Dye Pair Research Articles

Saxs Confirms that FRET Dyes Promote Collapse of an Otherwise Fully Disordered Protein

Significance: The dimensions of intrinsically disordered proteins (IDPs) remains controversial. Using SAXS, we previously found that even relatively hydrophobic IDPs remain expanded in the complete absence of denaturant, contrary to conclusions drawn from FRET studies that foldable sequences collapse in water. Here we use our methodology to demonstrate that labeling with Alexa488 causes the IDP to undergo significant dye-induced contraction. These results demonstrate that the addition of dyes influences the properties of IDPs and is likely the primary origin of the discrepancy between prior SAXS and FRET results. ABSTRACT: The origins of the discrepancy between SAXS and FRET measurements of IDP dimensions remain controversial. To address this, we recently developed a robust analysis for extracting Rg and ν (the Flory exponent; Rg∼Nν) from single SAXS experiments using a molecular form factor derived from simulations. Performing this analysis on a 334 AA IDP, we find that the addition of a dye pair promotes collapse, decreasing Rg from 51 to 45 Å and ν from 0.54 to 0.50. This observation is consistent with prior studies on PEG, which found the addition of dyes induces similar contraction1 despite the fact that, as we show here, the polymer behaves as a self-avoiding random walk. Recent studies have suggested that FRET and SAXS results can be reconciled if SAXS reports on Rg while remaining relatively insensitive to Rend-to-end, essentially decoupling the two. To the contrary, our analysis finds that the entire SAXS profile is sensitive to conformational ensembles enriched in shortened end-to-end distances, such as those produced by dye-dye interactions. More broadly, our simulations define a route to characterize the deviations from homopolymeric behavior inherent in disordered proteins. 1Watkins et al, PNAS 2015.

Quantification of Single-Molecule FRET between Quantum Dots and Organic Dyes

Single-molecule Förster resonance energy transfer (FRET) is a useful technique for studying inter- and intra-molecular dynamics in biophysics. FRET efficiency is highly sensitive to distance, with half-maximal energy transfer occurring at an inter-dye distance on the order of 5 nm, which makes it possible to quantify distances changes on the molecular scale. FRET efficiency also depends on the spectral overlap and quantum yield of the donor and acceptor molecules, and therefore the choice of dye pair is critical to the success of any experiment. Most organic dyes that are used for single-molecule imaging have bleaching lifetimes in the tens of seconds at most, which limits their use to relatively fast processes. Quantum dot nanocrystals can emit for thousands of seconds without entering a long-lived dark state, which should make them good candidates for use as a FRET donor for longer single-molecule experiments, but there has been little quantitative study of the energy transfer efficiency at the single-molecule level. In this work, we use dsDNA to couple individual quantum dot donors to fluorescent dye acceptors. We then immobilize these FRET pairs on a functionalized glass coverslip and image them using total internal fluorescence (TIRF) microscopy with additional optics that allow us to image the donor and acceptor simultaneously. The length of the DNA between the pair can be varied from 11 to 32 base pairs, allowing us to observe and quantify the energy transfer efficiency over a range of distances on the nanometer scale. Our goal is to develop the use of quantum dots as donors for quantitative single-molecule FRET experiments.

Defining Dynamics of Membrane-Bound Pyrophosphatases by Experimental and Computational Single-Molecule FRET.

Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular animals, making them a viable target for drug design against protozoan parasites such as Plasmodium falciparum. An excellent understanding of many of the catalytic states throughout the enzymatic cycle has already been afforded by crystallography. However, the dynamics and kinetics of the catalytic cycle between these static snapshots remain to be elucidated. Here, we employ single-molecule Förster resonance energy transfer (FRET) measurements to determine the dynamic range and frequency of conformations available to the enzyme in a lipid bilayer during the catalytic cycle. First, we explore issues related to the introduction of fluorescent dyes by cysteine mutagenesis; we discuss the importance of residue selection for dye attachment, and the balance between mutating areas of the protein that will provide useful dynamics while not altering highly conserved residues that could disrupt protein function. To complement and guide the experiments, we used all-atom molecular dynamics simulations and computational methods to estimate FRET efficiency distributions for dye pairs at different sites in different protein conformational states. We present preliminary single-molecule FRET data that points to insights about the binding modes of different membrane-bound pyrophosphatase substrates and inhibitors.

