Consideration of dipole orientation angles yields accurate rate equations for energy transfer in the rapid diffusion limit.

Dipole-dipole energy transfer between suitable donor and acceptor chromophores is an important luminescence quenching mechanism and has been shown to be useful for distance determination at the molecular level. In the rapid diffusion limit, where the excited-state lifetime of the donor is long enough to allow the donor and acceptor to diffuse many times their average separation before deexcitation, it is usually assumed that the relative dipolar orientation is completely averaged due to rotational Brownian motion. Under this simplifying assumption, analytical expressions have been derived earlier for the energy transfer rate between donor and acceptor characterized by different geometries. Most such expressions, however, are only approximate because complete angular averaging is permitted only in a geometry that possesses spherical symmetry surrounding each chromophore. In this paper analytical expressions that correctly account for incomplete angle averaging due to steric hindrance are presented for several geometries. Each of the equations reveals a dependence of the energy transfer rate on chromophore orientation. It is shown that correctly accounting for this effect can lead to improvements in estimates of the distance of closest approach from measured quenching rates based on energy transfer experiments.     

Cytochrome c location in phosphatidylcholine/cardiolipin model membranes: resonance energy transfer study

Resonance energy transfer between lipid-bound fluorescent probe 3-methoxybenzanthrone as a donor and heme group of cytochrome c as an acceptor has been examined to ascertain the protein disposition relative to the surface of model membranes composed of phosphatidylcholine and cardiolipin (10, 50 and 80 mol%). The model of energy transfer in membrane systems has been extended to the case of donors distributed between the two-bilayer leaflets and acceptors located at the outer monolayer taking into account the donor and acceptor orientational behavior. Assuming specific protein orientation relative to the membrane surface and varying lateral distance of the donor-acceptor closest approach in the range from 0 to 3.5 nm the limits for possible heme distances from the bilayer midplane have been found to be 0.8-3 nm (10 mol% CL), 0-2.6 nm (50 mol% CL), and 1.4-3.3 nm (80 mol% CL).     

Fluorescence energy transfer studies of transmembrane location of retinal in purple membrane

A diffusion-enhanced energy transfer technique was employed for the determination of transmembrane location of the retinal chromophore in the purple membrane. Theoretical considerations showed that the rate of energy transfer from an energy donor embedded within a membrane to acceptors dissolved in solvent could be described by an analytical function of the distance a of closest approach between the donor and acceptor, if the "rapid-diffusion limit" was attained. The criterion for this limit was given by the relation: (RO)6 much less than 20D tau Da4, where RO is the characteristic distance of energy transfer, D is the diffusion coefficient of the acceptor and tau D is the fluorescence lifetime of the donor in the absence of acceptor. By photo-reduction of the purple membrane with sodium borohydride, the retinal chromophore was converted to a highly fluorescent derivative, which showed a broad emission band in the visible region. From analysis of the fluorescence decay curves of the photo-reduced purple membrane in the presence of various concentrations of cobalt-ethylenediamine tetraacetate (Co-EDTA: energy acceptor), the depth of the chromophore from the membrane surface was estimated to be 8 (+/-3) A. This result was supported by investigations of energy transfer processes in a system where the native purple membranes and the photo-reduced membranes were stacked in parallel: the energy acceptor in this system was the native retinal chromophore.     

Fluorescence energy transfer studies on lima bean lectin. Distance between the subunit hydrophobic binding site and the thiol group essential for carbohydrate binding

Measurements of the efficiency of singlet-singlet energy transfer were used to determine the distance between the hydrophobic binding site and the thiol group required for carbohydrate-binding activity of lima bean lectin. 1-Anilino-8-naphthalenesulfonate, bound to the hydrophobic binding site by noncovalent interactions, was used as the donor. Two different nonfluorescent probes were used as the acceptors: a mercurial, 2-chloromercuri-4-nitrophenol, and a maleimide, 4-dimethylaminophenylazophenyl-4'-maleimide. Acceptor was covalently attached to the thiol group at the putative carbohydrate binding site. The efficiency of energy transfer in both the 1-anilino-8-naphthalenesulfonate/2-chloromercuri-4-nitrophenol and and 1-anilino-8-naphthalenesulfonate/4-dimethylaminophenylazophenyl-4' -maleimide donor-acceptor systems indicated an apparent distance of 28 A between the two sites, assuming that the transition dipole of the donor is not correlated with respect to that of the acceptor and that each donor is quenched by a single acceptor. Using an alternate model wherein each donor is equally quenched by two acceptors on adjacent subunits, an apparent distance of 33.4 A was calculated. The fact that two donor-acceptor pairs with different F?rster's critical distance parameters yielded the same distance between the sites is consistent with our assumption of uncorrelated donor-acceptor transition dipoles.     

