Optical Binding-Driven Micropatterning and Photosculpting with Silver Nanorods.

Controlling the nano- and micropatterning of metal structures is an important requirement for various technological applications in photonics and biosensing. This work presents a method for controllably creating silver micropatterns by laser-induced photosculpting. Photosculpting is driven by plasmonic interactions between pulsed laser radiation and silver nanorods (AgNRs) in aqueous suspension; this process leads to optical binding forces transporting the AgNRs in the surroundings, while electronic thermalization results in photooxidation, melting, and ripening of the AgNRs into well-defined 3D structures. This work call these structures Airy castles due to their structural similarity with a diffraction-limited Airy disk. The photosculpted Airy castles contain emissive Ag nanoclusters, allowing for the visualization and examination of the aggregation process using luminescence microscopy. This work comprehensively examines the factors that define the photosculpting process, namely, the concentration and shape of the AgNRs, as well as the energy, power, and repetition rate of the laser. Finally, this work investigates the potential applications by measuring the metal-enhanced luminescence of a europium-based luminophore using Airy castles.

A multicomponent reaction platform towards multimodal near-infrared BODIPY dyes for STED and fluorescence lifetime imaging.

We report a platform combining multicomponent reaction synthesis and automated cell-based screening to develop biocompatible NIR-BODIPY fluorophores. From a library of over 60 fluorophores, we optimised compound NIRBD-62c as a multimodal probe with suitable properties for STED super-resolution and fluorescence lifetime imaging. Furthermore, we employed NIRBD-62c for imaging trafficking inside cells and to examine how pharmacological inhibitors can alter the vesicular traffic between intracellular compartments and the plasma membrane.

Dynamic Excimer (DYNEX) Imaging of Lipid Droplets.

Unraveling cellular physiological processes via luminescent probes that target specific cellular microenvironments is quite challenging due to the uneven distribution of probes. Herein, we designed a new dynamic excimer (DYNEX) imaging method that involves the sensitive detection of nanosecond-scale dynamic molecular contacts of a fluorescent acridone derivative and reveals the cell microenvironment polarity. Using our method, we specifically tracked cell lipid droplets in fibroblast colon carcinoma cells. These organelles play a central role in metabolic pathways, acting as energy reservoirs in regulatory processes. DYNEX imaging provides the inner polarity of cell lipid droplets, which can be related to lipid contents and metabolic dysfunctions. This new methodology will inspire development of novel multidimensional fluorescent sensors that are able to provide target-specific and orthogonal information at the nanosecond scale.

Synthesis, biological, and photophysical studies of molecular rotor-based fluorescent inhibitors of the trypanosome alternative oxidase.

We have recently reported on the development and trypanocidal activity of a class of inhibitors of Trypanosome Alternative Oxidase (TAO) that are targeted to the mitochondrial matrix by coupling to lipophilic cations via C14 linkers to enable optimal interaction with the enzyme's active site. This strategy resulted in a much-enhanced anti-parasite effect, which we ascribed to the greater accumulation of the compound at the location of the target protein, i.e. the mitochondrion, but to date this localization has not been formally established. We therefore synthesized a series of fluorescent analogues to visualize accumulation and distribution within the cell. The fluorophore chosen, julolidine, has the remarkable extra feature of being able to function as a viscosity sensor and might thus additionally act as a probe of the cellular glycerol that is expected to be produced when TAO is inhibited. Two series of fluorescent inhibitor conjugates incorporating a cationic julolidine-based viscosity sensor were synthesized and their photophysical and biological properties were studied. These probes display a red emission, with a high signal-to-noise ratio (SNR), using both single- and two-photon excitation. Upon incubation with T. brucei and mammalian cells, the fluorescent inhibitors 1a and 2a were taken up selectively in the mitochondria as shown by live-cell imaging. Efficient partition of 1a in functional isolated (rat liver) mitochondria was estimated to 66 ± 20% of the total. The compounds inhibited recombinant TAO enzyme in the submicromolar (1a, 2c, 2d) to low nanomolar range (2a) and were effective against WT and multidrug-resistant trypanosome strains (B48, AQP1-3 KO) in the submicromolar range. Good selectivity (SI > 29) over mammalian HEK cells was observed. However, no viscosity-related shift could be detected, presumably because the glycerol was produced cytosolically, and released through aquaglyceroporins, whereas the probe was located, virtually exclusively, in the trypanosome's mitochondrion.