Probing the dynamics of restriction endonuclease NgoMIV-DNA interaction by single-molecule FRET.

Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single-molecule Förster resonance energy transfer (smFRET) of surface-immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein-induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA-NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA-protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites ("slow" trajectories) or by semi-specific interactions of two DNA-bound NgoMIV tetramers ("fast" trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules.

Helicase Stepping Investigated with One-Nucleotide Resolution Fluorescence Resonance Energy Transfer.

Single-molecule Förster resonance energy transfer is widely applied to study helicases by detecting distance changes between a pair of dyes anchored to overhangs of a forked DNA. However, it has been lacking single-base pair (1-bp) resolution required for revealing stepping kinetics of helicases. We designed a nanotensioner in which a short DNA is bent to exert force on the overhangs, just as in optical or magnetic tweezers. The strategy improved the resolution of Förster resonance energy transfer to 0.5bp, high enough to uncover differences in DNA unwinding by yeast Pif1 and E. coli RecQ whose unwinding behaviors cannot be differentiated by currently practiced methods. We found that Pif1 exhibits 1-bp-stepping kinetics, while RecQ breaks 1bp at a time but sequesters the nascent nucleotides and releases them randomly. The high-resolution data allowed us to propose a three-parameter model to quantitatively interpret the apparently different unwinding behaviors of the two helicases which belong to two superfamilies.

Confined-Space Mechanism Inspired by the Ingenious Fabrication of a Förster Resonance Energy Transfer System as a Ratiometric Probe for Ag+ Recognition

In this work, a facile and interesting fabrication of the Forster resonance energy transfer (FRET) system is presented for the first time by introducing an independent donor and acceptor into a confined space, which is different from the typical “donor–linker–acceptor” FRET system. Human serum albumin (HSA), as a proof-of-concept, is selected to load a pair of spectra-matchable but independent fluorescent dyes (ES1 and R1) in its different binding sites. Furthermore, this space-confined FRET system could act as a ratiometric sensor for the quantitative recognition of Ag+ in the water samples with good selectivity and high sensitivity.

Stress Detection Using Polymer Optical Fiber with FRET Pair Dyes

Noble fiber-optic strain sensor using dye-doped polymer optical fiber (POF) was proposed, and its ability to sense stress (strain) by simple viewing of the large-core cross section was proposed. Conventional well-known fiber-optic strain sensors such as BOTDR or FBG types require spectral analysis to determine the strain value. Our sensor does not require expensive equipments such as spectral analyzer or well-tuned light source that are required in conventional fiber-optic strain sensors. A pair of luminescent organic dyes, Coumarin 540A and Rhodamine 6G, that functions as donor and acceptor of excitation energy were doped in core and cladding of the fiber, respectively. By taking this structure, stress applied to the fiber could be detected as an increase in cladding intensity. This happens because emission of core dye leaks to the cladding due to the deformation, and it excites the cladding dye. Using the fabricated POF, change in near field pattern upon different fiber bending diameter was detected. Lasers with three different wavelengths were used; namely, 406 nm, 532 nm, and 635 nm. 406 nm is the optimum wavelength for exciting the Coumarin 540A of the core efficiently. 532 nm excites Rhodamin 6G well, but does not excite Coumarin 540A well. 635 nm excites neither dyes. Pigtail end was coupled to the test fiber with direct contact to the core center of the test fiber in which no cladding excitation was expected. Fibers were stressed in various magnitudes by bending them in different loop diameters, namely, 1.5 cm, 3 cm, and 6 cm. Single loop was created in the middle point of the whole length. Relaxed condition, which the fiber was naturally straightened with no loop, was also investigated. Output beam from the test fiber was analyzed using Near Field Pattern measuring system (Hamamatsu C9664-01G02). In addition, dye-doped POF with lower numerical aperture had shown enhancement in sensitivity. Therefore, it was suggested that sensitivity of the sensor is controllable by further optimization of the waveguide parameters. Therefore, the proposed results suggests a potential for an enhanced fiber-optic stress detection with simpler and inexpensive accessory components.

