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superbright, photostabLe and mUlticolour novel fluoresCent mEtal quaNtum clusters for super-resoluTion imaging

Periodic Reporting for period 1 - LUCENT (superbright, photostabLe and mUlticolour novel fluoresCent mEtal quaNtum clusters for super-resoluTion imaging)

Reporting period: 2019-05-01 to 2021-04-30

The advent of super resolution optical microscopy (SRM) revolutionized imaging technologies providing new tools to visualize cells and synthetic materials with unprecedented detail. SRM maintains some of the key features of fluorescence microscopy, e.g. multicolour ability and minimal invasiveness, but overcome the diffraction limit (~ 250 nm) offering a nanometric resolution – namely Nanoscopy. Since then, it has become possible to directly detect and image subcellular features and synthetic nanostructures, impacting the fields of cell biology, chemistry and nanotechnology.
Although working principles and instrumentation of nanoscopes differ among techniques, they all share the same basic idea: the super resolution does not arise from physical means (e.g. optics) but from the accurate control of the state of the fluorescent markers – i.e. from the photochemistry and photophysics of the labels. The great technological advancements over the last years resulted in a plethora of nanoscopy techniques such as: stochastic optical reconstruction microscopy (STORM), photo-activated localization microscopy (PALM), and points accumulation for imaging in nanoscale topography (PAINT). All these techniques rely on bright photostable fluorophores that can switched on and off in a controlled manner. Therefore, the development of probes suitable for super resolution microscopy is currently the limiting factor for the performances of state-of-the-art nanoscopes.
As a result, further advancements in the field critically depend on the ability to develop and manipulate fluorescent probes, which are still numbered. This project aims to provide novel model systems for improved single molecule imaging by developing photoluminescent metal quantum clusters (MQCs) as advanced optical probes for super-resolution microscopy. MQCs are extremely promising probes for nanoscopy because they effectively combine ultra-small sizes, brightness and high photoluminescence (PL) efficiency with good photostability and chemical inertness, which render them interesting candidates as highly biocompatible fluorescent markers. To this purpose, property-designed nanochemistry routes by combining quantum size effects and surface engineered strategies will be used for producing multicolour MQCs. Using advanced microscopy techniques we aim at fully understanding of the photophysical and photochemistry features of different nanoparticle based probes, revealing properties like brightness, stabilility and photoswitching behaviour at single particle level. Finally, the toxicity and selective targeting of the new probes will be investigated. This research will have a strong impact on broad scientific community, namely materials science, colloidal chemistry and nanoscopy fields.
The project “superbright, photostabLe and mUlticolour novel fluoresCent mEtal quaNtum clusters for super-resoluTion imaging” (LUCENT) aimed at developing new generation of advanced optical probes for super-resolution microscopy. The main idea was to use property-designed nanochemistry routes by combining bottom up and top-down approaches as well as surface engineered strategies for producing fluorescent nanoclusters emitting different colours that could ultimately serve as novel model systems for improving single molecule imaging.
For achieving this goal the research activity was structured in three main working packages dealing with a) the design and synthesis of metal nanoclusters with different emission colours together with their physicochemical characterization; b) to study their photophysical behaviour with focus on brightness, stability and photoswitching (properties required for super resolution imaging), and c) the toxicity investigation, selective targeting and implement nanoparticle based probes as fluorescent markers in Nanoscopy. For comparison, different types of semiconductor nanocrystal quantum dots with different emissions have been acquired through collaborations as well as from companies.

During the project it has been achieved the reproducible synthesis of multicolour advanced nanoclusters and their correspondent photophysical properties has been deeply investigated. An arsenal of varied nanoparticle based fluorophores potentially applied as markers in nanoscopy techniques were obtained. The variability on both fluorescent properties in terms of excitation and emission, as well as different quantum yields facilitate the implementation of these fluorescent particle based materials in super resolution techniques. For the photophysics studies, all the materials were tested under TIRF microscope. For this purpose, the sample preparation protocol was first optimized and afterwards, a series of different amount of frames were taken under TIRF microscope for obtaining the blinking dynamics that allowed to extract both bright and dark time, brightness and fluorescence emission spectra at single particle level. The innovation of these studies arises from the single particle level information obtained thanks to nanoscopy techniques. These photophysics studies are of paramount importance not only for determining which fluorescent particles can be used as novel fluorescent probes for super resolution microscopy but also because they can reveal heterogeneities on sample preparation. Importantly, this information can be used to identify both suitable probes for nanoscopy as well as the best synthesis procedures for obtaining homogenous fluorescent particles with desired optical properties. At the moment all these information has been analyzed and being prepared for exploiting the results both on social media and as well to publish them on journals of high impact factor.
The advancements of super resolution microscopy techniques will play key role in our society in the coming years thanks to its potential to impact many scientific fields. The capability of nanoscopy to obtain molecular insights into structure, dynamics and functions of complex synthetic and biologic systems with nanometric accuracy will reveal new discoveries in numerous fields, including supramolecular chemistry, biomaterials, catalysis, plasmonics, and many more. This ability will rely mostly on the synthesis of small, brighter, and photostable fluorophores with controlled photophysical and photochemical properties, which are still numbered. The results of this project demonstrates the potential of fluorescent nanoparticle based probes for this paradigm shift. Furthermore, super resolution microscopy techniques have been revealed as a robust method for characterizing the photophysical properties of nanoparticles at single particle level, providing multiparametric information that is not possible to achieve with other techniques. The correlation between various photophysical properties like blinking dynamics, brightness and emission spectra at single particle level have been achieved. This information will provide a remarkable advancement towards the development of novel nanoparticle based probes for nanoscopy. Colloidal chemists, materials scientists, nanotechnologists and spectroscopists will benefit from this information that will help to improve and optimize the rational design of fluorescent nanoparticles, paving the way to solve challenges like sample heterogeneity in nanoparticle formulation.
a) Schematic representation of MQCs and QDs samples b) Scheme of spectral PAINT setup and raw data