Fluorescence Count Rate Research Articles

UV fluorescence enhancement by aluminum and magnesium equilateral bowtie nanoantennas

The intrinsic fluorescence of biomolecules such as proteins and nucleic acids lies in the ultraviolet (UV) range of the spectrum. UV plasmonic nano-structures have been shown to enhance the fluorescence quantum yield and reduce the lifetimes of various biomolecules. Fluorescence enhancement is contributed to by both excitation rate and emission rate enhancement. Since biomolecules are prone to photon-degradation in the UV range, excitation rate enhancement should be minimized, while radiative rate enhancement should be maximized. Although numerous nano-structures have been proposed both numerically and experimentally to enhance the fluorescence of native biomolecules, very few studies have achieved more than 10x radiative rate enhancement. Here we report systematic studies of fluorescence enhancement by equilateral bowtie nano-antennas (BNA) made of aluminum (Al) or magnesium (Mg) in the ultraviolet region. We modeled the emission rate enhancement using the excitation and emission peak wavelength of tryptophan. The quantum yield of tryptophan is also taken into account. Our results show that with the optimal geometry, Al BNA with oxide yields an excitation enhancement of 21× at the excitation wavelength of tryptophan (270nm), a radiative enhancement of 37×, a quantum yield enhancement of 5×, and a net fluorescence count rate enhancement of 64× at the emission wavelength of tryptophan (340nm). Mg BNA with oxide sustains the highest Purcell factor enhancement, 14×. The effect of the native oxide layer on both metals is investigated. The studies reported here are meaningful in the design of better UV plasmonic nano-structures for label-free sensing of biomolecules.

High-performance frequency stabilization of ultraviolet diode lasers by using dichroic atomic vapor spectroscopy and transfer cavity**Project supported by the National Natural Science Foundation of China (Grant Nos. 11704408 and 91836106), the Beijing Natural Science Foundation, China (Grant No. Z180013), and the Joint Fund of the Ministry of Education, China (Grant No. 6141A020333xx).

We develop a high-performance ultraviolet (UV) frequency stabilization technique implemented directly on UV diode lasers by combining the dichroic atomic vapor laser lock and the resonant transfer cavity lock. As an example, we demonstrate a stable locking with measured frequency standard deviations of approximately 200 kHz and 300 kHz for 399 nm and 370 nm diode lasers in 20 min. We achieve a long-term frequency drift of no more than 1 MHz for the target 370 nm laser within an hour, which is further verified with fluorescence count rates of a single trapped 171Yb+ ion. We also find strong linear correlations between lock points and environmental factors such as temperature and atmospheric pressure. Our approach provides a simple and stable solution at a relatively low cost, and features flexible control, high feedback bandwidth and minimal power consumption of the target UV laser.

Extending Single-Molecule Förster Resonance Energy Transfer (FRET) Range beyond 10 Nanometers in Zero-Mode Waveguides.

Single-molecule Förster resonance energy transfer (smFRET) is widely used to monitor conformations and interaction dynamics at the molecular level. However, conventional smFRET measurements are ineffective at donor-acceptor distances exceeding 10 nm, impeding the studies on biomolecules of larger size. Here, we show that zero-mode waveguide (ZMW) apertures can be used to overcome the 10 nm barrier in smFRET. Using an optimized ZMW structure, we demonstrate smFRET between standard commercial fluorophores up to 13.6 nm distance with a significantly improved FRET efficiency. To further break into the classical FRET range limit, ZMWs are combined with molecular constructs featuring multiple acceptor dyes to achieve high FRET efficiencies together with high fluorescence count rates. As we discuss general guidelines for quantitative smFRET measurements inside ZMWs, the technique can be readily applied for monitoring conformations and interactions on large molecular complexes with enhanced brightness.

Optimizing the metric in sensorless adaptive optical microscopy with fluorescence fluctuations.

