[Imaging of In Vivo Oxygen Tension Based on Phosphorescence Lifetime Microscopy].

Molecular oxygen plays essential roles in aerobic organisms as a terminal electron acceptor in the electron transport chain in mitochondria. The intracellular oxygen concentration of the entire body is strictly regulated by a balance between the supply of oxygen from blood vessels and the consumption of oxygen in mitochondria. The disruption of oxygen homeostasis in the body often results in serious pathologies such as cancer, cerebral infarction, and chronic kidney disease, and thus considerable effort has been devoted to the development of suitable techniques allowing the qualitative and quantitative detection of tissue oxygen levels. This review focuses on recent advances in the visualization of oxygen levels in tissue based on phosphorescence lifetime measurements using exogenously small molecular oxygen probes. Specially, I introduce the principle of oxygen sensing by means of phosphorescence quenching, recent advances in intracellular and intravascular oxygen probes based on iridium(III) complexes, a system for measuring phosphorescence lifetime combined with confocal scanning microscopy, and the applications of these technologies to in vivo oxygen measurements, emphasizing the usefulness of iridium(III) complexes as biological oxygen probes.

A silyl porphyrin derivative conjugated with 6-deoxy-6-sulfo-α-d-glucopyranose functions as an efficient photosensitizer for photodynamic therapy.

We synthesized a new silyl porphyrin derivative conjugated with 6-deoxy-6-sulfo-α-d-glucopyranose (SGlc). Conjugation with SGlc improved A549 cellular uptake without significant changes in the photophysical and photochemical properties and subcellular localization. This improved cellular uptake led to enhanced photodynamic activity. Furthermore, conjugation with SGlc suppressed dark toxicity. These advantages were not observed for a conjugate with a glucose molecule. These results indicated that the conjugation with SGlc is a promising strategy for enhancing photodynamic efficacy.

An Optochemical Oxygen Scavenger Enabling Spatiotemporal Control of Hypoxia.

We present an optochemical O2 scavenging system that enables precise spatiotemporal control of the level of hypoxia in living cells simply by adjusting the light intensity in the illuminated region. The system employs rhodamine containing a selenium or tellurium atom as an optochemical oxygen scavenger that rapidly consumes O2 by photochemical reaction with glutathione as a coreductant upon visible light irradiation (560-590 nm) and has a rapid response time, within a few minutes. The glutathione-consuming quantum yields of the system were calculated as about 5 %. The spatiotemporal O2 consuming in cultured cells was visualized with a hypoxia-responsive fluorescence probe, MAR. Phosphorescence lifetime imaging was applied to confirmed that different light intensities could generate different levels of hypoxia. To illustrate the potential utility of this system for hypoxia research, we show that it can spatiotemporally control calcium ion (Ca2+ ) influx into HEK293T cells expressing the hypoxia-responsive Ca2+ channel TRPA1.

In Vivo 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 in vivo 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)2Ir(acac) (btp = benzothienylpyridine, acac = acetylacetone)) as the first probe that meets this requirement. BTP was efficiently taken up into cultured 3T3-L1 adipocytes and selectively accumulated into LDs. Quantifying oxygen levels in LDs based on the phosphorescence lifetime of BTP allowed us to track changes in cellular oxygen tension after treatment with metabolic stimulants. Phosphorescence lifetime imaging microscopy combined with intravenously administered BTP in mice enabled specific visualization of LDs in hepatic lobules and simultaneous imaging of the oxygen gradient that decreased from the portal vein (PV) to the central vein (CV). NAFL model mice were created by feeding a high-fat diet (HFD) to mice for 3 or 7 days. The mice fed an HFD showed a marked increase in the amount and size of LDs in hepatocytes compared with those fed a normal diet, leading to abnormal microvascular structures. In addition, HFD-fed mice also exhibited reduced oxygen tension in areas other than the CV. Multicolor imaging with the LD-accumulated oxygen probe BTP and vasculature-staining FITC-lectin suggested that structural distortions of the sinusoidal microvasculature caused by enlarged LDs were associated with partial hypoxia in NAFL.

