A Small‐Molecule Two‐Photon Probe for Nitric Oxide in Living Tissues

Two‐photon microscopy (TPM) has become an indispensable tool in the study of biology and medicine due to the capability of this method for molecular imaging deep inside intact tissues. For the maximum utilization of TPM, a variety of two‐photon (TP) probes for specific applications are needed. In this article, we report a small‐molecule TP probe (ANO1) for nitric oxide (NO) that shows a rapid and specific NO response, a 68‐fold fluorescence enhancement in response to NO, and a maximum TP‐action cross‐section of 170 GM (GM: 10^?50 cm⁴ photon^?1) upon reaction with excess NO. This probe can be easily loaded into cells and tissues and can real‐time monitor NO in living tissues at 100–180 μm depth for longer than 1200 s through the use of TPM, with minimum interference from other biologically relevant species.     

Two‐Photon STED Spectral Determination for a New V‐Shaped Organic Fluorescent Probe with Efficient Two‐Photon Absorption

Two‐photon stimulated emission depletion (STED) cross sections were determined over a broad spectral range for a novel two‐photon absorbing organic molecule, representing the first such report. The synthesis, comprehensive linear photophysical, two‐photon absorption (2PA), and stimulated emission properties of a new fluorene‐based compound, (E)‐2‐{3‐[2‐(7‐(diphenylamino)‐9,9‐diethyl‐9H‐fluoren‐2‐yl)vinyl]‐5‐methyl‐4‐oxocyclohexa‐2,5‐dienylidene} malononitrile ( 1 ), are presented. Linear spectral parameters, including excitation anisotropy and fluorescence lifetimes, were obtained over a broad range of organic solvents at room temperature. The degenerate two‐photon absorption (2PA) spectrum of 1 was determined with a combination of the direct open‐aperture Z‐scan and relative two‐photon‐induced fluorescence methods using 1 kHz femtosecond excitation. The maximum value of the 2PA cross section ～1700 GM was observed in the main, long wavelength, one‐photon absorption band. One‐ and two‐photon stimulated emission spectra of 1 were obtained over a broad spectral range using a femtosecond pump–probe technique, resulting in relatively high two‐photon stimulated emission depletion cross sections (～1200 GM). A potential application of 1 in bioimaging was demonstrated through one‐ and two‐photon fluorescence microscopy images of HCT 116 cells incubated with micelle‐encapsulated dye.     

Diketopyrrolopyrrole‐Porphyrin Conjugates with High Two‐Photon Absorption and Singlet Oxygen Generation for Two‐Photon Photodynamic Therapy

Two‐photon photodynamic therapy is a promising therapeutic method which requires the development of sensitizers with efficient two‐photon absorption and singlet‐oxygen generation. Reported here are two new diketopyrrolopyrrole‐porphyrin conjugates as robust two‐photon absorbing dyes with high two‐photon absorption cross‐sections within the therapeutic window. Furthermore, for the first time the singlet‐oxygen generation efficiency of diketopyrrolopyrrole‐containing systems is investigated. A preliminary study on cell culture showed efficient two‐photon induced phototoxicity.     

Black Pigment Gallstone Inspired Platinum‐Chelated Bilirubin Nanoparticles for Combined Photoacoustic Imaging and Photothermal Therapy of Cancers

Bilirubin (BR), a bile pigment that exerts potent antioxidant and anti‐inflammatory effects, is also a major constituent of black pigment gallstones found in bile ducts under certain pathological conditions. Inspired by the intrinsic metal‐chelating power of BR found in gallstones, herein we report a cisplatin‐chelated BR‐based nanoparticle (cisPt@BRNP) for use as a new photonic nanomedicine for combined photoacoustic imaging and photothermal therapy of cancers. The cisPt@BRNPs were prepared by simply mixing cisplatin with BRNPs, yielding ca. 150‐nm‐size NPs. Upon near‐IR laser irradiation at 808 nm, cisPt@BRNPs generated considerable heat and induced clear death of cancer cells in vitro. Following intravenous injection into human colon cancer‐bearing mice, cisPt@BRNPs allowed effective tumor visualization by photoacoustic imaging and remarkable antitumor efficacy by photothermal therapy, suggesting their potential for use as a new photonic nanomedicine for cancer therapy.     

