Fluorogenic Squaraine Dendrimers for Background-Free Imaging of Integrin Receptors in Cancer Cells.

To overcome the limited brightness of existing fluorogenic molecular probes for biomolecular targets, we introduce a concept of fluorogenic dendrimer probe, which undergoes polarity-dependent switching due to intramolecular aggregation-caused quenching of its fluorophores. Based on a rational design of dendrimers with four and eight squaraine dyes, we found that octamer bearing dyes through a sufficiently long PEG(8) linker displays >400-fold fluorescence enhancement from water to non-polar dioxane. High extinction coefficient (≈2,300,000 m-1 cm-1 ) resulted from eight squaraine dyes and quantum yield (≈25 %) make this octamer the brightest environment-sensitive fluorogenic molecule reported to date. Its conjugate with cyclic RGD used at low concentration (3 nm) enables integrin-specific fluorescence imaging of cancer cells with high signal-to-background ratio. The developed dendrimer probe is a "golden middle" between molecular probes and nanoparticles, combining small size, turn-on response and high brightness, important for bioimaging.

Ratiometric Nanoparticle Probe Based on FRET-Amplified Phosphorescence for Oxygen Sensing with Minimal Phototoxicity.

Luminescent oxygen probes enable direct imaging of hypoxic conditions in cells and tissues, which are associated with a variety of diseases, including cancer. Here, a nanoparticle probe that addresses key challenges in the field is developed, it: i) strongly amplifies room temperature phosphorescence of encapsulated oxygen-sensitive dyes; ii) provides ratiometric response to oxygen; and iii) solves the fundamental problem of phototoxicity of phosphorescent sensors. The nanoprobe is based on 40 nm polymeric nanoparticles, encapsulating ≈2000 blue-emitting cyanine dyes with fluorinated tetraphenylborate counterions, which are as bright as 70 quantum dots (QD525). It functions as a light-harvesting nanoantenna that undergoes efficient Förster resonance energy transfer to ≈20 phosphorescent oxygen-sensitive platinum octaethylporphyrin (PtOEP) acceptor dyes. The obtained nanoprobe emits stable blue fluorescence and oxygen-sensitive red phosphorescence, providing ratiometric response to dissolved oxygen. The light harvesting leads to ≈60-fold phosphorescence amplification and makes the single nanoprobe particle as bright as ≈1200 PtOEP dyes. This high brightness enables oxygen detection at a single-particle level and in cells at ultra-low nanoprobe concentration with no sign of phototoxicity, in contrast to PtOEP dye. The developed nanoprobe is successfully applied to the imaging of a microfluidics-generated oxygen gradient in cancer cells. It constitutes a promising tool for bioimaging of hypoxia.

A fluorogenic BODIPY molecular rotor as an apoptosis marker.

Based on a BODIPY molecular rotor and a zinc-dipicolylamine receptor, we designed a fluorogenic probe for the detection of apoptosis. Being poorly emissive in solution and with healthy cells, it selectively binds phosphatidylserine of early apoptotic cells and internalizes into late apoptotic cells, lighting up its green fluorescence.

Solvatochromic Near-Infrared Probe for Polarity Mapping of Biomembranes and Lipid Droplets in Cells under Stress.

Can polarity-sensitive fluorescent dyes monitor the response of live cells to fundamental stress conditions, such as deprivation from nutrition and oxidative stress? To address this question, we developed a push-pull dioxaborine probe (DXB-NIR) for biomembranes and lipid droplets featuring strong solvatochromism in the far-red to near-infrared region, high fluorescence brightness, photostability, and two-photon absorption cross section, reaching 13800 GM at 930 nm. In model membranes, DXB-NIR exhibits unprecedented 80 nm shift between liquid ordered and disordered membrane phases, allowing robust imaging of separated membrane microdomains. Two-color imaging of live cells with DXB-NIR enables polarity mapping in plasma membranes, endoplasmic reticulum, and lipid droplets, which reveals that starvation and oxidative stress produce an increase in the local polarity, and this change is different for each of the studied cell compartments. Thus, by pushing the limits of existing solvatochromic dyes, we introduce a concept of polarity mapping for monitoring the response of cells to stress.