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. Here, we devised a method to determine the oxygen tension in bone marrow monocytes using two-photon phosphorescence lifetime imaging microscopy with the cell-penetrating phosphorescent probe, BTPDM1. Phosphorescence lifetime imaging revealed the physiological level of oxygen tension in monocytes to be 5.3% in live mice exposed to normal air. When the mice inhaled hypoxic air, the level of oxygen tension in bone marrow monocytes decreased to 2.4%. By performing in vitro cell culture experiment within the physiological range of oxygen tension, hypoxia changed the molecular phenotype of monocytes, leading to enhanced the expression of CD169 and CD206, which are markers of a unique subset of macrophages in bone marrow, osteal macrophages. This current study enables the determination of the physiological range of oxygen tension in bone marrow with spatial resolution at a cellular level and application of this information on oxygen tension in vivo to in vitro assays. Quantifying oxygen tension in tissues can provide invaluable information on metabolism under physiological and pathophyisological conditions. This method will open new avenues for research on oxygen biology.

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

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 in vivo 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. This complex gave a phosphorescence spectrum with a maximum at 773 nm in the wavelength range of the so-called biological window and exhibited an exceptionally long lifetime (18.5 µs in degassed acetonitrile), allowing for excellent oxygen sensitivity even in the near-infrared window. PPYDM-BBMD showed a high intracellular uptake in cultured cells and mainly accumulated in the endoplasmic reticulum. We evaluated the oxygen sensitivity of PPYDM-BBMD phosphorescence in alpha mouse liver 12 (AML12) cells based on the Stern-Volmer analysis, which gave an O2-induced quenching rate constant of 1.42 × 103 mmHg-1 s-1. PPYDM-BBMD was administered in the tail veins of anesthetized mice, and confocal one-photon PLIM images of hepatic tissues were measured at different depths from the liver surfaces. The PLIM images visualized the oxygen gradients in hepatic lobules up to a depth of about 100 µm from the liver surfaces with a cellular-level resolution, allowing for the quantification of oxygen partial pressure based on calibration results using AML12 cells.

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 (O2) in normal and diseased tissues. Here, to achieve this goal, we developed green-emitting intracellular O2 probes based on the Ir(III) complex, PPY (tris(2-phenylpyridinato)iridium(III)), and investigated the possibility of multicolor O2 imaging by co-staining tissues with a red-emitting intravascular probe BTP-PEG48. 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. The photophysical and cellular properties of these complexes were systematically investigated to evaluate their ability as O2 probes. Among these complexes, PPYDM and PPY2OH, which have a dimethylamino group and two hydroxy groups, respectively, exhibited much higher cellular uptake efficiencies compared with PPY and showed high O2 sensitivity in HeLa cells. Phosphorescence lifetime imaging microscopy (PLIM) measurements of HeLa cells co-stained with PPYDM and hydrophilic BTP-PEG48 allowed for the evaluation of intracellular and extracellular O2 levels in cell culture. We took PLIM images of the pancreas following intravenous administration of PPYDM and BTP-PEG48 into anesthetized mice. The PLIM measurements using these probes allowed simultaneous O2 imaging of acinar cells and capillaries in the pancreas with cellular-level resolution. From the phosphorescence lifetimes of PPYDM and BTP-PEG48 and the calibration curves evaluated in rat pancreatic acinar cells and blood plasma, we found that the average oxygen partial pressures of acinar cells and capillaries were almost equal at about 30 mmHg.

Augmented Self-Association by Electrostatic Forces in Thienopyrrole-Fused Thiadiazoles that Contain an Ester instead of an Ether Linker.

During the self-assembly of π-conjugated molecules, linkers and substituents can potentially add supportive noncovalent intermolecular interactions to π-stacking interactions. Here, we report the self-assembly behavior of thienopyrrole-fused thiadiazole (TPT) fluorescent dyes that possess ester or ether linkers and dodecyloxy side chains in solution and the condensed phase. A comparison of the self-association behavior of the ester- and ether-bridged compounds in solution using detailed UV-vis, fluorescence, and NMR spectroscopic studies revealed that the subtle replacement of the ether linkers by ester linkers leads to a distinct increase in the association constant (ca. 3-4 fold) and the enthalpic contribution (ca. 3 kcal mol-1 ). Theoretical calculations suggest that the ester linkers, which are in close proximity to one another due to the π-stacking interactions, induce attractive electrostatic forces and augment self-association. The self-assembly of TPT dyes into well-defined 1D clusters with high aspect ratios was observed, and their morphologies and crystallinity were investigated using SEM and X-ray diffraction analyses. TPTs with ester linkers exhibit a columnar liquid crystalline mesophase in the condensed phase.

