D-luciferin analogues: a multicolor toolbox for bioluminescence imaging

Colorful mixture: Three types of luciferin analogues, that is, alkylaminoluciferins, aminoselenoluciferin, and luciferins with a benzimidazole scaffold, have been reported. These analogues show excellent bioluminescent properties and great potential in bioluminescence imaging.     

A Dual‐Color Far‐Red to Near‐Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging

Red‐shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red‐shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual‐color, far‐red to near‐infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far‐red to nIR emission maxima up to λ_max=706 nm with different Fluc mutants. This emission is the most red‐shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep‐tissue bioluminescence imaging.     

Bioluminescence Profiling of NanoKAZ/NanoLuc Luciferase Using a Chemical Library of Coelenterazine Analogues

We describe here an extensive structure-bioluminescence relationship study of a chemical library of analogues of coelenterazine, using nanoKAZ/NanoLuc, a mutated luciferase originated from the catalytic subunit of the deep-sea shrimp Oplophorus gracilirostris. Out of the 135 O-acetylated precursors that were prepared by using our recently reported synthesis and following their hydrolysis to give solutions of the corresponding luciferins, notable bioluminescence improvements were achieved in comparison with furimazine, which is currently amongst the best substrates of nanoKAZ/NanoLuc. For instance, the rather more lipophilic analogue 8-(2,3-difluorobenzyl)-2-((5-methylfuran-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one provided a 1.5-fold improvement of the total light output over a 2 h period, a close to threefold increase of the initial signal intensity and a signal-to-background ratio five times greater than furimazine. The kinetic parameters for the enzymatic reaction were obtained for a selection of luciferin analogues and provided unexpected insights into the luciferase activity. Most prominently, along with a general substrate-dependent and irreversible inactivation of this enzyme, in the case of the optimized luciferin mentioned above, the consumption of 2664 molecules was found to be required for the detection of a single Relative Light Unit (RLU; a luminometer-dependent fraction of a photon).     

Protein‐Specific,Multicolor and 3D STED Imaging in Cells with DNA‐Labeled Antibodies

Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal‐to‐noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed by using exchangeable labels, which transiently bind to and dissociate from a target, thereby replenishing the destroyed labels with intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore‐labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein‐specific antibodies. The constant exchange of fluorophore labels in DNA‐based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two‐color STED imaging of whole cells.     

Synthetic Routes to Coelenterazine and Other Imidazo[1,2‐a]pyrazin‐3‐one Luciferins: Essential Tools for Bioluminescence‐Based Investigations

In the last few decades, bioluminescent systems based on the expression of a luciferase and the addition of a luciferin to monitor the emission of light have become very important tools for biological investigations. A growing proportion of these systems use coelenterazine or analogues of imidazo[1,2‐a]pyrazine luciferins along with photoproteins or luciferases from sea creatures such as Aequorea, Renilla, Gaussia or Oplophorus. Central to the success of these tools are the synthetic pathways developed not only to prepare the naturally occurring luciferins, but also to design altered compounds that exhibit improved bioluminescence. Current work is indeed focused on the design of systems exhibiting extended luminescence (“glow” systems) or redshifted wavelengths, as well as constructions better adapted to conditions in cells or in vivo. This review describes the synthetic pathways used to prepare imidazo[1,2‐a]pyrazine luciferins along with the research efforts aimed at preparing analogues even better suited to the design of assays.     

Polynuclear SmIII Polyamidoamine‐Based Dendrimer: A Single Probe for Combined Visible and Near‐Infrared Live‐Cell Imaging

We report herein the synthesis of a luminescent polynuclear dendritic structure (Sm^III‐G3P‐2,3Nap) in which eight Sm^III ions are sensitized by thirty‐two 2,3‐naphthalimide chromophores. Upon a single excitation wavelength, the dendrimer complex exhibits two types of emission in the visible and in the near‐infrared (NIR) ranges. Sm^III‐G3P‐2,3Nap was non‐cytotoxic after 24 h of incubation and up to 2.5 μM . The ability of the Sm^III‐based probe to be taken up by cells was confirmed by confocal microscopy. Epifluorescence microscopy validated Sm^III‐G3P‐2,3Nap as a versatile probe, capable of performing interchangeably in the visible or NIR for live‐cell imaging. As both emissions are obtained from a single complex, the cytotoxicity and biodistribution are inherently the same. The possibility for discriminating the sharp Sm^III signals from autofluorescence in two spectral ranges increases the reliability of analysis and reduces the probability of artifacts and instrumental errors.     