Anomalous Surplus Energy Transfer Observed with Multiple FRET Acceptors

Background

Förster resonance energy transfer (FRET) is a mechanism where energy is transferred from an excited donor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecular complex. Thus, the use of FRET as a tool for measuring protein-protein interactions inside living cells requires an understanding of how FRET changes with multiple acceptors. When multiple FRET acceptors are present it is assumed that a quantum of energy is either released from the donor, or transferred in toto to only one of the acceptors present. The rate of energy transfer between the donor and a specific acceptor (k_D→A) can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates.

Methodology/Principal Findings

The generality of this approach was tested by measuring the ensemble FRET efficiency in two constructs, each containing a single fluorescent-protein donor (Cerulean) and either two or three FRET acceptors (Venus). FRET transfer rates between individual donor-acceptor pairs within these constructs were calculated from FRET efficiencies measured after systematically introducing point mutations to eliminate all other acceptors. We find that the amount of energy transfer observed in constructs having multiple acceptors is significantly greater than the FRET efficiency predicted from the sum of the individual donor to acceptor transfer rates.

Conclusions/Significance

We conclude that either an additional energy transfer pathway exists when multiple acceptors are present, or that a theoretical assumption on which the kinetic model prediction is based is incorrect.     

Fluorescence energy transfer from diphenylhexatriene to bacteriorhodopsin in lipid vesicles

Fluorescence energy transfer between the donor diphenylhexatriene (DPH) and the acceptor retinal and fluorescence depolarization of DPH are used to test current theories for fluorescence energy transfer in two-dimensional systems and to obtain information on the effect of the intrinsic membrane protein, bacteriorhodopsin, on the order and dynamics of the lipid phase. Increasing the surface concentration of acceptors by raising the protein to lipid ratio leads to a decrease in the mean fluorescence lifetime by up to a factor of four. When the acceptor concentration is reduced at a fixed protein to lipid ratio by photochemical destruction of retinal, the lifetime increases and reaches approximately the value observed in protein-free vesicles when the bleaching is complete. The shape of the decay curve and the dependency of the mean lifetime on the surface concentration of acceptors are in agreement with theoretical predictions for a two-dimensional random distribution of donors and acceptors. From this analysis a distance of closest approach between donors and acceptors of approximately 18 A is obtained, which is close to the effective radius of bacteriorhodopsin (17 A) and consistent with current ideas about the location of retinal in the interior of the protein. In the absence of energy transfer (bleached vesicles), the steady-state fluorescence anisotropy, -r, of DPH is considerably lower than in the corresponding unbleached vesicles, indicating that the effect of energy transfer must be taken into account when interpreting -r in terms of order and dynamics.     

Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations

We studied fluorescence resonance energy transfer between donors and acceptors bound to double-helical DNA. The donor Hoechst 33258 binds to the minor groove of DNA and the acceptor propidium iodide (PI) is an intercalator. The time-resolved donor decays were measured in the frequency domain. The donor decays were consistent with a random 1-dimensional distribution of acceptors. The decays were analyzed in terms of three 1-dimensional models: a random continuous acceptor distribution; acceptors placed on discrete lattice sites; and a cylindrical model with the acceptor in the center, and the donors on a cylinder surface. The data were well described by all three models. Interpretation in terms of continuous distribution of acceptors revealed a minimum donor to acceptor distance of 13 A, which is 3 bp from the center of Hoechst 33252. These results suggest that PI is excluded from the 4 bp covered by Hoechst 33252 when it is bound to the minor groove of DNA.     

Engineering resonance energy transfer for advanced immunoassays: the case of celiac disease

Celiac disease (CD) is an immune-mediated disorder affecting genetically predisposed subjects. It is caused by the ingestion of wheat gluten and related prolamins. A final diagnosis for this disease can be obtained by examination of jejunal biopsies. Nevertheless, different analytical approaches have been established to detect the presence of anti-tissue transglutaminase antibodies that represent a serological hallmark of the disease. In this work, we explored a new method for the diagnosis of CD based on the detection of serum anti-transglutaminase antibodies by resonance energy transfer (RET) between donor molecules and acceptor molecules. In particular, we labeled the liver transglutaminase (tTG) enzyme from guinea pig and the rabbit anti-tTG antibodies with a couple of fluorescence probes that are able to make RET if they are located within with Förster distance. We labeled tTG with the fluorescence probe DyLight 594 as donor and the anti-tTG antibodies with the fluorescence probe DyLight 649 as acceptor. However, due to the large size of the formed complex (tTG/anti-tTG), and consequently to the low efficiency energy transfer process between the donor–acceptor molecules, we explored a new experimental approach that allows us to extend the utilizable range of RET between donor:acceptor pairs by using one single molecule as donor and multiple molecules as energy acceptors, instead of using a single acceptor molecule as usually occurs in RET experiments. The obtained results clearly show that the use of one donor and multiacceptor strategy enables for a simple and rapid detection of serum anti-transglutaminase antibodies. In addition, our results point out that it is possible to consider this approach as a new method for a wide variety of analytical assays.     