Cyclometalated Iridium(III) Complex-Cationic Peptide Hybrids Trigger Paraptosis in Cancer Cells via an Intracellular Ca2+ 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. Our initial mechanistic studies of this cell death suggest that IPHs would bind to the calcium (Ca2+)-calmodulin (CaM) complex and induce an overload of intracellular Ca2+, resulting in the induction of non-apoptotic programmed cell death. In this work, we conduct a detailed mechanistic study of cell death induced by ASb-2, a typical example of IPHs, and describe how ASb-2 induces paraptotic programmed cell death in a manner similar to that of celastrol, a naturally occurring triterpenoid that is known to function as a paraptosis inducer in cancer cells. It is suggested that ASb-2 (50 µM) induces ER stress and decreases the mitochondrial membrane potential (ΔΨm), thus triggering intracellular signaling pathways and resulting in cytoplasmic vacuolization in Jurkat cells (which is a typical phenomenon of paraptosis), while the change in ΔΨm values is negligibly induced by celastrol and curcumin. Other experimental data imply that both ASb-2 and celastrol induce paraptotic cell death in Jurkat cells, but this induction occurs via different signaling pathways.

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. We find that oxygen tension ranges from 17.4 to 36.4 mmHg, under hypoxic and normoxic conditions, respectively. Physiological normoxia thus corresponds to 5% and hypoxia to 2% oxygen in osteoclasts. Hypoxia in this range severely limits osteoclastogenesis, independent of energy metabolism and hypoxia-inducible factor activity. We observe that hypoxia decreases ten-eleven translocation (TET) activity. Tet2/3 cooperatively induces Prdm1 expression via oxygen-dependent DNA demethylation, which in turn activates NFATc1 required for osteoclastogenesis. Taken together, our results reveal that TET enzymes, acting as functional oxygen sensors, regulate osteoclastogenesis within the physiological range of oxygen tension, thus opening new avenues for research on in vivo response to oxygen perturbation.

S,C,C- and O,C,C-Bridged Triarylamines and Their Persistent Radical Cations.

This work reports the synthesis, crystal structures, and electronic properties of structurally constrained S,C,C- and O,C,C-bridged triarylamine derivatives and their persistent radical cations. O,C,C-Bridged triphenylamines and a dinaphthylphenylamine were obtained through a straightforward synthetic protocol. Similar to a previously reported S,C,C-bridged triphenylamine, the O,C,C-bridged triarylamines were easily oxidized to afford the corresponding radical cations, which were obtained as hexachloroantimonate salts. X-ray crystallographic analyses showed almost planar structures for these O,C,C-bridged triarylamine radical cations, which represent new members of the family of planar triarylamine radical cations without substituents on the aryl rings. Detailed investigations of the electronic properties of the S,C,C- and O,C,C-bridged triarylamine radical cations demonstrated that the spin and positive charge are sufficiently delocalized over the planar triarylamine scaffolds. The results provide the following insights into the effects of the bridging unit (sulfur vs oxygen) and the dibenzo-annulation on the spin delocalization in the bridged triarylamine radical cations: (1) An effective decrease of the spin density on the nitrogen atom is observed for the sulfur bridge relative to the oxygen bridge; and (2) a moderate decrease of the spin density on the oxygen atom rather than the nitrogen atom is induced by the dibenzo-annulation.

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-Rn (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). Intravenous administration of BTQ-R12 to mice and subsequent confocal luminescence microscope measurements enabled in vivo vascular imaging of tumors and various organs, including kidney, liver and pancreas. Dual color imaging of hepatic tissues of living mice fed a high-fat diet using BTQ-R12 and the lipid droplet-specific probe PC6S revealed small and large lipid droplets in the hepatocytes, causing distortion of the sinusoidal structure. BTQ-R12 selectively stains vascular endothelium and thus allows longer-term vascular network imaging compared to fluorescent dextran with a molecular weight of 70 kDa that circulate in the bloodstream. Furthermore, time-gated measurements using this phosphorescent vascular probe enabled imaging of blood vessel structures without interference from autofluorescence.

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)2), 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)2 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. Nonetheless, the fluorescent dye leakage from inside the AC-(di-O-C14PC)2 vesicles in the fluid phase is highly suppressed. The origin of the membrane properties characteristic of AC-(di-O-C14PC)2 monolayer is discussed in terms of the single long transmembrane hydrophobic linkage and the diffusional motion of the lipid molecules.

