Intracellular Zn2+ detection with quantum dot-based FLIM nanosensors

Fluorescence Lifetime Imaging Microscopy (FLIM) has been employed for the detection of intracellular Zn2+ levels, implicated in various signalling pathways, using a family of quantum dot (QD) nanosensors. The sensing mechanism was based on photoinduced electron transfer (PET) between an azacycle receptor group and the QD nanoparticles.This work was supported by Fundación Ramon Areces and grant CTQ2014-56370-R from Ministerio de Economia y Competitividad of Spain

Metallofluorescent Nanoparticles for Multimodal Applications

Herein, we describe the synthesis and application of cross-linked polystyrene-based dual-function nano- and microparticles containing both fluorescent tags and metals. Despite containing a single dye, these particles exhibit a characteristic dual-band fluorescence emission. Moreover, these particles can be combined with different metal ions to obtain hybrid metallofluorescent particles. We demonstrate that these particles are easily nanofected into living cells, allowing them to be used for effective fingerprinting in multimodal fluorescence-based and mass spectrometry-based flow cytometry experiments. Likewise, the in situ reductions of the metal ions enable other potential uses of the particles as heterogeneous catalysts

Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies.This work is funded by grant P10-FQM-6154 from the Consejeria de Innovacion, Ciencia y Empresa (Junta de Andalucia)

Breast Cancer Cell Subtypes Display Different Metabolic Phenotypes That Correlate with Their Clinical Classification

Metabolic reprogramming of cancer cells represents an orchestrated network of evolving molecular and functional adaptations during oncogenic progression. In particular, how metabolic reprogramming is orchestrated in breast cancer and its decisive role in the oncogenic process and tumor evolving adaptations are well consolidated at the molecular level. Nevertheless, potential correlations between functional metabolic features and breast cancer clinical classification still represent issues that have not been fully studied to date. Accordingly, we aimed to investigate whether breast cancer cell models representative of each clinical subtype might display different metabolic phenotypes that correlate with current clinical classifications. In the present work, functional metabolic profiling was performed for breast cancer cell models representative of each clinical subtype based on the combination of enzyme inhibitors for key metabolic pathways, and isotope-labeled tracing dynamic analysis. The results indicated the main metabolic phenotypes, so-called 'metabophenotypes', in terms of their dependency on glycolytic metabolism or their reliance on mitochondrial oxidative metabolism. The results showed that breast cancer cell subtypes display different metabophenotypes. Importantly, these metabophenotypes are clearly correlated with the current clinical classifications.This research, including APC charges, was funded by the Spanish Agencia Estatal de Investigación (Ministry of Science and Innovation) and the European Regional Development Fund [grant numbers CTQ2014-56370-R and CTQ2017-85658-R]; the Fundación Ramón Areces; and the initiative Solidaridad Entre Montañas.Ye

Fluorescence Lifetime Imaging Microscopy for the Detection of Intracellular pH with Quantum Dot Nanosensors

While the use of quantum dot (QD) nanoparticles for bioimaging and sensing has been improved and exploited during the last several years, most studies have used emission intensity-based techniques. Fluorescence lifetime imaging microscopy (FLIM) can also be employed for sensing purposes, overcoming many of the limitations of the aforementioned systems. Herein, we show that the photoluminescence (PL) lifetime of mercaptopropionic acid-capped QDs (MPA-QDs) collected from FLIM images can be used to determine intracellular pH. The PL average lifetime of MPA-QDs varied from 8.7 ns (pH < 5) to 15.4 ns (pH > 8) in media mimicking the intracellular environment. These long decay times of QD nanoparticles make them easily distinguishable from intrinsic cell autofluorescence, improving selectivity in sensing applications. We demonstrate, for the first time, the successful detection of changes in the intracellular pH of different cell types by examining the PL decay time of QDs. In particular, the combination of FLIM methodologies with QD nanoparticles exhibits greatly improved sensitivity compared with other fluorescent dyes for pH imaging. A detailed description of the advantages of the FLIM technique is presented

The First Step of Amyloidogenic Aggregation

The structural and dynamic characterization of the on-pathway intermediates involved in the mechanism of amyloid fibril formation is one of the major remaining biomedical challenges of our time. In addition to mature fibrils, various oligomeric structures are implicated in both the rate-limiting step of the nucleation process and the neuronal toxicity of amyloid deposition. Single-molecule fluorescence spectroscopy (SMFS) is an excellent tool for extracting most of the relevant information on these molecular systems, especially advanced multiparameter approaches, such as pulsed interleaved excitation (PIE). In our investigations of an amyloidogenic SH3 domain of α-spectrin, we have found dynamic oligomerization, even prior to incubation. Our single-molecule PIE experiments revealed that these species are small, mostly dimeric, and exhibit a loose and dynamic molecular organization. Furthermore, these experiments have allowed us to obtain quantitative information regarding the oligomer stability. These pre-amyloidogenic oligomers may potentially serve as the first target for fibrillization-prevention strategies

Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

Abstract: Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies. Int. J. Mol. Sci. 2012, 13 940

Mitochondrial pH Nanosensors for Metabolic Profiling of Breast Cancer Cell Lines.

The main role of mitochondria, as pivotal organelles for cellular metabolism, is the production of energy (ATP) through an oxidative phosphorylation system. During this process, the electron transport chain creates a proton gradient that drives the synthesis of ATP. One of the main features of tumoral cells is their altered metabolism, providing alternative routes to enhance proliferation and survival. Hence, it is of utmost importance to understand the relationship between mitochondrial pH, tumoral metabolism, and cancer. In this manuscript, we develop a highly specific nanosensor to accurately measure the intramitochondrial pH using fluorescence lifetime imaging microscopy (FLIM). Importantly, we have applied this nanosensor to establish differences that may be hallmarks of different metabolic pathways in breast cancer cell models, leading to the characterization of different metabophenotypes

Two-Step Amyloid Aggregation: Sequential Lag Phase Intermediates.

The self-assembly of proteins into fibrillar structures called amyloid fibrils underlies the onset and symptoms of neurodegenerative diseases, such as Alzheimer's and Parkinson's. However, the molecular basis and mechanism of amyloid aggregation are not completely understood. For many amyloidogenic proteins, certain oligomeric intermediates that form in the early aggregation phase appear to be the principal cause of cellular toxicity. Recent computational studies have suggested the importance of nonspecific interactions for the initiation of the oligomerization process prior to the structural conversion steps and template seeding, particularly at low protein concentrations. Here, using advanced single-molecule fluorescence spectroscopy and imaging of a model SH3 domain, we obtained direct evidence that nonspecific aggregates are required in a two-step nucleation mechanism of amyloid aggregation. We identified three different oligomeric types according to their sizes and compactness and performed a full mechanistic study that revealed a mandatory rate-limiting conformational conversion step. We also identified the most cytotoxic species, which may be possible targets for inhibiting and preventing amyloid aggregation