Optimizing the Fluorescence Properties of Nanoemulsions for Single Particle Tracking in Live Cells.

Nanoemulsions (NEs) are biocompatible lipid nanoparticles composed of an oily core stabilized by a surfactant shell. It is acknowledged that the surface decoration with poly(ethylene glycol), through the use of nonionic surfactants, confers high stealth in biological medium with reduced nonspecific interactions. Tracking individual NE by fluorescence microscopy techniques would lead to a better understanding of their behavior in cells and thus require the development of bright single particles with enhanced photostability. However, the understanding of the relationship between the physicochemical properties and chemical composition of the NEs, on the one hand, and its fluorescence properties of encapsulated dyes, on the other hand, remains limited. Herein, we synthesized three new dioxaborine barbituryl styryl (DBS) dyes that displayed high molar extinction coefficients (up to 120 000 M-1 cm-1) with relatively low quantum yields in solvents and impressive fluorescence enhancement when dissolved in viscous oils (up to 0.98). The reported screening of nine different oils allowed disclosing a range of efficient "oil/dye" couples and understanding the main parameters that lead to the brightest NEs. We determine vitamin E acetate/DBS-C8 as the representative most efficient couple, combining high dye loading capabilities and low aggregation-induced quenching, leading to <50 nm ultrabright NEs (with brightness as high as 30 × 106 M-1 cm-1) with negligible dye leakage in biological media. Beyond a comprehensive optical and physicochemical characterization of fluorescent NEs, cellular two-photon excitation imaging was performed with polymer-coated cell penetrating NEs. Thanks to their impressive brightness and photostability, NEs displaying different charge surfaces were microinjected in HeLa cells and were individually tracked in the cytosol to study their relative velocity.

MemBright: A Family of Fluorescent Membrane Probes for Advanced Cellular Imaging and Neuroscience.

The proper staining of the plasma membrane (PM) is critical in bioimaging as it delimits the cell. Herein, we developed MemBright, a family of six cyanine-based fluorescent turn-on PM probes that emit from orange to near infrared when reaching the PM, and enable homogeneous and selective PM staining with excellent contrast in mono- and two-photon microscopy. These probes are compatible with long-term live-cell imaging and immunostaining. Moreover, MemBright label neurons in a brighter manner than surrounding cells, allowing identification of neurons in acute brain tissue sections and neuromuscular junctions without any use of transfection or transgenic animals. In addition, MemBright probes were used in super-resolution imaging to unravel the neck of dendritic spines. 3D multicolor dSTORM in combination with immunostaining revealed en-passant synapse displaying endogenous glutamate receptors clustered at the axonal-dendritic contact site. MemBright probes thus constitute a universal toolkit for cell biology and neuroscience biomembrane imaging with a variety of microscopy techniques. VIDEO ABSTRACT.

Rational design of boron-dipyrromethene (BODIPY) reporter dyes for cucurbit[7]uril.

We introduce herein boron-dipyrromethene (BODIPY) dyes as a new class of fluorophores for the design of reporter dyes for supramolecular host-guest complex formation with cucurbit[7]uril (CB7). The BODIPYs contain a protonatable aniline nitrogen in the meso-position of the BODIPY chromophore, which was functionalized with known binding motifs for CB7. The unprotonated dyes show low fluorescence due to photoinduced electron transfer (PET), whereas the protonated dyes are highly fluorescent. Encapsulation of the binding motif inside CB7 positions the aniline nitrogen at the carbonyl rim of CB7, which affects the pKa value, and leads to a host-induced protonation and thus to a fluorescence increase. The possibility to tune binding affinities and pKa values is demonstrated and it is shown that, in combination with the beneficial photophysical properties of BODIPYs, several new applications of host-dye reporter pairs can be implemented. This includes indicator displacement assays with favourable absorption and emission wavelengths in the visible spectral region, fluorescence correlation spectroscopy, and noncovalent surface functionalization with fluorophores.

Ultrabright and Fluorogenic Probes for Multicolor Imaging and Tracking of Lipid Droplets in Cells and Tissues.

