Effects of Photodynamic Therapy on Tumor Metabolism and Oxygenation Revealed by Fluorescence and Phosphorescence Lifetime Imaging.

This work was aimed at the complex analysis of the metabolic and oxygen statuses of tumors in vivo after photodynamic therapy (PDT). Studies were conducted on mouse tumor model using two types of photosensitizers-chlorin e6-based drug Photoditazine predominantly targeted to the vasculature and genetically encoded photosensitizer KillerRed targeted to the chromatin. Metabolism of tumor cells was assessed by the fluorescence lifetime of the metabolic redox-cofactor NAD(P)H, using fluorescence lifetime imaging.

Imaging of Lipid Droplets and Oxygen Status in Hepatic Tissues of Nonalcoholic Fatty Liver Model Mice Using a Lipophilic Ir(III) Complex.

Nonalcoholic fatty liver disease (NAFLD) is becoming common worldwide. In pathophysiological studies of NAFLD, an optical probe that enables visualization of lipid droplets (LDs) and imaging of oxygen status in hepatic tissues simultaneously would be very useful. Here, we present the phosphorescent Ir(III) complex BTP ((btp)Ir(acac) (btp = benzothienylpyridine, acac = acetylacetone)) as the first probe that meets this requirement.

Determination of the physiological range of oxygen tension in bone marrow monocytes using two-photon phosphorescence lifetime imaging microscopy.

Oxygen is a key regulator of both development and homeostasis. To study the role of oxygen, a variety of in vitro and ex vivo cell and tissue models have been used in biomedical research. However, because of ambiguity surrounding the level of oxygen that cells experience in vivo, the cellular pathway related to oxygenation state and hypoxia have been inadequately studied in many of these models.

Intracellular and Intravascular Oxygen Sensing of Pancreatic Tissues Based on Phosphorescence Lifetime Imaging Microscopy Using Lipophilic and Hydrophilic Iridium(III) Complexes.

Simultaneous imaging of intracellular and blood oxygen levels in tissues provides valuable information on the dynamic behavior of molecular oxygen (O) in normal and diseased tissues. Here, to achieve this goal, we developed green-emitting intracellular O probes based on the Ir(III) complex, PPY (tris(2-phenylpyridinato)iridium(III)), and investigated the possibility of multicolor O imaging by co-staining tissues with a red-emitting intravascular probe BTP-PEG. The newly synthesized complexes possess modified 2-phenylpyridinato ligand(s) with a cationic or hydrophilic substituent, such as a dimethylamino group, triphenylphosphonium cation, or hydroxy group, in order to enhance cellular uptake efficiency.

Near-Infrared Emitting Ir(III) Complexes Bearing a Dipyrromethene Ligand for Oxygen Imaging of Deeper Tissues .

Phosphorescence lifetime imaging microscopy (PLIM) using a phosphorescent oxygen probe is an innovative technique for elucidating the behavior of oxygen in living tissues. In this study, we designed and synthesized an Ir(III) complex, PPYDM-BBMD, that exhibits long-lived phosphorescence in the near-infrared region and enables oxygen imaging in deeper tissues. PPYDM-BBMD has a π-extended ligand based on a meso-mesityl dipyrromethene structure and phenylpyridine ligands with cationic dimethylamino groups to promote intracellular uptake.

Cyclometalated Iridium(III) Complex-Cationic Peptide Hybrids Trigger Paraptosis in Cancer Cells via an Intracellular Ca Overload from the Endoplasmic Reticulum and a Decrease in Mitochondrial Membrane Potential.

In our previous paper, we reported that amphiphilic Ir complex-peptide hybrids (IPHs) containing basic peptides such as KK(K)GG (K: lysine, G: glycine) (e.g., ASb-2) exhibited potent anticancer activity against Jurkat cells, with the dead cells showing a strong green emission.

Osteoclasts adapt to physioxia perturbation through DNA demethylation.