Strain sensing based on radiative emission-absorption mechanism using dye-doped polymer optical fiber

A stress sensor based on a dye-doped polymeric optical fiber is able to detect stress by simple comparison of two luminescence peaks from a pair of energy transfer organic dyes. Coumarin 540A (donor) and Rhodamine 6G (acceptor) were doped in the core and cladding of the fiber, respectively. For various laser wavelengths, the change in the near-field pattern and visible emission spectrum upon variation in the fiber bending diameter was evaluated. From a comparison with a low-numerical-aperture fiber, it is shown that the sensitivity of the sensor is controllable by optimization of the waveguide parameters.

Spectral and Structural Comparisons on Four Pairs of Isomeric 4- and 2-Aminobenzoic Acid Based Aromatic Heterocyclic Dyes

Four pairs of isomeric aromatic heterocyclic dyes 1 – 8, synthesized from the diazotization and subsequent coupling reactions between 4 or 2-aminobenzoic acid as the diazo component and different N-substituted pyridone precursors as the coupling component, have been characterized and compared including UV–Vis and 1H NMR spectra, X-ray single-crystal structures and related DFT computations. It is noted that X-ray single-crystal diffraction analyses reveal that all of them adopt the same hydrazone-tautomeric form and planar molecular conformation between the pyridine and phenyl rings. However, different intramolecular and intermolecular hydrogen bonding interactions are observed because of the introduction of an electron-withdrawing carboxylic acid group at the ortho or para position in the phenyl ring. 4-Aminobenzoic acid and 2-aminobenzoic acid have been used as the diazo components to couple with different N-substituted pyridone precursors to produce four pairs of isomeric aromatic heterocyclic dyes, for which 1H NMR spectral, UV–Vis spectral and single-crystal structural comparisons have been made.

Complexes of DNA with fluorescent dyes are effective reagents for detection of autoimmune antibodies

To date, there are multiple assays developed that detect and quantify antibodies in biofluids. Nevertheless, there is still a lack of simple approaches that specifically detect autoimmune antibodies to double-stranded DNA. Herein we investigate the potential of novel nucleic acid complexes as targets for these antibodies. This is done in a simple, rapid and specific immunofluorescence assay. Specifically, employing 3D nanostructures (DNA origami), we present a new approach in the detection and study of human antibodies to DNA. We demonstrate the detection of anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We tested the most potent non-covalent pairs of DNA and fluorescent dyes. Several complexes showed specific recognition of autoimmune antibodies in human samples of lupus patients using a simple one-step immunofluorescence method. This makes the novel assay developed herein a promising tool for research and point-of-care monitoring of anti-DNA antibodies. Using this method, we for the first time experimentally confirm that the disease-specific autoimmune antibodies are sensitive to the 3D structure of nucleic acids and not only to the nucleotide sequence, as was previously thought.

Tracking the Energy Flow on Nanoscale via Sample-Transmitted Excitation Photoluminescence Spectroscopy.

Tracking the energy flow in nanoscale materials is an important yet challenging goal. Experimental methods for probing the intermolecular energy transfer (ET) are often burdened by the spectral crosstalk between donor and acceptor species, which complicates unraveling their individual contributions. This issue is particularly prominent in inorganic nanoparticles and biological macromolecules featuring broad absorbing profiles. Here, we demonstrate a general spectroscopic strategy for measuring the ET efficiency between nanostructured or molecular dyes exhibiting a significant donor-acceptor spectral overlap. The reported approach is enabled through spectral shaping of the broadband excitation light with solutions of donor molecules, which inhibits the excitation of respective donor species in the sample. The resulting changes in the acceptor emission induced by the spectral modulation of the excitation beam are then used to determine the quantum efficiency and the rate of ET processes between arbitrary fluorophores (molecules, nanoparticles, polymers) with high accuracy. The feasibility of the reported method was demonstrated using a control donor-acceptor system utilizing a protein-bridged Cy3-Cy5 dye pair and subsequently applied for studying the energy flow in a CdSe560-CdSe600 binary nanocrystal film.