Lipid droplets (LDs) are intracellular lipid-rich organelles that regulate the storage of neutral lipids and were recently found to be involved in many physiological processes, metabolic disorders, and diseases including obesity, diabetes, and cancers. Herein we present a family of new fluorogenic merocyanine fluorophores based on an indolenine moiety and a dioxaborine barbiturate derivative. These so-called StatoMerocyanines (SMCy) fluoresce from yellow to the near-infrared (NIR) in oil with an impressive fluorescence enhancement compared to aqueous media. Additionally, SMCy display remarkably high molar extinction coefficients (up to 390 000 M-1 cm-1) and high quantum yield values (up to 100%). All the members of this new family specifically stain the LDs in live cells with very low background noise. Unlike Nile Red, a well-known lipid droplet marker, SMCy dyes possess narrow absorption and emission bands in the visible, thus allowing multicolor imaging. SMCy proved to be compatible with fixation and led to high-quality 3D images of lipid droplets in cells and tissues. Their high brightness allowed efficient tissue imaging of adipocytes and circulating LDs. Moreover their remarkably high two-photon absorption cross-section, especially SMCy5.5 (up to 13 300 GM), as well as their capacity to efficiently fluoresce in the NIR region led to two-photon multicolor tissue imaging (liver). Taking advantage of the available color palette, lipid droplet exchange between cells was tracked and imaged, thus demonstrating intercellular communication.

PEGylated Red-Emitting Calcium Probe with Improved Sensing Properties for Neuroscience.

Monitoring calcium concentration in the cytosol is of main importance as this ion drives many biological cascades within the cell. To this end, molecular calcium probes are widely used. Most of them, especially the red emitting probes, suffer from nonspecific interactions with inner membranes due to the hydrophobic nature of their fluorophore. To circumvent this issue, calcium probes conjugated to dextran can be used to enhance the hydrophilicity and reduce the nonspecific interaction and compartmentalization. However, dextran conjugates also feature important drawbacks including lower affinity, lower dynamic range, and slow diffusion. Herein, we combined the advantage of molecular probes and dextran conjugate without their drawbacks by designing a new red emitting turn-on calcium probe based on PET quenching, Rhod-PEG, in which the rhodamine fluorophore bears four PEG4 units. This modification led to a high affinity calcium probe (Kd = 748 nM) with reduced nonspecific interactions, enhanced photostability, two-photon absorbance, and brightness compared to the commercially available Rhod-2. After spectral characterizations, we showed that Rhod-PEG quickly and efficiently diffused through the dendrites of pyramidal neurons with an enhanced sensitivity (ΔF/F0) at shorter time after patching compared to Rhod-2.

Optical pH Sensor Covering the Range from pH 0-14 Compatible with Mobile-Device Readout and Based on a Set of Rationally Designed Indicator Dyes.

In this work, a family of pH-responsive fluorescent probes has been designed in a rational manner with the aid of quantum chemistry tools, covering the entire pH range from 0-14. Relying on the boron-dipyrromethene (BODIPY) core, all the probes as well as selected reference dyes display very similar spectroscopic properties with ON-OFF fluorescence switching responses, facilitating optical readout in simple devices used for detection and analysis. Embedding of the probes and reference dyes into hydrogel spots on a plastic strip yielded a test strip that reversibly indicates pH with a considerably small uncertainty of ∼0.1 pH units. These strips are not only reusable but, combined with a 3D-printed case that can be attached to a smartphone, the USB port of which drives the integrated LED used for excitation, allows for autonomous operation in on-site or in-the-field applications; the developed Android application software ("app") further simplifies operation for unskilled users.

Test-strip-based fluorometric detection of fluoride in aqueous media with a BODIPY-linked hydrogen-bonding receptor.