Oxygen plays an important role in diverse biological processes. However, since quantitation of the partial pressure of cellular oxygen in vivo is challenging, the extent of oxygen perturbation in situ and its cellular response remains underexplored. Using two-photon phosphorescence lifetime imaging microscopy, we determine the physiological range of oxygen tension in osteoclasts of live mice.

Phosphorescent Ir(III) complexes conjugated with oligoarginine peptides serve as optical probes for in vivo microvascular imaging.

Imaging the vascular structures of organ and tumor tissues is extremely important for assessing various pathological conditions. Herein we present the new vascular imaging probe BTQ-R (n = 8, 12, 16), a phosphorescent Ir(III) complex containing an oligoarginine peptide as a ligand. This microvasculature staining probe can be chemically synthesized, unlike the commonly used tomato lectins labeled with a fluorophore such as fluorescein isothiocyanate (FITC).

Membrane properties of amacrocyclic tetraether bisphosphatidylcholine lipid: Effect of a single membrane-spanning polymethylene cross-linkage between two head groups of ditetradecylphosphatidylcholine membrane.

The plasma membranes of archaea are abundant in macrocyclic tetraether lipids that contain a single or double long transmembrane hydrocarbon chains connecting the two glycerol backbones at both ends. In this study, a novel amacrocyclic bisphosphatidylcholine lipid bearing a single membrane-spanning octacosamethylene chain, 1,1'-O-octacosamethylene-2,2'-di-O-tetradecyl-bis-(sn-glycero)-3,3'-diphosphocholine (AC-(di-O-C14PC)), was synthesized to elucidate effects of the interlayer cross-linkage on membrane properties based on comparison with its corresponding diether phosphatidylcholine, 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (DTPC), that forms bilayer membrane. Several physicochemical techniques demonstrated that while AC-(di-O-C14PC) monolayer, which adopts a particularly high-ordered structure in the gel phase, shows remarkably high thermotropic transition temperature compared to DTPC bilayer, the fluidity of both phospholipids above the transition temperature is comparable.

A distinctive distribution of hypoxia-inducible factor-1α in cultured renal tubular cells with hypoperfusion simulated by coverslip placement.

Chronic hypoxia in the renal tubulointerstitium plays a key role in the progression of chronic kidney disease (CKD). It is therefore important to investigate tubular hypoxia and the activity of hypoxia-inducible factor (HIF)-1α in response to hypoxia. Rarefaction of the peritubular capillary causes hypoperfusion in CKD; however, the effect of hypoperfusion on HIFs has rarely been investigated.

In vivo O imaging in hepatic tissues by phosphorescence lifetime imaging microscopy using Ir(III) complexes as intracellular probes.

Phosphorescence lifetime imaging microscopy (PLIM) combined with an oxygen (O)-sensitive luminescent probe allows for high-resolution O imaging of living tissues. Herein, we present phosphorescent Ir(III) complexes, (btp)Ir(acac-DM) (Ir-1) and (btp-OH)Ir (Ir-2), as useful O probes for PLIM measurement. These small-molecule probes were efficiently taken up into cultured cells and accumulated in specific organelles.

Design of spontaneously blinking fluorophores for live-cell super-resolution imaging based on quantum-chemical calculations.

Spontaneously blinking fluorophores are powerful tools for live-cell super-resolution imaging under physiological conditions. Here we show that quantum-chemical calculations can predict key parameters for fluorophore design. We applied this methodology to develop a spontaneously blinking fluorophore with yellow fluorescence for super-resolution imaging of microtubules in living cells.

Spontaneously Blinking Fluorophores Based on Nucleophilic Addition/Dissociation of Intracellular Glutathione for Live-Cell Super-resolution Imaging.

Single-molecule localization microscopy (SMLM) allows the reconstruction of super-resolution images but generally requires prior intense laser irradiation and in some cases additives to induce blinking of conventional fluorophores. We previously introduced a spontaneously blinking rhodamine fluorophore based on an intramolecular spirocyclization reaction for live-cell SMLM under physiological conditions. Here, we report a novel principle of spontaneous blinking in living cells, which utilizes reversible ground-state nucleophilic attack of intracellular glutathione (GSH) upon a xanthene fluorophore.