Adaptive optics (AO) strategies using optimization-based, sensorless approaches are widely used, especially for microscopy applications. To converge rapidly to the best correction, such approaches require that a quality metric and a set of modes be chosen optimally. Fluorescence fluctuations microscopy, a family of methods that provides quantitative measurements of molecular concentration and mobility in living specimen, is in particular need of adaptive optics, since its results can be strongly biased by optical aberrations. We examined two possible metrics for sensorless AO, measured in a solution of fluorophores diffusing in 3D: the fluorescence count rate and the molecular brightness (or number of photons detected per molecule in the observation volume). We studied their respective measurement noise and sensitivity to aberrations. Then, AO correction accuracy was experimentally assessed by measuring the residual aberration after correcting a known wavefront. We proposed a theoretical framework to predict the correction accuracy, knowing the metric measurement noise and sensitivity. In the small aberration range, the brightness allows more accurate corrections when fluorophores are few but bright, whereas the count rate performs better in more concentrated solutions. When correcting large aberrations, the count rate is expected to be a more reliable metric.

Advancing 3D Single Molecule Tracking by Time-Gating and Fast Simultaneous Spinning Disk Imaging for Contextual Information

Three dimensional single molecule tracking is an essential technique for investigating biomolecular trafficking and signaling. Here we report the latest improvements to our confocal based tracking system. First, we added a Nipkow spinning disk imaging system. This system takes images of the plane of the tracked molecule, thus yielding contextual information about the molecule's environment. Laser illumination is used for the spinning disk system which together with an acousto-optic modulator allows for exposure times of 10 to 50 ms, thus greatly minimizing motion blur effects due to the feedback motion during tracking. Second, we introduced real-time photon time-gating to our tracking setup which allows tracking using only those photons detected within a defined time-window relative to the excitation laser pulse. Gating out detected photons within the first few nanoseconds after the excitation laser pulse greatly reduces Raman scattering and short lived fluorescence, and significantly increases the signal-to-noise ratio for tracked molecules with a long fluorescence lifetime. We also present a model for choosing the optimal time-gate based upon the fluorescence count rates and life times of the background and the tracked molecule.

Prospects for laser spectroscopy of highly charged ions with high-harmonic XUV and soft x-ray sources

We present novel high photon flux XUV and soft x-ray sources based on high harmonic generation (HHG). The sources employ femtosecond fiber lasers, which can be operated at very high (MHz) repetition rate and average power (>100 W). HHG with such lasers results in ∼1013 photons s−1 within a single harmonic line at ∼40 nm (∼30 eV) wavelength, a photon flux comparable to what is typically available at synchrotron beam lines. In addition, resonant enhancement of HHG can result in narrow-band harmonics with high spectral purity—well suited for precision spectroscopy. These novel light sources will enable seminal studies on electronic transitions in highly-charged ions. For example, at the experimental storage ring 2s1/2–2p1/2 transitions in Li-like ions can be excited up to Z = 47 (∼100 eV transition energy), which provides unique sensitivity to quantum electro-dynamical effects and nuclear corrections. We estimate fluorescence count rates of the order of tens per second, which would enable studies on short-lived isotopes as well. In combination with the Doppler up-shift available in head-on excitation at future heavy-ion storage rings, such as the high energy storage ring, even multi-keV transitions can potentially be excited. Pump–probe experiments with femtosecond resolution could also be feasible and access the lifetime of short-lived excited states, thus providing novel benchmarks for atomic structure theory.

Photonic nano-structures on (111)-oriented diamond

We demonstrate the fabrication of single-crystalline diamond nanopillars on a (111)-oriented chemical vapor deposited diamond substrate. This crystal orientation offers optimal coupling of nitrogen-vacancy (NV) center emission to the nanopillar mode and is thus advantageous over previous approaches. We characterize single native NV centers in these nanopillars and find one of the highest reported saturated fluorescence count rates in single crystalline diamond in excess of 106 counts per second. We show that our nano-fabrication procedure conserves the preferential alignment as well as the spin coherence of the NVs in our structures. Our results will enable a new generation of highly sensitive probes for NV magnetometry and pave the way toward photonic crystals with optimal orientation of the NV center's emission dipole.