Analysis of resonance energy transfer in model membranes: role of orientational effects

The model of resonance energy transfer (RET) in membrane systems containing donors randomly distributed over two parallel planes separated by fixed distance and acceptors confined to a single plane is presented. Factors determining energy transfer rate are considered with special attention being given to the contribution from orientational heterogeneity of the donor emission and acceptor absorption transition dipoles. Analysis of simulated data suggests that RET in membranes, as compared to intramolecular energy transfer, is substantially less sensitive to the degree of reorientational freedom of chromophores due to averaging over multiple donor-acceptor pairs. The uncertainties in the distance estimation resulting from the unknown mutual orientation of the donor and acceptor are analyzed.     

Detection of actin assembly by fluorescence energy transfer

Fluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide). Donor- labeled actin and acceptor-labeled actin were coassembled. The dependence of the transfer efficiency on the mole fraction of acceptor- labeled actin showed that the radial coordinate of the label at cysteine 373 is approximately 35 A, which means that this site is located near the outer surface of the filament. The distance between a donor and the closest acceptor in such a filament is 58 A. The increase in fluorescence after the mixing of actin filaments containing both donor and acceptor with unlabeled filaments showed that there is a slow continuous exchange of actin units. The rate of exchange was markedly accelerated when the filaments were sonicated. The rapid loss of energy transfer caused by mechanical shear probably resulted from an increase in the number of filament ends, which in turn accelerated the exchange of monomeric actin units. Energy transfer promises to be a valuable tool in characterizing the assembly and dynamics of actin and other cytoskeletal and contractile proteins in vitro and in intact cells.     

A domain of membrane-bound blood coagulation factor Va is located far from the phospholipid surface. A fluorescence energy transfer measurement

The larger subunit of blood coagulation factor Va was covalently labeled with iodoacetamido derivatives of fluorescein and rhodamine without loss of functional activity, as measured by either the one-stage clotting assay or the ability to accelerate prothrombin activation in a purified system. The spectral properties of the dyes were not altered by the presence or absence of the smaller subunit of factor Va, Ca2+, prothrombin, factor Xa, or phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) vesicles. When fluorescein-labeled protein (factor VaF) was titrated with PC/PS vesicles containing either octadecylrhodamine or 5-(N-hexadecanoylamino)eosin, fluorescence energy transfer was observed between the protein-bound donor dyes and the acceptor dyes at the outer surface of the phospholipid bilayer. The extent of energy transfer correlated directly with the extent of protein binding to the vesicles monitored by light scattering. The distance of closest approach between the fluorescein on factor Va and the bilayer surface averaged 90 A for the two different acceptors. Association of factor VaF with factor Xa on the phospholipid surface reduced this separation by 7 A, but association with prothrombin did not alter the distance between the labeled domain on factor VaF and the surface. The efficiency of diffusion-enhanced energy transfer between rhodamine-labeled factor Va and terbium dipicolinate entrapped inside PC/PS vesicles was less than 0.01, consistent with the location of the dye far above the inner surface of the vesicle. Thus, a domain of membrane-bound factor Va is located a minimum of 90 A above the phospholipid surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

A flexible approach to the calculation of resonance energy transfer efficiency between multiple donors and acceptors in complex geometries

Resonance energy transfer provides a practical way to measure distances in the range of 10-100 A between sites in biological molecules. Although the relationship between the efficiency of energy transfer and the distance between sites is well described for a single pair of fluorophores, the situation is more difficult when more than two fluorophores are present. Using a Monte Carlo calculation scheme, we demonstrate how resonance energy transfer can be used to measure distances between fluorophores in complex geometries. We demonstrate the versatility of the approach by calculating the efficiency of energy transfer for individual fluorophores randomly distributed in two and three dimensions, for linked pairs of donors and acceptors and pentameric structures of five linked fluorophores. This approach can be used to relate the efficiency of energy transfer to the distances between fluorophores, R0, molecular concentrations, laser power, and donor/acceptor ratios in ensembles of molecules or when many fluorophores are attached to a single molecule such as in multimeric proteins.     