Chemical transformations of push-pull fluorenones: push-pull dibenzodicyanofulvenes as well as fluorenone- and dibenzodicyanofulvene-tetracyanobutadiene conjugates.

Push-pull fluorenones (FOs) were synthesized by treating a benzopentalenequinone (BPO) derivative with alkynes that bear an electron-rich aniline moiety via a regioselective [4 + 2] cycloaddition (CA) followed by a [4 + 1] retrocycloaddition (RCA). The resulting FOs were readily converted into dibenzodicyanofulvenes (DBDCFs) by treatment with malononitrile in the presence of TiCl4 and pyridine. The FOs and DBDCFs exhibit intramolecular charge-transfer (ICT) that manifests in absorptions at 350-650 nm and amphoteric electrochemical behavior.

Visualization of Lipid Droplets in Living Cells and Fatty Livers of Mice Based on the Fluorescence of π-Extended Coumarin Using Fluorescence Lifetime Imaging Microscopy.

Lipid droplets (LDs) are closely related to lipid metabolism in living cells and are highly associated with diverse diseases such as fatty liver, diabetes, and cancer. Herein we describe a π-extended fluorescent coumarin (PC6S) for visualizing LDs in living cells and in the tissues of living mice using confocal fluorescence lifetime imaging microscopy (FLIM). PC6S showed a large positive solvatochromic shift and high fluorescence quantum yield (>0.

Synthesis, Structures, and Properties of Neutral and Radical Cationic S,C,C-Bridged Triphenylamines.

A structurally constrained S,C,C-bridged triphenylamine was synthesized, and the corresponding radical cation was obtained as a hexachloroantimonate by chemical oxidation. An X-ray crystallographic analysis revealed an almost planar structure for this radical cation, which thus represents the first example of a planar, -unsubstituted triphenylamine radical cation analogue with a sulfur bridge. The electronic properties of the radical cation were examined by UV-vis-NIR and ESR spectroscopy as well as DFT calculations.

Absolute Quantum Yield Measurements of Near-Infrared Emission with Correction for Solvent Absorption.

In this work, we evaluated the effect of solvent absorption during photoluminescence quantum yield (PLQY) measurements of near-infrared (NIR) emission with an integrating sphere (IS) instrument, and propose an effective correction method. Transmittance spectra of representative solvents measured with an IS instrument showed significant absorption bands in the first NIR region (NIR-I; 700-950 nm), and more prominently in the second NIR (NIR-II; 1000-1700 nm) region due to overtones and a combination of fundamental vibrations involving C-H and O-H stretching modes. The emission spectra of typical NIR-I and NIR-II emitting compounds exhibited dips owing to solvent absorption, resulting in somewhat reduced PLQY values.

Fluorescence Quenching-based Assay for Measuring Golgi endo-α-Mannosidase.

Golgi endo-α-mannosidase (G-EM) catalyzes an alternative deglucosylation process for N-glycans and plays important roles in the post-endoplasmic reticulum (ER) quality control pathway. To understand the post-ER quality control mechanism, we synthesized a tetrasaccharide probe for the detection of the hydrolytic activity of G-EM based on a fluorescence quenching assay. The probe was labeled with an N-methylanthraniloyl group as a reporter dye at the non-reducing end and a 2,4-dinitrophenyl group as a quencher at the reducing end.

Structure-efficiency relationship of photoinduced electron transfer-triggered nitric oxide releasers.

Spatiotemporally controllable nitric oxide (NO) releasers are required for biological studies and as candidate therapeutic agents. Here, we investigate the structure-efficiency relationship of a series of photoinduced electron transfer-triggered NO releasers based on our reported yellowish-green light-controllable NO releaser, NO-Rosa. The distance between the NO-releasing N-nitrosoaminophenol moiety and the rosamine antenna moiety was critical for efficient NO release.