Dinuclear Ruthenium(II) Complexes as Two‐Photon,Time‐Resolved Emission Microscopy Probes for Cellular DNA

The first transition‐metal complex‐based two‐photon absorbing luminescence lifetime probes for cellular DNA are presented. This allows cell imaging of DNA free from endogenous fluorophores and potentially facilitates deep tissue imaging. In this initial study, ruthenium(II) luminophores are used as phosphorescent lifetime imaging microscopy (PLIM) probes for nuclear DNA in both live and fixed cells. The DNA‐bound probes display characteristic emission lifetimes of more than 160 ns, while shorter‐lived cytoplasmic emission is also observed. These timescales are orders of magnitude longer than conventional FLIM, leading to previously unattainable levels of sensitivity, and autofluorescence‐free imaging.     

Phosphorus‐Modified Tungsten Nitride/Reduced Graphene Oxide as a High‐Performance,Non‐Noble‐Metal Electrocatalyst for the Hydrogen Evolution Reaction

Phosphorus‐modified tungsten nitride/reduced graphene oxide (P‐WN/rGO) is designed as a high‐efficient, low‐cost electrocatalyst for the hydrogen evolution reaction (HER). WN (ca. 3 nm in size) on rGO is first synthesized by using the H₃[PO₄(W₃O₉)₄] cluster as a W source. Followed by phosphorization, the particle size increase slightly to about 4 nm with a P content of 2.52 at %. The interaction of P with rGO and WN results in an obvious increase of work function, being close to Pt metal. The P‐WN/rGO exhibits low onset overpotential of 46 mV, Tafel slope of 54 mV dec⁻¹, and a large exchange current density of 0.35 mA cm⁻² in acid media. It requires overpotential of only 85 mV at current density of 10 mA cm⁻², while remaining good stability in accelerated durability testing. This work shows that the modification with a second anion is powerful way to design new catalysts for HER.     

Glycosylated Star‐Shaped Conjugated Oligomers for Targeted Two‐Photon Fluorescence Imaging

A glucopyranose functionalized star‐shaped oligomer, N‐tris{4,4′,4′′‐[(1E)‐2‐(2‐{(E)‐2‐[4‐(benzo[d]thiazol‐2‐yl)phenyl]vinyl}‐9,9‐bis(6‐2‐amido‐2‐deoxy‐1‐thio‐β‐D ‐glucopyranose‐hexyl)‐9H‐fluoren‐7‐yl)vinyl]phenyl}phenylamine (TVFVBN‐S‐NH₂), is synthesized for two‐photon fluorescence imaging. In water, TVFVBN‐S‐NH₂ self‐assembles into nanoparticles with an average diameter of ～49 nm and shows a fluorescence quantum yield of 0.21. Two‐photon fluorescence measurements reveal that TVFVBN‐S‐NH₂ has a two‐photon absorption cross‐section of ～1100 GM at 780 nm in water. The active amine group on the glucopyranose moiety allows further functionalization of TVFVBN‐S‐NH₂ with folic acid to yield TVFVBN‐S‐NH₂FA with similar optical and physical properties as those for TVFVBN‐S‐NH₂. Cellular imaging studies reveal that TVFVBN‐S‐NH₂FA has increased uptake by MCF‐7 cells relative to that for TVFVBN‐S‐NH₂, due to specific interactions between folic acid and folate receptors on the MCF‐7 cell membrane. This study demonstrates the effectiveness of glycosylation as a molecular engineering strategy to yield water‐soluble materials with a large two‐photon absorption (TPA) cross‐section for targeted cancer‐cell imaging.     

Two‐Photon Polymerization as a Tool for Studying 3D Printed Topography‐Induced Stem Cell Fate

Geometric topographies are known to influence cellular differentiation toward specific phenotypes, but to date the range of features and type of substrates that can be easily fabricated to study these interactions is somewhat limited. In this study, an emerging technology, two‐photon polymerization, is used to print topological patterns with varying feature‐size and thereby study their effect on cellular differentiation. This technique offers rapid manufacturing of topographical surfaces with good feature resolution for shapes smaller than 3 µm. Human‐induced pluripotent stem cells, when attached to these substrates or a non‐patterned control for 1 week, express an array of genetic markers that suggest their differentiation toward a heterogeneous population of multipotent progenitors from all three germ layers. Compared to the topographically smooth control, small features (1.6 µm) encourage differentiation toward ectoderm while large features (8 µm) inhibit self‐renewal. This study demonstrates the potential of using two‐photon polymerization to study and control stem cell fate as a function of substrate interactions. The ability to tailor and strategically design biomaterials in this way can enable more precise and efficient generation or maintenance of desired phenotypes in vitro and in vivo.     

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.