Hydrophilic,Upconverting, Multicolor,Lanthanide‐Doped NaGdF4 Nanocrystals as Potential Multifunctional Bioprobes

Monodisperse water‐soluble hexagonal phase Ln³⁺‐doped NaGdF₄ upconverting nanocrystals (UCNCs) have been successfully fabricated by means of a fast, facile, and environmentally friendly microwave‐assisted route with polyethylenimine as the surfactant. Fine‐tuning of the UC emission from visible to near‐IR and finally to white light has been achieved. Furthermore, studies of the magnetic resonance imaging as well as the magnetization (magnetization–magnetic field curves) and the targeted recognition properties of FA‐coupled amine‐functionalized NaGdF₄@SiO₂ UCNCs indicate that the obtained NaGdF₄ UCNCs can be potential candidates for dual‐mode optical/magnetic bioapplications.     

DMAP‐BODIPY Alkynes: A Convenient Tool for Labeling Biomolecules for Bimodal PET–Optical Imaging

Several new boron dipyrromethene/N,N‐dimethylaminopyridine (BODIPY‐DMAP) assemblies were synthesized as precursors for bimodal imaging probes (optical imaging, OI/positron emission tomography, PET). The photophysical properties of the new compounds were also studied. The first proof‐of‐concept was obtained with the preparation of several new BODIPY‐labeled bombesins and evaluation of the affinity for bombesin receptors by using a competition binding assay. Fluorination reactions were investigated on DMAP‐BODIPY precursors as well as on DMAP‐BODIPY‐labeled bombesins. Chemical modifications on the BODIPY core were also performed to obtain luminescent dyes emitting in the therapeutic window (650–900 nm), suitable for in vivo imaging, making these compounds promising precursors for PET/optical dual‐modality imaging agents.     

Multimodal‐Luminescence Core–Shell Nanocomposites for Targeted Imaging of Tumor Cells

Light on target : Silica‐coated upconverting nanophosphor (UCNP) nanocomposites have been synthesized with organic dye (fluorescein isothiocyanate, FITC) incorporated in the silica shell. The nanocomposites are well dispersed and have good photostability and biocompatibility. These nanocomposites readily conjugated with folic acid for targeted multimodal bioimaging (see schematic representation).

     

A novel planar D‐A alternating copolymer with D‐A integrated structures exhibiting H‐aggregate behaviors for polymer solar cells

With the D‐A integrated structure concept, a new donor–acceptor (D‐A) copolymer poly{(N‐dodecyl‐carbazole[3,4‐c:5,6‐c]bis[1,2,5]thiadiazole‐alt‐4,8‐di(2‐ethylhexy‐loxyl)benzo[1,2‐b:4,5‐b′]dithiophene)} has been designed and synthesized using a novel architecture N‐dodecyl‐carbazole[3,4‐c:5,6‐c]bis[1,2,5]thiadiazole, and di(2‐ethylhexy‐loxyl)benzo[1,2‐b:4,5‐b′]dithiophene) as the basic building blocks. The copolymer has a low‐lying highest occupied molecular orbital energy level of ?5.41 eV and a broaden absorption matching well with the main solar photon flux. Note that an H‐aggregation beneficial for charge transportation and collection is formed in the macromolecules film, which implies that the planar D‐A integrated structure favors the strong intermolecular interaction to render molecules aggregated via face‐to‐face self‐assembly. The aggregation becomes larger scale after thermal annealing. Additionally, obvious intramolecular charge transfer and energy transfer have occurred in created D‐A integration. Without any treatment, the resulting polymer achieved a efficiency of 2.0% and relatively high open‐circuit voltage (V_oc) value of 0.77 V when blended with [6,6]‐phenyl‐C61‐butyric acid methyl ester in a typical bulk heterojunction. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

Phosphorescent Polymeric Nanoparticles by Coordination Cross‐Linking as a Platform for Luminescence Imaging and Photodynamic Therapy

Water‐soluble phosphorescent polymeric nanoparticles with an average diameter of approximately 100 nm were synthesized by a coordination cross‐linking reaction. The pyridine blocks in poly(4‐vinyl pyridine‐b‐ethylene oxide) (P4VP‐b‐PEO) were cross‐linked by the iridium chloride‐bridged dimer in DMF solution. Owing to the presence of an iridium complex with different ligands in the core of the polymeric nanoparticles, NP‐1, NP‐2, and NP‐3 showed bright green, yellow, and red phosphorescence, respectively. PEG chains in the shell gave the polymeric nanoparticles solubility and biocompatibility, which was confirmed by an MTT assay using HeLa cells as a model cancer cell line. The flow cytometry and laser confocal fluorescence microscopy results revealed NP‐2, as an example, could be effectively uptaken by HeLa cells. Therefore, these polymeric nanoparticles can be used as luminescent probes for living cells. In addition, ¹O₂ could be effectively generated in the presence of NP‐2 upon irradiation with visible light (λ>400 nm, 300 mW cm^?2), which was confirmed by a clear decrease in the fluorescence intensity of 9,10‐dimethylanthracene (DMA). After incubation with NP‐2 at a concentration of 200 μg mL^?1 for 6 h, approximately 90 % of HeLa cells were effectively ablated upon irradiation with visible light for only 10 min, indicating the potential for photodynamic therapy with polymeric nanoparticles.