Fluorescence Screening of DNA-AgNCs with Pulsed White Light Excitation.

DNA-stabilized silver nanoclusters (DNA-AgNCs) are a class of fluorophores with interesting photophysical properties dominated by the choice of DNA sequence. Screening methods with ultraviolet excitation and steady state well plate readers have previously been used for deepening the understanding between DNA sequence and emission color of the resulting DNA-AgNCs. Here, we present a new method for screening DNA-AgNCs by using pulsed white light excitation (λex ≈ 490-900 nm). By subtraction and time gating we are able to circumvent the dominating scatter of the white excitation light and extract both temporally and spectrally resolved emission of DNA-AgNCs over the visible to near-infrared range. Additionally, we are able to identify weak long-lived emission, which is often buried underneath the intense nanosecond fluorescence. This new approach will be useful for future screening of DNA-AgNCs (or other novel emissive materials) and aid machine-learning models by providing a richer training data set.

Bioconjugation of a Near-Infrared DNA-Stabilized Silver Nanocluster to Peptides and Human Insulin by Copper-Free Click Chemistry.

DNA-stabilized silver nanoclusters (DNA-AgNCs) are biocompatible emitters with intriguing properties. However, they have not been extensively used for bioimaging applications due to the lack of structural information and hence predictable conjugation strategies. Here, a copper-free click chemistry method for linking a well-characterized DNA-AgNC to molecules of interest is presented. Three different peptides and a small protein, human insulin, were tested as labeling targets. The conjugation to the target compounds was verified by MS, HPLC, and time-resolved anisotropy measurements. Moreover, the spectroscopic properties of DNA-AgNCs were found to be unaffected by the linking reactions. For DNA-AgNC-conjugated human insulin, fluorescence imaging studies were performed on Chinese hamster ovary (CHO) cells overexpressing human insulin receptor B (hIR-B). The specific staining of the CHO cell membranes demonstrates that DNA-AgNCs are great candidates for bioimaging applications, and the proposed linking strategy is easy to implement when the DNA-AgNC structure is known.

Chloride Ligands on DNA-Stabilized Silver Nanoclusters.

DNA-stabilized silver nanoclusters (AgN-DNAs) are known to have one or two DNA oligomer ligands per nanocluster. Here, we present the first evidence that AgN-DNA species can possess additional chloride ligands that lead to increased stability in biologically relevant concentrations of chloride. Mass spectrometry of five chromatographically isolated near-infrared (NIR)-emissive AgN-DNA species with previously reported X-ray crystal structures determines their molecular formulas to be (DNA)2[Ag16Cl2]8+. Chloride ligands can be exchanged for bromides, which red-shift the optical spectra of these emitters. Density functional theory (DFT) calculations of the 6-electron nanocluster show that the two newly identified chloride ligands were previously assigned as low-occupancy silvers by X-ray crystallography. DFT also confirms the stability of chloride in the crystallographic structure, yields qualitative agreement between computed and measured UV-vis absorption spectra, and provides interpretation of the 35Cl-nuclear magnetic resonance spectrum of (DNA)2[Ag16Cl2]8+. A reanalysis of the X-ray crystal structure confirms that the two previously assigned low-occupancy silvers are, in fact, chlorides, yielding (DNA)2[Ag16Cl2]8+. Using the unusual stability of (DNA)2[Ag16Cl2]8+ in biologically relevant saline solutions as a possible indicator of other chloride-containing AgN-DNAs, we identified an additional AgN-DNA with a chloride ligand by high-throughput screening. Inclusion of chlorides on AgN-DNAs presents a promising new route to expand the diversity of AgN-DNA structure-property relationships and to imbue these emitters with favorable stability for biophotonics applications.

A DNA-Stabilized Ag18 12+ Cluster with Excitation-Intensity-Dependent Dual Emission.