Wide-Field Multispectral Super-Resolution Imaging Using Spin-Dependent Fluorescence in Nanodiamonds

Recent advances in fluorescence microscopy have enabled spatial resolution below the diffraction limit by localizing multiple temporally or spectrally distinguishable fluorophores. Here, we introduce a super-resolution technique that deterministically controls the brightness of uniquely addressable, photostable emitters. We modulate the fluorescence brightness of negatively charged nitrogen-vacancy (NV(-)) centers in nanodiamonds through magnetic resonance techniques. Using a CCD camera, this "deterministic emitter switch microscopy" (DESM) technique enables super-resolution imaging with localization down to 12 nm across a 35 × 35 μm(2) area. DESM is particularly well suited for biological applications such as multispectral particle tracking since fluorescent nanodiamonds are not only cytocompatible but also nonbleaching and bright. We observe fluorescence count rates exceeding 1.5 × 10(6) photons per second from single NV(-) centers at saturation. When combined with emerging NV(-)-based techniques for sensing magnetic and electric fields, DESM opens the door to rapid, super-resolution imaging for tracking and sensing applications in the life and physical sciences.

Correcting for spectral cross-talk in dual-color fluorescence cross-correlation spectroscopy.

Dual-color fluorescence cross-correlation spectroscopy (dcFCCS) allows one to quantitatively assess the interactions of mobile molecules labeled with distinct fluorophores. The technique is widely applied to both reconstituted and live-cell biological systems. A major drawback of dcFCCS is the risk of an artifactual false-positive or overestimated cross-correlation amplitude arising from spectral cross-talk. Cross-talk can be reduced or prevented by fast alternating excitation, but the technology is not easily implemented in standard commercial setups. An experimental strategy is devised that does not require specialized hardware and software for recognizing and correcting for cross-talk in standard dcFCCS. The dependence of the cross-talk on particle concentrations and brightnesses is quantitatively confirmed. Moreover, it is straightforward to quantitatively correct for cross-talk using quickly accessible parameters, that is, the measured (apparent) fluorescence count rates and correlation amplitudes. Only the bleed-through ratio needs to be determined in a calibration measurement. Finally, the limitations of cross-talk correction and its influence on experimental error are explored.

Large molecular fluorescence enhancement by a nanoaperture with plasmonic corrugations

We investigate the influence of circular corrugations surrounding a central nanoaperture to further enhance the fluorescence count rate per emitter and control its emission directionality. Adding a single corrugation already allows to significantly increase the fluorescence signal as compared to a bare nanoaperture. A complete fluorescence characterization quantifies the excitation and emission gains contributing to the fluorescence enhancement process as the number of corrugations is increased.

Bright Unidirectional Fluorescence Emission of Molecules in a Nanoaperture with Plasmonic Corrugations

Controlling the fluorescence emission from nanoscale quantum emitters is a key element for a wide range of applications, from efficient analytical sensing to quantum information processing. Enhancing the fluorescence intensity and narrowing the emission directivity are both essential features to achieve a full control of fluorescence, yet this is rarely obtained simultaneously with optical nanoantennas. Here we report that gold nanoapertures surrounded by periodic corrugations transform standard fluorescent molecules into bright unidirectional sources. We obtain enhancement factors of the fluorescence count rate per molecule up to 120 fold simultaneously with a directional emission of the fluorescence into a narrow angular cone in the direction normal to the sample plane. The bright emission and narrow directionality enable the detection of single molecules with a low numerical aperture objective, and improve the effectiveness of fluorescence-based applications. We thoroughly quantify the increased light-matter coupling as well as the radiation pattern intensity. These results are highly relevant for the development of single molecule sensing, single-photon sources, and light emitting devices.