Intravital phosphorescence lifetime imaging of the renal cortex accurately measures renal hypoxia.

Renal tubulointerstitial hypoxia is recognized as a final common pathway of chronic kidney disease and is considered a promising drug target. However, hypoxia in the tubules is not well examined because of limited detection methods. Here, we devised a method to visualize renal tubular oxygen tension with spatial resolution at a cellular level using the cell-penetrating phosphorescent probe, BTPDM1 (an iridium-based cationic lipophilic dye), and confocal phosphorescence lifetime imaging microscopy to precisely assess renal hypoxia.

Interrogation of metabolic and oxygen states of tumors with fiber-based luminescence lifetime spectroscopy.

The study of metabolic and oxygen states of cells in a tumor in vivo is crucial for understanding of the mechanisms responsible for tumor development and provides background for the relevant tumor's treatment. Here, we show that a specially designed implantable fiber-optic probe provides a promising tool for optical interrogation of metabolic and oxygen states of a tumor in vivo. In our experiments, the excitation light from a ps diode laser source is delivered to the sample through an exchangeable tip via a multimode fiber, and the emission light is transferred to the detector by another multimode fiber.

Novel One-Tube-One-Step Real-Time Methodology for Rapid Transcriptomic Biomarker Detection: Signal Amplification by Ternary Initiation Complexes.

We have developed a novel RNA detection method, termed signal amplification by ternary initiation complexes (SATIC), in which an analyte sample is simply mixed with the relevant reagents and allowed to stand for a short time under isothermal conditions (37 °C). The advantage of the technique is that there is no requirement for (i) heat annealing, (ii) thermal cycling during the reaction, (iii) a reverse transcription step, or (iv) enzymatic or mechanical fragmentation of the target RNA. SATIC involves the formation of a ternary initiation complex between the target RNA, a circular DNA template, and a DNA primer, followed by rolling circle amplification (RCA) to generate multiple copies of G-quadruplex (G4) on a long DNA strand like beads on a string.

Intracellular and in vivo oxygen sensing using phosphorescent iridium(III) complexes.

Molecular oxygen plays an indispensable role as a terminal electron acceptor in the electron transport chain in mitochondria. Acute or chronic oxygen deprivation (hypoxia) in organisms results in various diseases, and the elucidation of the pathogenic mechanism of hypoxia-related diseases and various cellular responses to hypoxia is an urgent issue. Optical oxygen imaging methods using phosphorescent probes have opened up techniques for noninvasive imaging of the intracellular and tissue oxygen status, and oxygen-sensitive probes play a key role in the development of this approach.

Quantitating intracellular oxygen tension in vivo by phosphorescence lifetime measurement.

Hypoxia appears to have an important role in pathological conditions in many organs such as kidney; however, a method to quantify intracellular oxygen tension in vivo has not been well established. In this study, we established an optical method to quantify oxygen tension in mice kidneys using a cationic lipophilic phosphorescence probe, BTPDM1, which has an intracellular oxygen concentration-sensitive phosphorescence lifetime. Since this probe is distributed inside the tubular cells of the mice kidney, we succeeded in detecting acute renal hypoxic conditions and chronic kidney disease.

Ratiometric Molecular Probes Based on Dual Emission of a Blue Fluorescent Coumarin and a Red Phosphorescent Cationic Iridium(III) Complex for Intracellular Oxygen Sensing.

Ratiometric molecular probes RP1 and RP2 consisting of a blue fluorescent coumarin and a red phosphorescent cationic iridium complex connected by a tetra- or octaproline linker, respectively, were designed and synthesized for sensing oxygen levels in living cells. These probes exhibited dual emission with good spectral separation in acetonitrile. The photorelaxation processes, including intramolecular energy transfer, were revealed by emission quantum yield and lifetime measurements.