Systematic study of the hybrid plasmonic-photonic band structure underlying lasing action of diffractive plasmon particle lattices

We study lasing in distributed feedback lasers made from square lattices of silver particles in a dye-doped waveguide. We present a systematic analysis and experimental study of the band structure underlying the lasing process as a function of the detuning between the particle plasmon resonance and the lattice Bragg diffraction condition. To this end, as gain medium we use either a polymer doped with Rh6G only, or polymer doped with a pair of dyes (Rh6G and Rh700) that act as a F\orster energy transfer (FRET) pair. This allows for gain, respectively, at 590 or 700 nm when pumped at 532 nm, compatible with the achievable size tunability of silver particles embedded in the polymer. By polarization-resolved spectroscopic Fourier microscopy, we are able to observe the plasmonic/photonic band structure of the array, unraveling both the stop gap width, as well as the loss properties of the four involved bands at fixed lattice Bragg diffraction condition and as a function of detuning of the plasmon resonance. To explain the measurements we derive an analytical model that sheds insights on the lasing process in plasmonic lattices, highlighting the interaction between two competing resonant processes, one localized at the particle level around the plasmon resonance, and one distributed across the lattice. Both are shown to contribute to the lasing threshold and the overall emission properties of the array.

Simulations and Experiments Provide a Convergent View of Protein Unfolded States under Folding Conditions

An accurate description of unfolded states under folding conditions is important for quantitative studies of protein stability, protein folding kinetics, and an understanding of how features of the unfolded state may have contributed to the convergent evolution toward foldable sequences. Here, we present results from multi-pronged biophysical investigations that uncover a comprehensive description of conformational ensembles accessed by unfolded states of proteins under folding conditions. We combined results from experiments based on time-resolved Forster resonance energy transfer, using multiple non-perturbing, amino-acid-sized dye pairs, and time-resolved small angle X-ray scattering, with novel molecular simulations and polymer physics theories. Previous work showed that aqueous milieus are poor solvents for polypeptide backbones. This should drive chain collapse and globule formation. Our current results, obtained for the N-terminal domain of the ribosomal protein L9, show that under folding conditions, native and non-native intra-chain interactions lead to transient globule formation, while large conformational fluctuations brought about by favorable sidechain-solvent interactions lead to an unfolded ensemble that behaves statistically like a flexible polymer would in a theta-solvent. Therefore, the unfolded state under folding conditions may be described as an ensemble with fluctuating elements of native and non-native non-random elements of structure that are averaged over to yield random coil-like statistics of chains in theta solvents. This behavior derives from the sequence-encoded interplay between backbone- and sidechain-mediated intra-chain and chain-solvent interactions. Our results have broad implications for interactions of unfolded proteins in crowded milieus, for folding-unfolding transitions, and for tilting the balance toward heterogeneous ensembles as is the case with intrinsically disordered proteins.

Environment sensitive fluorescent analogue of biologically active oxazoles differentially recognizes human serum albumin and bovine serum albumin: Photophysical and molecular modeling studies

An environment sensitive fluorophore, 4-(5-(4-(dimethylamino)phenyl)oxazol-2-yl)benzoic acid (DMOBA), that closely mimics biologically active 2,5-disubstituited oxazoles has been designed to probe two homologous serum proteins, human serum albumin (HSA) and bovine serum albumin (BSA) by means of photophysical and molecular modeling studies. This fluorescent analogue exhibits solvent polarity sensitive fluorescence due to an intramolecular charge transfer in the excited state. In comparison to water, the steady state emission spectra of DMOBA in BSA is characterized by a greater blue shift (~10nm) and smaller Stokes' shift (~5980cm−1) in BSA than HSA (Stokes'shift~6600cm−1), indicating less polar and more hydrophobic environment of the dye in the former than the latter. The dye-protein binding interactions are remarkably stronger for BSA than HSA which is evident from higher value of the association constant for the DMOBA-BSA complex (Ka~5.2×106M−1) than the DMOBA-HSA complex (Ka~1.0×106M−1). Fӧrster resonance energy transfer studies revealed remarkably less efficient energy transfer (8%) between the donor tryptophans in BSA and the acceptor DMOBA dye than that (30%) between the single tryptophan moiety in HSA and the dye, which is consistent with a much larger distance between the donor (tryptophan)-acceptor (dye) pair in BSA (34.5Å) than HSA (25.4Å). Site specific competitive binding assays have confirmed on the location of the dye in Sudlow's site II of BSA and in Sudlow's site I of HSA, respectively. Molecular modeling studies have shown that the fluorescent analogue is tightly packed in the binding site of BSA due to strong steric complementarity, where, binding of DMOBA to BSA is primarily dictated by the van der Waals and hydrogen bonding interactions. In contrast, in HSA the steric complementarity is less significant and binding is primarily guided by polar interactions and van der Waals interactions appear to be less significant in the formation of the HSA-DMOBA complex. Electrostatic interactions contribute significantly in the binding of DMOBA to HSA (−2.09kcal/mol) compared to BSA (−0.47kcal/mol). Electrostatic surface potential calculation reveals that the DMOBA binding site within HSA is highly charged compared to BSA.