Impurity spectroscopy at its ultimate limit: relation between bulk spectrum, inhomogeneous broadening, and local disorder by spectroscopy of (nearly) all individual dopant molecules in solids

We present a technique for the measurement of the low-temperature fluorescence excitation spectra and imaging of a substantial fraction of all single chromophore molecules (hundreds of thousands and even more) embedded in solid bulk samples as nanometre-sized probes. An important feature of our experimental studies is that the full information about the lateral coordinates and spectral parameters of all individual molecules is stored for detailed analysis. This method enables us to study a bulk sample in a broad spectral region with ultimate sensitivity, combining excellent statistical accuracy and the capability of detecting rare events. From the raw data we determined the distributions of several parameters of the chromophore spectra and their variations across the inhomogeneous absorption band, including the frequencies of the electronic zero-phonon lines, their spectral linewidths, and fluorescence count rates. Relationships between these distributions and the disorder of the matrix were established for the examples of two polycrystalline solids with very different properties, n-hexadecane and o-dichlorobenzene, and the amorphous polymer polyisobutylene. We also found spatially inhomogeneous distributions of some parameters.

Nanoaperture Fluorescence Enhancement in the Ultraviolet

We study fluorescence excitation and emission enhancement in the ultraviolet regime for molecules confined within sub-wavelength metal apertures. Calculations are performed across a range of excitation wavelengths for individual apertures constructed in gold, silver, and aluminum. As expected, enhancement in the ultraviolet is greatest with aluminum. Using excitation and emission wavelengths appropriate for tryptophan, we find that more than 10× net increase in fluorescence count rate should be obtainable for aluminum apertures of ~75 nm diameter. These results suggest that many applications utilizing native protein fluorescence could become practical, even to the single molecule level.

Measuring, in Solution, Multiple-Fluorophore Labeling by Combining Fluorescence Correlation Spectroscopy and Photobleaching

Determining the number of fluorescent entities that are coupled to a given molecule (DNA, protein, etc.) is a key point of numerous biological studies, especially those based on a single molecule approach. Reliable methods are important, in this context, not only to characterize the labeling process but also to quantify interactions, for instance within molecular complexes. We combined fluorescence correlation spectroscopy (FCS) and photobleaching experiments to measure the effective number of molecules and the molecular brightness as a function of the total fluorescence count rate on solutions of cDNA (containing a few percent of C bases labeled with Alexa Fluor 647). Here, photobleaching is used as a control parameter to vary the experimental outputs (brightness and number of molecules). Assuming a Poissonian distribution of the number of fluorescent labels per cDNA, the FCS-photobleaching data could be easily fit to yield the mean number of fluorescent labels per cDNA strand (approximately = 2). This number could not be determined solely on the basis of the cDNA brightness, because of both the statistical distribution of the number of fluorescent labels and their unknown brightness when incorporated in cDNA. The statistical distribution of the number of fluorophores labeling cDNA was confirmed by analyzing the photon count distribution (with the cumulant method), which showed clearly that the brightness of cDNA strands varies from one molecule to the other. We also performed complementary continuous photobleaching experiments and found that the photobleaching decay rate of Alexa Fluor 647 in the excited state decreases by about 30% when incorporated into cDNA, while its nonradiative decay rate is increased such that the brightness of individual Alexa labels is decreased by 25% compared to free Alexa dyes.

Independence of Maximum Single Molecule Fluorescence Count Rate on the Temporal and Spectral Laser Pulse Width in Two-Photon FCS

We investigate the fluorescence emission characteristics of standard dye tetramethylrhodamine (TMR) in two-photon fluorescence correlation spectroscopy for different temporal and spectral properties of the femtosecond excitation pulses. After determining the second-order dispersion of our setup, including the microscope objective, a pulse stretcher was employed to control the temporal width at the location of the specimen. As expected, the fluorescence per molecule and therefore the signal-to-noise ratio of an FCS-measurement can be improved at constant average excitation power by altering either the temporal or spectral width of the excitation pulses. We found however, that the maximum achievable molecular brightness is largely independent of the temporal and spectral width in the regime analyzed. This observation confirms the current working hypothesis for two-photon fluorescence correlation spectroscopy that bleaching and saturation, and thus, the inherent properties of the dye system, are the dominant effects limiting the quality of measurements. As a practical consequence, elaborate optimization of temporal and spectral laser pulse width, e.g. by introducing pulse stretchers in the beam path, is less critical than previously expected.