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. We induced hypoperfusion caused by coverslip placement in human kidney-2 cells, and observed an oxygen gradient under the coverslip. Immunocytochemistry of HIF-1α showed a doughnut-shaped formation on the edge of a pimonidazole-positive area, which we named the "HIF-ring". The oxygen tension of the HIF-ring was estimated to be between approximately 4 mmHg and 20 mmHg. This result was not compatible with those of past research showing HIF-1α accumulation in the anoxic range with homogeneous oxygen tension. We further observed the presence of a pH gradient under a coverslip, as well as a shift of the HIF ring due to changes in the pH of the culture medium, suggesting that the HIF ring was formed by suppression of HIF-1α related to low pH. This research demonstrated that HIF-1α activation mimics the physiological state in cultured cells with hypoperfusion.

In vivo O2 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 (O2)-sensitive luminescent probe allows for high-resolution O2 imaging of living tissues. Herein, we present phosphorescent Ir(III) complexes, (btp)2Ir(acac-DM) (Ir-1) and (btp-OH)3Ir (Ir-2), as useful O2 probes for PLIM measurement. These small-molecule probes were efficiently taken up into cultured cells and accumulated in specific organelles. Their excellent cell-permeable properties allowed for efficient staining of three-dimensional cell spheroids, and thereby phosphorescence lifetime measurements enabled the evaluation of the O2 level and distribution in spheroids, including the detection of alterations in O2 levels by metabolic stimulation with an effector. We took PLIM images of hepatic tissues of living mice by intravenously administrating these probes. The PLIM images clearly visualized the O2 gradient in hepatic lobules with cellular-level resolution, and the O2 levels were derived based on calibration using cultured cells; the phosphorescence lifetime of Ir-1 gave reasonable O2 levels, whereas Ir-2 exhibited much lower O2 levels. Intravenous administration of NH4Cl to mice caused the hepatic tissues to experience hypoxia, presumably due to O2 consumption to produce ATP required for ammonia detoxification, suggesting that the metabolism of the probe molecule might affect liver O2 levels.

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. Structural optimization afforded two pyronine fluorophores with different colors, both of which exhibit equilibrium (between the fluorescent dissociated form and the nonfluorescent GSH adduct form) and blinking kinetics that enable SMLM of microtubules or mitochondria in living cells. Furthermore, by using spontaneously blinking fluorophores working in the near-infrared (NIR) and green ranges, we succeeded in dual-color live-cell SMLM without the need for optimization of the imaging medium.

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. Furthermore, FOs and DBDCFs that contain a C[triple bond, length as m-dash]C bond react with tetracyanoethylene in a formal [2 + 2] CA followed by a retro-electrocyclization to afford sterically congested tetracyanobutadiene (TCBD) conjugates. The substituent (H or Me) on the aromatic ring adjacent to the butadiene moiety thereby determines whether the butadiene adopts an s-cis or s-trans conformation, and thus controls the physicochemical properties of the resulting TCBDs. The TCBD conjugates exhibit ICT absorptions (≤800 nm) together with up to four reversible reduction steps.

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 (>0.80) in both nonpolar and polar solvents. Additionally, the fluorescence lifetimes of PC6S were largely dependent on solvent polarity. The excellent spectral and photophysical properties of PC6S allowed its selective staining of LDs in living and fixed cells, and multicolor imaging. Fluorescence lifetime measurements of PC6S allowed estimation of the apparent polarity of LDs. The high photostability and long intracellular retention of PC6S supported in situ visualization of the formation processes of LDs resulting from the accumulation of fatty acid. Furthermore, intravenous administration of PC6S and use of the FLIM system allowed the imaging of LDs in hepatocytes in living normal mice and the growth of LDs resulting from the excess accumulation of lipids in high-fat-diet-fed mice (fatty liver model mice). Taking advantage of the high selectivity and sensitivity of PC6S for LDs in liver, we could visualize the adipocytes of lipid-rich tissues and LDs in kidney peritubular cells by PC6S fluorescence. These results demonstrated that PC6S combined with a FLIM system can be useful for monitoring and tracking the formation of LDs in both cultured cells and specific tissues and organs.

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. We utilized the transmittance spectrum of the solvent to correct the observed emission spectrum. Distortion due to solvent absorption was properly corrected, and additional corrections for the reabsorption/reemission effect gave more reliable PLQY values.