DNA-stabilized silver nanoclusters (DNA-AgNCs) are easily tunable emitters with intriguing photophysical properties. Here, a DNA-AgNC with dual emission in the red and near-infrared (NIR) regions is presented. Mass spectrometry data showed that two DNA strands stabilize 18 silver atoms with a nanocluster charge of 12+. Besides determining the composition and charge of DNA2 [Ag18 ]12+ , steady-state and time-resolved methods were applied to characterize the picosecond red fluorescence and the relatively intense microsecond-lived NIR luminescence. During this process, the luminescence-to-fluorescence ratio was found to be excitation-intensity-dependent. This peculiar feature is very rare for molecular emitters and allows the use of DNA2 [Ag18 ]12+ as a nanoscale excitation intensity probe. For this purpose, calibration curves were constructed using three different approaches based either on steady-state or time-resolved emission measurements. The results showed that processes like thermally activated delayed fluorescence (TADF) or photon upconversion through triplet-triplet annihilation (TTA) could be excluded for DNA2 [Ag18 ]12+ . We, therefore, speculate that the ratiometric excitation intensity response could be the result of optically activated delayed fluorescence.

Single-molecule excitation-emission spectroscopy.

Single-molecule spectroscopy (SMS) provides a detailed view of individual emitter properties and local environments without having to resort to ensemble averaging. While the last several decades have seen substantial refinement of SMS techniques, recording excitation spectra of single emitters still poses a significant challenge. Here we address this problem by demonstrating simultaneous collection of fluorescence emission and excitation spectra using a compact common-path interferometer and broadband excitation, which is implemented as an extension of a standard SMS microscope. We demonstrate the technique by simultaneously collecting room-temperature excitation and emission spectra of individual terrylene diimide molecules and donor-acceptor dyads embedded in polystyrene. We analyze the resulting spectral parameters in terms of optical lineshape theory to obtain detailed information on the interactions of the emitters with their nanoscopic environment. This analysis finally reveals that environmental fluctuations between the donor and acceptor in the dyads are not correlated.

Frequency Encoding of Upconversion Nanoparticle Emission for Multiplexed Imaging of Spectrally and Spatially Overlapping Lanthanide Ions.

We present frequency encoded upconversion (FE-UPCON) widefield microscopy, an imaging approach that allows for multiplexed signal recovery based on frequency encoding of selected upconverted lanthanide ion emission rather than separation based on energy or time. FE-UPCON allows for the separation of luminescence from spectrally and spatially overlapping trivalent lanthanide ions (Ln3+) in upconversion nanoparticles (UCNPs). Utilizing the numerous electronic energy levels of Ln3+, one can generate a frequency encoded signal by periodic coexcitation with a secondary light source (modulated at a chosen frequency) that, for a particular wavelength, enhances the luminescence of the Ln3+ of interest. We demonstrate that it is possible to selectively image spectrally overlapping UCNPs co-doped with Yb3+/Ho3+ or Yb3+/Er3+ by FE-UPCON in cells up to 10 frames per second on a conventional widefield microscope with the simple extension of an additional secondary light source and a chopper wheel for modulation. Additionally, we show that FE-UPCON does not compromise sensitivity and that single UCNP detection is obtainable. FE-UPCON adds a new dimension (frequency space) for multiplexed imaging with UCNPs.

Rational Design of Bright Long Fluorescence Lifetime Dyad Fluorophores for Single Molecule Imaging and Detection.

Increasing demand for detecting single molecules in challenging environments has raised the bar for the fluorophores used. To achieve better resolution and/or contrast in fluorescence microscopy, it is now essential to use bright and stable dyes with tailored photophysical properties. While long fluorescence lifetime fluorophores offer many advantages in time-resolved imaging, their inherently lower molar absorption coefficient has limited applications in single molecule imaging. Here we propose a generic approach to prepare bright, long fluorescence lifetime dyad fluorophores comprising an absorbing antenna chromophore with high absorption coefficient linked to an acceptor emitter with a long fluorescence lifetime. We introduce a dyad consisting of a perylene antenna and a triangulenium emitter with 100% energy transfer from donor to acceptor. The dyad retained the long fluorescence lifetime (∼17 ns) and high quantum yield (75%) of the triangulenium emitter, while the perylene antenna increased the molar absorption coefficient (up to 5 times) in comparison to the free triangulenium dye. These triangulenium based dyads with significantly improved brightness can now be detected at the single molecule level and easily discriminated from bright autofluorescence by time-gated and other lifetime-based detection schemes.