Accounting for Triplet and Saturation Effects in FCS Measurements

Fluorescence correlation spectroscopy (FCS) is an increasingly important tool for determining low concentrations and dynamics of molecules in solution. Oftentimes triplet transitions give rise to fast blinking effects, which are accounted for by including an exponential term in the fitting of the autocorrelation function (ACF). In such cases, concomitant saturation effects also modify the amplitude and shape of the remaining parts of the ACF. We review studies of triplet and saturation effects in FCS and present a simple procedure to obtain more accurate results of particle concentrations and diffusional dynamics in experiments where triplet kinetics are evident, or where moderate laser powers approaching saturation levels are used, for example, to acquire sufficient photon numbers when observation times are limited. The procedure involves use of a modified function for curve-fitting the ACF, but there are no additional fitting parameters. Instead, a simple calibration of the total fluorescence count rate as a function of relative laser power is fit to a polynomial, and the non-linear components of this fit, together with the relative laser power used for the FCS measurement, are used to specify the magnitude of additional terms in the fitting function. Monte Carlo simulations and experiments using Alexa dyes and quantum dots, with continuous and pulsed laser excitation, demonstrate the application of the modified fitting procedure with first order correction terms, in the regime where distortions in the ACF due to photobleaching and detector dead time are small compared to those of fluorescence saturation and triplet photophysics.

A Five-coordinate Metal Center in Co(II)-substituted VanX

In an effort to structurally probe the metal binding site in VanX, electronic absorption, EPR, and extended x-ray absorption fine structure (EXAFS) spectroscopic studies were conducted on Co(II)-substituted VanX. Electronic spectroscopy revealed the presence of Co(II) ligand field transitions that had molar absorptivities of approximately 100 m(-1) cm(-1), which suggests that Co(II) is five-coordinate in Co(II)-substituted VanX. Low temperature EPR spectra of Co(II)-substituted VanX were simulated using spin Hamiltonian parameters of M(S) = |+/-1/2), E/D = 0.14, g(real(x,y)) = 2.37, and g(real(z)) = 2.03. These parameters lead to the prediction that Co(II) in the enzyme is five-coordinate and that there may be at least one solvent-derived ligand. Single scattering fits of EXAFS data indicate that the metal ions in both native Zn(II)-containing and Co(II)-substituted VanX have the same coordination number and that the metal ions are coordinated by 5 nitrogen/oxygen ligands at approximately 2.0 angstroms. These data demonstrate that Co(II) (and Zn(II) from EXAFS studies) is five-coordinate in VanX in contrast to previous crystallographic studies (Bussiere, D. E., Pratt, S. D., Katz, L., Severin, J. M., Holzman, T., and Park, C. H. (1998) Mol. Cell 2, 75-84). These spectroscopic studies also demonstrate that the metal ion in Co(II)-substituted VanX when complexed with a phosphinate analog of substrate D-Ala-D-Ala is also five-coordinate.

Terrylene in p-terphenyl: a novel single crystalline system for single molecule spectroscopy at low temperatures

Terrylene in p-terphenyl represents a promising novel single crystalline system for optical spectroscopy of single impurity molecules at low temperatures. The optical spectra of terrylene in p-terphenyl show four origins in absorption and emission, indicating four crystalline sites. In one of the sites saturated fluorescence count rates of up to 600000 s −1 were measured for single terrylene molecules. From a measurement of the fluorescence autocorrelation function for single terrylene molecules we could determine the populating and depopulating rates of the triplet state. Using the very small populating rate the triplet quantum yield was calculated to be only ~ 10 −5.

Lifetime and collisional depopulation of the metastable 5D 3/2-state of Yb+

The lifetime and collisional depopulation rates of the metastable 5D 3/2 state of Yb+ have been determined in a radiofrequency ion trap by observation of the fluorescence count rate after ion excitation by a short laser pulse. From measurements using He, N2 and H2 as buffer gases between 10−8 and 10−6 mbar pressure and linear extrapolation to zero pressure we obtain a lifetime of τ=52.15±1.00 ms and rate constants ofR(H2)=(1.02±0.10)×10−9 cm3/s andR(N2)=(1.78±0.19)×10−10 cm3/s. The lifetime is in fair agreement with a calculated value of 74 ms.