Accurate Quantitative Sensing of Intracellular pH based on Self-ratiometric Upconversion Luminescent Nanoprobe

Accurate quantitation of intracellular pH (pHi) is of great importance in revealing the cellular activities and early warning of diseases. A series of fluorescence-based nano-bioprobes composed of different nanoparticles or/and dye pairs have already been developed for pHi sensing. Till now, biological auto-fluorescence background upon UV-Vis excitation and severe photo-bleaching of dyes are the two main factors impeding the accurate quantitative detection of pHi. Herein, we have developed a self-ratiometric luminescence nanoprobe based on förster resonant energy transfer (FRET) for probing pHi, in which pH-sensitive fluorescein isothiocyanate (FITC) and upconversion nanoparticles (UCNPs) were served as energy acceptor and donor, respectively. Under 980 nm excitation, upconversion emission bands at 475 nm and 645 nm of NaYF4:Yb3+, Tm3+ UCNPs were used as pHi response and self-ratiometric reference signal, respectively. This direct quantitative sensing approach has circumvented the traditional software-based subsequent processing of images which may lead to relatively large uncertainty of the results. Due to efficient FRET and fluorescence background free, a highly-sensitive and accurate sensing has been achieved, featured by 3.56 per unit change in pHi value 3.0–7.0 with deviation less than 0.43. This approach shall facilitate the researches in pHi related areas and development of the intracellular drug delivery systems.

Comment on the article “Investigation of Fluorescence Resonance Energy Transfer between Fluorescein and Rhodamine 6G”

In this comment we, report the missing of relevant literature regarding Forster energy transfer (FRET) between fluorescein and rhodamine 6G in a recent paper (Spectrochim. Acta A, 149 (2015) 143–149). In this paper, the authors claim that “a new FRET pair” has been identified, which is absolutely incorrect. In fact, studies on FRET in this dye pair under different conditions have been done earlier. Further, the estimated critical transfer distance may have uncertainty because of donor quantum yield which is not clarified in the paper.

A supramolecular indicator displacement assay for acetyl amantadine, a proxy biomarker for spermidine/spermine N1-acetyltransferase (SSAT) activity

Acetyl amantadine (AcAm) is produced from amantadine (Am) in vivo upon catalysis by spermidine/spermine N1-acetyl transferase (SSAT). SSAT is a biomarker for multiple aggressive cancers, and the analysis of AcAm in urine has been promoted as a proxy measure for the early detection of cancer. We report here the development and optimization of cucurbit[7]uril–dye pair based indicator displacement assay (IDA) for the detection of AcAm in solution. In deionized water, using Rhodamine B as the dye, the limit of detection of AcAm was 0.087 μM with a linear response range from 0 to 1 μM. Using berberine as the dye, the limit of detection was 0.077 μM with the same range of linear response. Our efforts and difficulties in translating this assay to function in human urine are also described. We achieve a partial response of the berberine IDA to the presence of AcAm in urine that has undergone a simple PD-10 desalting step.

Molecular-Based Fluorescent Nanoparticles Built from Dedicated Dipolar Thienothiophene Dyes as Ultra-Bright Green to NIR Nanoemitters.