The measurement of biologically relevant anions, such as fluoride, is an important task in analytical chemistry, in particular, for dental health and osteoporosis. Although a large number of fluoride probes are known, the applicability under relevant conditions is limited to a few examples. To improve this situation, BODIPY-amidothiourea dyes with varying hydrogen-bond donating strengths were developed, the most H-acidic of which (1 c) could detect F(-) from an inorganic source (NaF) in 50 % aqueous solution (DMSO/water 1:1, v/v) with 0.01 ppm sensitivity through selective fluorescence quenching by a photoinduced electron-transfer (PET) process. Use of the probe and a reference dye with a test-strip assay and a portable and rapidly recording lateral-flow fluorescence reader made determination of F(-) in neat aqueous solutions, such as spiked water samples and toothpaste extracts, possible in a self-referenced manner, achieving a detection limit of 0.2 ppm.

Highly selective, sensitive and quantitative detection of Hg2+ in aqueous medium under broad pH range.

Amidothiourea linked acridinedione derivatives selectively detect Hg(2+) in unbuffered aqueous solution under broad pH range with both single- and two-photon excitation. The observed linear fluorescence intensity change allows the quantitative detection of Hg(2+) in the concentration range of 22 nM-0.33 µM with the lower detection limit of 2 nM.

Head-to-tail intermolecular hydrogen bonding of OH and NH groups with fluoride.

To explore the anion-recognition ability of the phenolic hydroxyl group and the amino hydrogen, we synthesized three different acridinedione (ADD) based anion receptors, 1, 2 and 3, having OH, NH, and combination of OH and NH groups, respectively. Absorption, emission and (1)H NMR spectral studies revealed that receptor 1, having only a phenolic OH group, shows selective deprotonation of the hydroxyl proton towards F(-), which results in an "ON-OFF"-type signal in the fluorescence spectral studies. Receptor 2, which only has an amino hydrogen, also shows deprotonation of the amino hydrogen with F(-), whereas receptor 3 (having both OH and NH groups) shows head-to-tail intermolecular hydrogen bonding of OH and NH groups with F(-) prior to deprotonation. The observation of hydrogen bonding of the OH and NH groups in a combined solution of 1 and 2 with F(-) in a head-to-tail hetero-intermolecular fashion, and the absence of head-to-head and tail-to-tail intermolecular hydrogen bonding in 1 and 2 with F(-), prove that the difference in the acidity of the OH and NH protons leads to the formation of an intermolecular hydrogen-bonding complex with F(-) prior to deprotonation. The presence of this hydrogen-bonding complex was confirmed by absorption spectroscopy, 3D emission contour studies, and (1)H NMR titration.

Fluorescence spectroscopic evidence for hydrogen bonding and deprotonation equilibrium between fluoride and a thiourea derivative.

Interaction of anions with thiourea-linked acridinedione fluorophore was studied by absorption, (1)H NMR, steady-state and time-resolved fluorescence techniques. Addition of AcO(-) and H(2)PO(4)(-) shows a genuine H-bonded complex with thiourea receptor; whereas, F(-) shows stepwise H-bonding and deprotonation of thiourea NH as confirmed by (1)H NMR titration. Free receptor 1 shows emission maximum at 418 nm; whereas, H-bonded complex of 1·F(-) shows a new redshifted emission maximum at 473 nm and the deprotonated 1 exhibits an emission peak at 502 nm. Presence of these three different emitting species was probed by 3D emission spectroscopic studies. Equilibrium between the free receptor 1, 1·F(-) H-bonded complex and deprotonated 1 was confirmed by time-resolved fluorescence studies. Time-resolved area normalised emission spectra (TRANES) of 1 in the presence of F(-) shows two isoemissive points at 456 and 479 nm between time delays of 0-0.5 ns and 1-20 ns, respectively, due to the existence of three emitting species in equilibrium. Observation of such an equilibrium based on fluorescence spectroscopic studies further proves the earlier reported absorption and (1)H NMR spectroscopic studies of H-bonding and deprotonation processes and also illustrates the dynamics of anion-receptor interactions.

Unprecedented formation of an N-benzamidobisthiourea derivative and its role in the formation of a new CT state specific towards fluoride ion.

A novel N-benzamidobisthiourea derivative (1) was synthesized. Tuning of its emission from the locally excited (LE) state to the charge transfer (CT) state was observed specifically for fluoride ion.