeling enzyme. Here, we use molecular evolution to optimize the self-labeling enzyme HaloTag7 for improved performance with a designed fluorogenic substrate. Screening over 250 million variants, we isolated the optimized enzyme BenzoHTag which shows low background and rapid turn-on fluorescence in live mammalian cells.
Non-radiative traps and structures are present on the graphene quantum dots doped with nitrogen and functionalized with amino groups (amino-N-GQDs) and multiple crystalline layers because of cross-link-enhanced emission. Secondary and tertiary amines, which are potential fluorophores, have also been observed on the polyethylenimine (PEI) coating of amino-N-GQDs. Cross-linked PEI coating reduced rotation and vibration, thereby enhancing the photoluminescence quantum yield (PL QY). Introduced nitrogen atoms from N dopants, amino-functionalized groups and PEI, as well as sulfur from polystyrene sulfonate (PSS) enhanced the cooperative effect on the properties of heteroatom-doped materials among electrons captured by new surface states. This enhanced radiative recombination and subsequently enhanced PL QY, therefore, surface conjugation improved the amino-N-GQD surfaces by increasing the quantum confinement of their emissive energy, evidenced by the increased PL QY of amino-N-GQD-PSS-PEI (or amino-N-GQD-polymer composites). In some situations, the maximum available power required for delivery to the two-photon imaging plane without damaging tissues limits imaging depth but the additional brightness provided by amino-N-GQD-polymer composites in this study extended the maximum imaging depth to 240 μm. Amino-N-GQD-polymer composites had favorable two-photon properties under two-photon excitation (self-developed femtosecond Ti–sapphire laser optical system; power: 23.93 nJ pixel−1, 160 scans, approximately 1.09 s of total exposure time; excitation wavelength: 980 nm, near-infrared-II region), indicating that cells treated with amino-N-GQD-polymer composites and the anti-epidermal growth factor receptor antibody can achieve two-photon luminescence with 1/81 of the power required for similar-intensity two-photon autofluorescence (1938.33 nJ pixel−1 with 800 scans, total exposure time of ∼5.44 s). The materials can serve as contrast agents for the non-invasive detection of biological specimens and interior tissues using a two-photon excitation wavelength in the near-infrared region.
Much effort is being employed for designing "green" environmental emissive materials that are capable of color-tuning, i.e., down-converting the emission, and white-light generation (WLG). Here, we introduce a protein-based elastomer that can noncovalently bind a variety of chromophores while preventing their aggregation. Such binding capabilities are unique to the albumin-based materials that we use here in a process we refer to as "molecular doping". In the first part of this study, we explore the energy transfer across five different chromophores within the protein matrix, where the closely packed chromophore organization enables high energy-transfer efficiencies among them. In the second part, we show the easy control of blue, green, and red chromophores within the biopolymer, resulting in tunable emission properties of the film and WLG. The highly affordable chosen protein and the straightforward molecular doping strategy make our protein elastomers an attractive choice for an emissive material, as either a scaffold for investigating energy transfer in proteins or possible integration in light-emitting applications.