Fluorescent Organic Nanoparticles (FONs), prepared by self-aggregation of dedicated dyes in water, represent a promising green alternative to the toxic quantum dots (QDs) for bioimaging purposes. In the present paper, we describe the synthesis and photophysical properties of new dipolar push-pull derivatives built from thieno[3,2-b]thiophene as a π-conjugated bridge that connects a triphenylamine moiety bearing various bulky substituents as electron-releasing moiety to acceptor end-groups of increasing strength (i.e., aldehyde, dicyanovinyl and diethylthiobarbiturate). All dyes display fluorescence properties in chloroform, which shifts from the green to the NIR range depending on the molecular polarization (i.e., strength of the end-groups) as well as a large two-photon absorption (TPA) band response in the biological spectral window (700–1000 nm). The TPA bands show a bathochromic shift and hyperchromic effect with increasing polarization of the dyes with maximum TPA cross-section reaching 2000 GM for small size chromophore. All dyes are found to form stable and deeply colored nanoparticles (20–45 nm in diameter) upon nanoprecipitation in water. Although their fluorescence is strongly reduced upon aggregation, all nanoparticles show large one-photon (up to 108 M−1·cm−1 in the visible region) and two-photon (up to 106 GM in the NIR) brightness. Interestingly, both linear and non-linear optical properties are significantly affected by interchromophoric interactions, which are promoted by the molecular confinement and modulated by both the dipolar strength and the presence of the bulky groups. Finally, we exploited the photophysical properties of the FONs to design optimized core-shell nanoparticles built from a pair of complementary dipolar dyes that promotes an efficient core-to-shell FRET process. The resulting molecular-based core-shell nanoparticles combine large two-photon absorption and enhanced emission both located in the NIR spectral region, thanks to a major amplification (by a factor of 20) of the core fluorescence quantum yield. These novel nanoparticles, which combine huge one-and two-photon brightness, hold major promise for in vivo optical bioimaging.

Tautomerism, crystal structure, and copper(II) complexation of isomeric pyridonylazo dyes derived from 2‐ and 4‐aminobenzoic acids

A pair of isomeric aromatic heterocyclic dyes, bearing the same N‐methyl pyridine‐2,6‐dione coupling component but a different 2‐ or 4‐aminobenzoic acid diazo component, were characterised structurally and spectroscopically. X‐ray single‐crystal diffraction analyses revealed that they both adopt the same hydrazone‐tautomeric form and planar molecular conformation between the pyridine and phenyl rings. Furthermore, azo–hydrazone transformation was achieved for the 2‐aminobenzoic‐acid‐based dye after Cu(II) ion complexation, as verified by the formation of a neutral Cu(II) dye–metal complex. The coordination geometry of the central Cu(II) ion exhibits a slightly distorted pyramid with a value of 0.028, where the ligand loses a proton during complexation and serves as the tridentate coordination mode. In addition, thermogravimetric analysis and corresponding differential thermal analysis for the isomeric pair showed that they both have excellent thermal stability, and 4‐aminobenzoic‐acid‐based dye has a higher decomposition temperature (319 °C) than the 2‐aminobenzoic‐acid‐based dye (312 °C). To the best of our knowledge, this is the first structural study on aromatic heterocyclic dyes having aminobenzoic acid and pyridine‐2,6‐dione components at the same time.

Simultaneous uptake of a Förster transfer dye pair by diatoms: Application in determination of staining density

The simultaneous uptake of PDMPO and Rhodamine B as two fluorescent dyes forming a Förster transfer pair by the diatom Cyclotella meneghiniana is demonstrated by in vivo-fluorochromation. The incorporation density in the cell walls was high enough for achieving resonant energy transfer between the two dyes as detected by fluorescence and excitation spectroscopy. The mean fluorescence lifetime of the donor is shortened in the presence of the acceptor by a factor of 0.75. By determining the mean lifetime from the fluorescence decay fitted by three eponentials, the efficiency of the energy transfer and the acceptor concentration is calculated assuming a homogenous distribution. For an initial concentration of both dyes of 5μM in the culture medium which is at the saturation limit of incorporation, an acceptor incorporation density of 0.6mM is obtained. In addition to such quantitative determinations, efficient emitting systems based on resonant energy transfer between two laser dyes may be useful in photonic applications of the hybrid biomineral. By achieving stimulated emission, the presence of optical modes in diatom frustules, which may act as photonic resonators due to the refractive index contrast to the environment in combination with the more or less regular pore pattern, may be characterized further.