Observation of microsecond luminescence while studying two DNA-stabilized silver nanoclusters emitting in the 800-900 nm range.

We investigated two DNA-stabilized silver nanoclusters (DNA-AgNCs) that show multiple absorption features in the visible region, and emit around 811 nm (DNA811-AgNC) and 841 nm (DNA841-AgNC). Both DNA-AgNCs have large Stokes shifts and can be efficiently excited with red light. A comparison with the commercially available Atto740 yielded fluorescence quantum yields in the same order of magnitude, but a higher photon output above 800 nm since both DNA-AgNCs are more red-shifted. The study of both DNA-AgNCs also revealed previously unobserved photophysical behavior for this class of emitters. The fluorescence quantum yield and decay time of DNA841-AgNC can be increased upon consecutive heating/cooling cycles. DNA811-AgNC has an additional absorption band around 470 nm, which is parallel in orientation to the lowest energy transition at 640 nm. Furthermore, we observed for the first time a DNA-AgNC population (as part of the DNA811-AgNC sample) with green and near-infrared emissive states with nanosecond and microsecond decay times, respectively. A similar dual emissive DNA-AgNC stabilized by a different 10-base DNA strand is also reported in the manuscript. These two examples highlight the need to investigate the presence of red-shifted microsecond emission for this class of emitters.

Solvent-Dependent Growth and Stabilization Mechanisms of Surfactant-Free Colloidal Pt Nanoparticles.

Understanding the formation of nanoparticles (NPs) is key to develop materials by sustainable routes. The Co4CatTM process is a new synthesis of precious metal NPs in alkaline mono-alcohols well-suited to develop active nanocatalysts. The synthesis is 'facile', surfactant-free and performed under mild conditions like low temperature. The reducing properties of the solvent are here shown to strongly influence the formation of Pt NPs. Based on the in situ formation of CO adsorbed on the NP surface by solvent oxidation, a model is proposed that accounts for the different growth and stabilization mechanisms as well as re-dispersion properties of the surfactant-free NPs in different solvents. Using in situ and ex situ characterizations, it is established that in methanol, a slow nucleation with a limited NP growth is achieved. In ethanol, a fast nucleation followed by continuous and pronounced particle sintering occurs.

Spectral shifts of BODIPY derivatives: a simple continuous model.

The BODIPY dyes are a versatile family of chromophores that have found use in fluorescence based bioimaging and other applications. The BODIPY core can be substituted in a vast number of ways, but the photophysical changes, such as shifts in absorption spectra, are not always immediately obvious from the molecular structure. We introduce a simple model that let you vary the electron withdrawing or electron donating character of each substituent continuously to get an overview of the landscape of possible spectral shifts. The features of substituted BODIPY cores are compared to the corresponding linear system, giving a new perspective on BODIPY photophysics. Using the model, we are able to rationalize the trend seen in a family of BODIPY, with chalcogen-containing substituents, as being due to a change in electronegativity.

Crystal structure of a NIR-Emitting DNA-Stabilized Ag16 Nanocluster.

DNA has been used as a scaffold to stabilize small, atomically monodisperse silver nanoclusters, which have attracted attention due to their intriguing photophysical properties. Herein, we describe the X-ray crystal structure of a DNA-encapsulated, near-infrared emitting Ag16 nanocluster (DNA-Ag16 NC). The asymmetric unit of the crystal contains two DNA-Ag16 NCs and the crystal packing between the DNA-Ag16 NCs is promoted by several interactions, such as two silver-mediated base pairs between 3'-terminal adenines, two phosphate-Ca2+ -phosphate interactions, and π-stacking between two neighboring thymines. Each Ag16 NC is confined by two DNA decamers that take on a horse-shoe-like conformation and is almost fully shielded from the solvent environment. This structural insight will aid in the determination of the structure/photophysical property relationship for this class of emitters and opens up new research opportunities in fluorescence imaging and sensing using noble-metal clusters.

Upconversion Cross-Correlation Spectroscopy of a Sandwich Immunoassay.

Fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) have enabled biologists to study processes of transport, binding, and enzymatic reactions in living cells. However, applying FCS and FCCS to samples such as whole blood and plasma is complicated as the fluorescence bursts of diffusing labels can be swamped by strong autofluorescence. Here we present cross-correlation spectroscopy based on two upconversion nanoparticles emitting at different wavelengths on the anti-Stokes side of a single excitation laser. This upconversion cross-correlation spectroscopy (UCCS) approach allows us to completely remove all Stokes shifted autofluorescence background in biological material such as plasma. As a proof of concept, we evaluate the applicability of UCCS to a homogeneous sandwich immunoassay for thyroid stimulating hormone measured in buffer solution and in plasma.

Luminescence from Lanthanide(III) Ions Bound to the Glycocalyx of Chinese Hamster Ovary Cells.

Lanthanide(III) ions bind to the glycocalyx of Chinese Hamster Ovary (CHO) cells and give rise to a unique luminescent fingerprint. Following direct excitation of terbium(III) and europium(III) ions in the visible part of the spectrum, we are able to collect emission spectra pixel-by-pixel in images of CHO cells. Following data analysis that removes the background signal, the fine structure of the europium(III) luminescence indicate that the lanthanide(III) ions are bound to a single structure of the CHO cell glycocalyx. This was deduced from the fact that the structure-sensitive emission spectrum of europium is unchanged throughout the investigated samples.

Probing heterogeneity of NIR induced secondary fluorescence from DNA-stabilized silver nanoclusters at the single molecule level.

In this communication, we investigate optically activated delayed fluorescence (OADF) from DNA-stabilized silver nanoclusters (DNA-AgNCs) at the single molecule level, and we probe the heterogeneity in the primary fluorescence (PF) intensity, NIR induced secondary fluorescence (SF) intensity and SF/PF ratio. Our experiments reveal a heterogeneous distribution in the SF/PF ratio, indicating that engineering of DNA-AgNCs towards a high SF/PF ratio and high OADF signal for background-free imaging might be possible.

Probing the Absorption and Emission Transition Dipole Moment of DNA Stabilized Silver Nanoclusters.

Using single molecule polarization measurements, we investigate the excitation and emission polarization characteristics of DNA stabilized silver nanoclusters (C24-AgNCs). Although small changes in the polarization generally accompany changes to the emission spectrum, the emission and excitation transition dipoles tend to be steady over time and aligned in a similar direction, when immobilized in PVA. The emission transition dipole patterns, observed for C24-AgNCs in defocused wide field imaging, match that of a single emitter. The small changes to the polarization and spectral shifting that were observed could be due to changes to the conformation of the AgNC or the DNA scaffold. Although less likely, an alternative explanation could be that several well aligned spectrally similar emitters are present within the DNA scaffold which, due to Förster resonance energy transfer (FRET) processes such as energy hopping, energy transfer, and singlet-singlet annihilation, behave as a single emitter. The reported results can provide more insight in the structural and photophysical properties of DNA-stabilized AgNCs.

Peptide-Stabilized, Fluorescent Silver Nanoclusters: Solid-Phase Synthesis and Screening.

Few-atom silver nanoclusters (AgNCs) can exhibit strong fluorescence; however, they require ligands to prevent aggregation into larger nanoparticles. Fluorescent AgNCs in biopolymer scaffolds have so far mainly been synthesized in solution, and peptides have only found limited use compared to DNA. Herein, we demonstrate how solid-phase methods can increase throughput dramatically in peptide ligand screening and in initial evaluation of fluorescence intensity and chemical stability of peptide-stabilized AgNCs (P-AgNCs). 9-Fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis on a hydroxymethyl-benzoic acid (HMBA) polyethylene glycol polyacrylamide copolymer (PEGA) resin enabled on-resin screening and evaluation of a peptide library, leading to identification of novel peptide-stabilized, fluorescent AgNCs. Using systematic amino acid substitutions, we synthesized and screened a 144-member library. This allowed us to evaluate the effect of length, charge, and Cys content in peptides used as ligands for AgNC stabilization. The results of this study will enable future spectroscopic studies of these peptide-stabilized AgNCs for bioimaging and other applications.