Extending the range of FRET—the Monte Carlo study of the antenna effect

The problem of extending the utilizable range of Förster resonance energy transfer (FRET) is of great current interest, due to the demand of conformation studies of larger biological structures at distances exceeding typical limiting distance of 100 Å. One of the ways to address this issue is the use of so-called antenna effect. In the present work, the influence of the antenna effect on the FRET efficiency is investigated by the Monte Carlo analysis. The previously published results Bojarski et al. (J Phys Chem B 115:10120–10125, 2011) indicate that using a simple model of donor linked with a protein labeled with multiple acceptors, significantly increases the transfer efficiency in comparison with donor–single acceptor system. The effect is stronger if the transition moments of acceptors are mutually parallel. In this work, to extend the scope of possible biological systems to be analyzed, different distributions of donor–acceptors distance are analyzed, as well as the size and shape of the attached molecule.     

Experimental Verification of the Kinetic Theory of FRET Using Optical Microspectroscopy and Obligate Oligomers

Förster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photoinsensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells.     

Transverse location of the retinal chromophore of rhodopsin in rod outer segment disc membranes

Diffusion-enhanced fluorescence energy transfer was used to study the structure of photoreceptor membranes from bovine retinal rod outer segments. The fluorescent energy donor was Tb³⁺ chelated to dipicolinate and the acceptor was the 11-cis retinal chromophore of rhodopsin in vesicles made from disc membranes. The rapid-diffusion limit for energy transfer was attained in these experiments because of the long excited state lifetime of the terbium donor (~2 ms). Under these conditions, energy transfer is very sensitive to a, the distance of closest approach between the donor and acceptor (Thomas et al., 1978). Vesicles containing terbium dipicolinate in their inner aqueous space were prepared by sonicating disc membranes in the presence of this chelate and chromatographing this mixture on a gel filtration column. The sidedness of rhodopsin in these vesicles was the same as in native disc membranes. The transfer efficiency from terbium to retinal in this sample was 43%. For an R₀ value of 46.7 Å and an average vesicle diameter of 650 Å, this corresponds to an a value of 22 Å from the inner aqueous space of the vesicle. The distance of closest approach from the external aqueous space, determined by adding terbium dipicolinate to a suspension of already formed vesicles, was found to be 28 Å. These values of a show that the retinal chromophore is far from both aqueous surfaces of the disc membrane. Hence, the transverse location of the retinal chromophore is near the center of the hydrophobic core of the disc membrane. These findings suggest that conformational changes induced by photoisomerization are transmitted through a distance of at least 20 Å within rhodopsin to trigger subsequent events in visual excitation.     

Resonance energy transfer between the active sites of rabbit muscle creatine kinase: analysis by steady-state and time-resolved fluorescence

Resonance energy transfer between the reactive thiols of rabbit muscle creatine kinase was evaluated. The reactive thiols are located at the active site, one occurring on each subunit of the dimeric protein that is known to be a constituent of the M-line structure of the myofibril. Transfer efficiency was evaluated from energy donor quenching of fluorescence by steady-state and phase-modulation lifetime measurements and determination of sensitized emission of the acceptor. Several sulfhydryl-specific donor fluorophores were used including 5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid, 7-(dimethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin, and 2-[4-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS). Energy transfer acceptors included 5-(iodoacetamido)fluorescein and the nonfluorescent dye [4-[[4-(dimethylamino)phenyl]azo]phenyl]iodoacetamide. In order to prepare the necessary homodimer labeled with both donor and acceptor, advantage was taken of the biphasic reaction between creatine kinase and IAANS. In some instances, donor/acceptor hybrids were prepared by denaturation/renaturation procedures, and possible deviations from expected hybridization stoichiometry were considered. Disproportionation of singly labeled dimers (to unlabeled and doubly labeled dimers) was not observed when the brain isozyme of creatine kinase was used to trap dissociated dye-conjugated or unlabeled muscle-type subunits of creatine kinase. From studies of five different donor/acceptor combinations, the efficiency of energy transfer was found to occur over a range of 5-14%, indicating that the reactive thiols are well separated. Overlap integrals and quantum yields were evaluated, and estimates of the range of orientation factor were obtained to determine a range for the distance between the active sites of creatine kinase. When the ranges are overlapped, a limited distance of 48.6-60.4 A is obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer

Bioluminescence resonance energy transfer (BRET), which relies on nonradiative energy transfer between luciferase-coupled donors and GFP-coupled acceptors, is emerging as a useful tool for analyzing the quaternary structures of cell-surface molecules. Conventional BRET analyses are generally done at maximal expression levels and single acceptor/donor ratios. We show that under these conditions substantial energy transfer arises from random interactions within the membrane. The dependence of BRET efficiency on acceptor/donor ratio at fixed surface density, or expression level at a defined acceptor/donor ratio, can nevertheless be used to correctly distinguish between well-characterized monomeric and oligomeric proteins, including a very weak dimer. The pitfalls associated with the nonrigorous treatment of BRET data are illustrated for the case of G protein-coupled receptors (GPCRs) proposed to form homophilic and/or mixed oligomers on the basis of previous, conventional BRET experiments.     

