Miguel R. Carro-Temboury

Ultrafast coherence transfer in DNA-templated silver nanoclusters

DNA-templated silver nanoclusters of a few tens of atoms or less have come into prominence over the last several years due to very strong absorption and efficient emission. Applications in microscopy and sensing have already been realized, however little is known about the excited-state structure and dynamics in these clusters. Here we report on a multidimensional spectroscopy investigation of the energy-level structure and the early-time relaxation cascade, which eventually results in the population of an emitting state. We find that the ultrafast intramolecular relaxation is strongly coupled to a specific vibrational mode, resulting in the concerted transfer of population and coherence between excited states on a sub-100 fs timescale.

Excited-State Relaxation and Förster Resonance Energy Transfer in an Organic Fluorophore/Silver Nanocluster Dyad

A single-stranded DNA-based (ssDNA) dyad was constructed comprising 15 silver atoms stabilized by a ssDNA scaffold (DNA-AgNC) and an Alexa 546 fluorophore bound to the 5′ end. The Alexa 546 was chosen to function as a Förster resonance energy transfer (FRET) donor for the AgNC. Time-correlated single photon counting (TCSPC) experiments allowed unraveling the excited-state relaxation processes of the purified DNA-AgNC-only system. The TCSPC results revealed slow relaxation dynamics and a red shift of the emission spectrum during the excited-state lifetime. The results from the model systems were needed to understand the more complicated decay pathways present in the collected high-performance liquid chromatography fraction, which contained the dyad (37% of the emissive population). In the dyad system, the FRET efficiency between donor and acceptor was determined to be 94% using TCSPC, yielding a center-to-center distance of 4.6 nm. To date, only limited structural information on DNA-AgNCs is available and the use of TCSPC and FRET can provide information on the center-to-center distance between chromophores and provide positional information in nanostructures composed of AgNCs.

Unusually large Stokes shift for a near-infrared emitting DNA-stabilized silver nanocluster

In this paper we present a new near-IR emitting silver nanocluster (NIR-DNA-AgNC) with an unusually large Stokes shift between absorption and emission maximum (211 nm or 5600 cm-1). We studied the effect of viscosity and temperature on the steady state and time-resolved emission. The time-resolved results on NIR-DNA-AgNC show that the relaxation dynamics slow down significantly with increasing viscosity of the solvent. In high viscosity solution, the spectral relaxation stretches well into the nanosecond scale. As a result of this slow spectral relaxation in high viscosity solutions, a multi-exponential fluorescence decay time behavior is observed, in contrast to the more mono-exponential decay in low viscosity solution.

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.

Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission

The popularity of fluorescence microscopy arises from the inherent mode of action, where the fluorescence emission from probes is used to visualize selected features on a presumed dark background. However, the background is rarely truly dark, and image processing and analysis is needed to enhance the fluorescent signal that is ascribed to the selected feature. The image acquisition is facilitated by using considerable illumination, bright probes at a relatively high concentration in order to make the fluorescent signal significantly more intense than the background signal. Here, we present two methods for completely removing the background signal in spectrally resolved fluorescence microscopy. The methodology is applicable for all probes with narrow and well-defined emission bands (Full width half-maximum < 20 nm). Here, we use two lanthanide based probes exploiting the narrow emission lines of europium(III) and terbium(III) ions. We used a model system with zeolites doped with lanthanides immobilized in a polymer stained with several fluorescent dyes regularly used in bioimaging. After smoothing the spectral data recorded in each pixel, they are differentiated. Method I is based on the direct sum of the gradient, while method II resolves the fluorescent signal by subtracting a background calculated via the gradient. Both methods improve signal-to-background ratio significantly and we suggest that spectral imaging of lanthanide-centered emission can be used as a tool to obtain absolute contrast in bioimaging.

Investigating dye performance and crosstalk in fluorescence enabled bioimaging using a model system

Detailed imaging of biological structures, often smaller than the diffraction limit, is possible in fluorescence microscopy due to the molecular size and photophysical properties of fluorescent probes. Advances in hardware and multiple providers of high-end bioimaging makes comparing images between studies and between research groups very difficult. Therefore, we suggest a model system to benchmark instrumentation, methods and staining procedures. The system we introduce is based on doped zeolites in stained polyvinyl alcohol (PVA) films: a highly accessible model system which has the properties needed to act as a benchmark in bioimaging experiments. Rather than comparing molecular probes and imaging methods in complicated biological systems, we demonstrate that the model system can emulate this complexity and can be used to probe the effect of concentration, brightness, and cross-talk of fluorophores on the detected fluorescence signal. The described model system comprises of lanthanide (III) ion doped Linde Type A zeolites dispersed in a PVA film stained with fluorophores. We tested: F18, MitoTracker Red and ATTO647N. This model system allowed comparing performance of the fluorophores in experimental conditions. Importantly, we here report considerable cross-talk of the dyes when exchanging excitation and emission settings. Additionally, bleaching was quantified. The proposed model makes it possible to test and benchmark staining procedures before these dyes are applied to more complex biological systems.

An optical authentication system based on imaging of excitation-selected lanthanide luminescence

Random patterns of lanthanide(III) ion–doped zeolites were used to create a tamper-proof optical authentication system. Secure data encryption relies heavily on one-way functions, and copy protection relies on features that are difficult to reproduce. We present an optical authentication system based on lanthanide luminescence from physical one-way functions or physical unclonable functions (PUFs). They cannot be reproduced and thus enable unbreakable encryption. Further, PUFs will prevent counterfeiting if tags with unique PUFs are grafted onto products. We have developed an authentication system that comprises a hardware reader, image analysis, and authentication software and physical keys that we demonstrate as an anticounterfeiting system. The physical keys are PUFs made from random patterns of taggants in polymer films on glass that can be imaged following selected excitation of particular lanthanide(III) ions doped into the individual taggants. This form of excitation-selected imaging ensures that by using at least two lanthanide(III) ion dopants, the random patterns cannot be copied, because the excitation selection will fail when using any other emitter. With the developed reader and software, the random patterns are read and digitized, which allows a digital pattern to be stored. This digital pattern or digital key can be used to authenticate the physical key in anticounterfeiting or to encrypt any message. The PUF key was produced with a staggering nominal encoding capacity of 73600. Although the encoding capacity of the realized authentication system reduces to 6 × 10104, it is more than sufficient to completely preclude counterfeiting of products.

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.