Probing DNA-stabilized fluorescent silver nanocluster spectral heterogeneity by time-correlated single photon counting.

DNA-stabilized silver nanoclusters (DNA-AgNCs) are promising fluorophores whose photophysical properties and synthesis procedures have received increased attention in the literature. However, depending on the preparation conditions and the DNA sequence, the DNA-AgNC samples can host a range of different emitters, which can influence the reproducibility of the optical response and the evolution over time of the populations of these emitters. We have developed a simple method to characterize the spectral heterogeneity and time evolution of these emissive species at any given point in time after preparation, by plotting the average decay time as a function of emission wavelength. These so-called average decay time spectra were acquired for different excitation wavelengths of AgNCs stabilized by an oligonucleotide containing 24 cytosines (C24-AgNCs). The average decay time spectra allowed the comparison of sample preparation and the judgment of reproducibility. Therefore, we propose the use of the average decay time spectra as a robust and easy tool to characterize and compare different as-synthesized DNA-AgNC samples. The average decay time spectra can in general also be used to characterize the spectral heterogeneity of other fluorophores, such as luminescent colloidal nanoparticles, and to assess the reproducibility of a synthetic procedure containing an unknown distribution of emissive species.

Morphology and composition of oxidized InAs nanowires studied by combined Raman spectroscopy and transmission electron microscopy.

Any device exposed to ambient conditions will be prone to oxidation. This may be of particular importance for semiconductor nanowires because of the high surface-to-volume ratio and only little is known about the consequences of oxidation for these systems. Here, we study the properties of indium arsenide nanowires which were locally oxidized using a focused laser beam. Polarization dependent micro-Raman measurements confirmed the presence of crystalline arsenic, and transmission electron microscopy diffraction showed the presence of indium oxide. The surface dependence of the oxidation was investigated in branched nanowires grown along the [Formula: see text] and [Formula: see text] wurtzite crystal directions exhibiting different surface facets. The oxidation did not occur at the [Formula: see text] direction. The origin of this selectivity is discussed in terms transition state kinetics of the free surfaces of the different crystal families of the facets and numerical simulations of the laser induced heating.

A Comparison of Single-Molecule Emission in Aluminum and Gold Zero-Mode Waveguides.

The effect of gold and aluminum zero-mode waveguides (ZMWs) on the brightness of immobilized single emitters was characterized by probing fluorophores that absorb in the green and red regions of the visible spectrum. Aluminum ZMWs enhance the emission of Atto565 fluorophores upon green excitation, but they do not enhance the emission of Atto647N fluorophores upon red excitation. Gold ZMWs increase emission of both fluorophores with Atto647N showing enhancement that is threefold higher than that observed for Atto565. This work indicates that 200 nm gold ZMWs are better suited for single-molecule fluorescence studies in the red region of the visible spectrum, while aluminum appears more suited for the green region of the visible spectrum.

Emissive Photoconversion Products of an Amino-triangulenium Dye.

Upon prolonged exposure to intense blue light, the tris(diethylamino)-trioxatriangulenium (A3-TOTA(+)) fluorophore can undergo a photochemical reaction to form either a blue-shifted or a red-shifted fluorescent photoproduct. The formation of the latter depends on the amount of oxygen present during the photoconversion. The A3-TOTA(+) fluorophore is structurally similar to rhodamine, with peripheral amino groups on a cationic aromatic system. The photoconversion products were identified by UV-vis absorption and steady-state and time-resolved fluorescence spectroscopy, and further characterized by HPLC, LC-MS, and (1)H NMR. Two reaction pathways were identified: a dealkylation reaction and an oxidation leading to formation of one or more amide groups on the peripheral donor groups. The photoconversion is controlled by the experimental conditions, in particular the presence of oxygen and water, and the choice of solvent. The results highlight the need to characterize the formation of fluorescent photoproducts of commonly used fluorescent probes, since these could give rise to false positives in multicolor/multilabel imaging, colocalization studies, and FRET based assays. Finally, an improved understanding of the photochemical reaction leading to bleaching of fluorescent dyes can lead to the creation of specific probes for fluorescence based monitoring of chemical reactions.