FRET detection of cellular alpha4-integrin conformational activation

Integrins are cell adhesion receptors, expressed on every cell type, that have been postulated to undergo conformational changes upon activation. Here, different affinity states were generated by exposing alpha4-integrins to divalent ions or by inside-out activation using a chemokine receptor. We probed the dynamic structural transformation of the integrin on live cells using fluorescence resonance energy transfer (FRET) between a peptide donor, which specifically binds to the alpha4-integrin, and octadecyl rhodamine B acceptors incorporated into the plasma membrane. We analyzed the data using a model that describes FRET between a random distribution of donors and acceptors in an infinite plane. The distance of closest approach was found to vary with the affinity of the integrin. The change in distance of closest approach was approximately 50 A between resting and Mn2+ activated receptors and approximately 25 A after chemokine activation. We used confocal microscopy to probe the lateral organization of donors and acceptors subsequent to integrin activation. Taken together, FRET and confocal results suggest that changes in FRET efficiencies are primarily due to the vertical extension of the integrin. The coordination between the extension of alpha4-integrin and its affinity provides a mechanism for Dembo's catch-bond concept.     

Detection of receptor clustering by flow cytometric fluorescence anisotropy measurements

BACKGROUND: Perrin equation suggests an alternative way for the accurate energy transfer determination on a cell-by-cell basis by measuring polarized donor intensities in a conventional flow cytometer. METHODS: The relationship between energy transfer and fluorescence anisotropy of the donor was investigated by flow cytometric generation of Perrin-lifetime plots of fluorescent antibody-labeled MHC class I and class II molecules on the surface of living cells. The energy transfer reduced the fluorescence lifetime of the donor. RESULTS: Perrin plots have proven to be sensitive to the segmental mobility of the labeling dye and that of antibodies of different isotypes, and homo-transfer due to the multiple labeling of antibodies. A method demonstrating the feasibility of energy transfer determination by measuring anisotropy enhancement of the donor is presented. Flow cytometric histograms of the donor anisotropy and of the deduced energy transfer efficiency are shown, indicating clustering of MHC class I and class II molecules on the surface of human T lymphoblasts. In the Appendix, a method for the simultaneous determination of both energy transfer efficiency and donor fluorescence anisotropy, without need for G-factor measurement, is also presented. CONCLUSIONS: We demonstrate that energy transfer efficiency, i.e., proximity, between suitably selected donor and acceptor, and the rotational relaxation of the donor, i.e., donor mobility, can be simultaneously measured in a flow cytometer.     

On the possibility of long-wavelength long-lifetime high-quantum-yield luminophores

We describe an approach to creating a new class of luminophores which display both long wavelength emissions exceeding 600 nm and long lifetimes. These luminophores are based on resonance energy transfer (RET) from a long lifetime donor to a short lifetime but long wavelength acceptor. We demonstrated the possibility of obtaining these desirable spectral properties using donors and acceptors noncovalently bound to DNA. The donor was a ruthenium (Ru) metal-ligand complex in which one of the diimine ligands intercalated into double-helix DNA. The acceptors were either nile blue, TOTO-3, or TO-PRO-3. Upon binding of the acceptor to donor-labeled DNA, we found that the acceptor quantum yield was remarkably enhanced so that the wavelength-integrated intensities of the donor and acceptor bound to DNA were many-fold greater than the intensity of the donor and acceptor alone when separately bound to DNA. The origin of this effect is efficient energy transfer from the donor. Under these conditions the effective overall quantum yield approaches that of the acceptor. Importantly, the increased quantum yield can be obtained while maintaining usefully long apparent acceptor lifetimes of 30 to 80 ns. The effect of an increased quantum yield from a low quantum yield donor may find use in assays to detect macromolecular binding interactions. These results suggest the synthesis of covalently linked donor-acceptor pairs with the desirable spectral properties of long wavelength emission, high quantum yield, and moderately long lifetimes for gated detection.