Chimeric Drug Design with a Noncharged Carrier for Mitochondrial Delivery.

Recently, it was proposed that the thiophene ring is capable of promoting mitochondrial accumulation when linked to fluorescent markers. As a noncharged group, thiophene presents several advantages from a synthetic point of view, making it easier to incorporate such a side moiety into different molecules. Herein, we confirm the general applicability of the thiophene group as a mitochondrial carrier for drugs and fluorescent markers based on a new concept of nonprotonable, noncharged transporter. We implemented this concept in a medicinal chemistry application by developing an antitumor, metabolic chimeric drug based on the pyruvate dehydrogenase kinase (PDHK) inhibitor dichloroacetate (DCA). The promising features of the thiophene moiety as a noncharged carrier for targeting mitochondria may represent a starting point for the design of new metabolism-targeting drugs.

Building Accurate Intracellular Polarity Maps through Multiparametric Microscopy.

The precise knowledge of intracellular polarity, a physiological parameter that involves complex and intertwined intracellular mechanisms, may be relevant in the study of important diseases like cancer or Alzheimer's. In this technical note, we illustrate our recently developed, accurate method for obtaining intracellular polarity maps employing potent fluorescence microscopy techniques. Our method is based on the selection of appropriate luminescent probes, in which several emission properties vary with microenvironment polarity, specifically spectral shifts and luminescence lifetime. A multilinear calibration is performed, correlating polarity vs. spectral shift vs. luminescence lifetime, to generate a powerful and error-free 3D space for reliable interpolation of microscopy data. Multidimensional luminescence microscopy is then used to obtain simultaneously spectral shift and luminescence lifetime images, which are then interpolated in the 3D calibration space, resulting in accurate, quantitative polarity maps.

miR-122 direct detection in human serum by time-gated fluorescence imaging.

A simple method for direct detection of microRNAs (miRs) in human serum without the use of polymerase amplification is presented, achieving low miR-122 concentrations and importantly, discerning effectively single-base sequence mutations. The method is based on the capture of target miRs with synthetic peptide nucleic acid oligomers, dynamic chemical labelling, separation with quaternary amine microplatforms and detection using time-gated fluorescence imaging.

Coupled Excited-State Dynamics in N-Substituted 2-Methoxy-9-Acridones.

Fluorophores of the acridone family have been widely employed in many applications, such as DNA sequencing, the detection of biomolecules, and the monitoring of enzymatic systems, as well as being the bases of intracellular sensors and even antitumoral agents. They have been widely used in fluorescence imaging due to their excellent photophysical properties, in terms of quantum yield and stability. However, frequently, the fluorescence emission data from acridones are not easily interpretable due to complex excited-state dynamics. The formation of π-stacking aggregates and excimers and excited-state proton transfer (ESPT) reactions usually result in emission features that are dependent on the experimental conditions. Therefore, an in-depth understanding of the dynamics involved in the excited-state transients of these dyes is mandatory for their appropriate application. Herein, we synthesized and fully characterized different 2-methoxy-9-acridone dyes. Their transient fluorescence emission spectra exhibited a complex dynamic behavior that can be linked to several excited-state reactions. We performed a thorough study of the excited-state dynamics of these dyes by means of time-resolved fluorimetry supported by computational calculations. All this allowed us to establish a multistate kinetic scheme, involving an ESPT reaction coupled to an excimer formation process. We have unraveled the rich dynamics behind this complex behavior, which provides a better understanding of the excited states of these dyes.