Photoluminescence Lifetime Imaging Microscopy Research Articles

Multiple pathways for lanthanide sensitization in self-assembled aqueous complexes

Lanthanide photoluminescence (PL) emission has attracted much attention for technological and bioimaging applications because of its particularly interesting features, such as narrow emission bands and very long PL lifetimes. However, this emission process necessitates a preceding step of energy transfer from suitable antennas. While biocompatible applications require luminophores that are stable in aqueous media, most lanthanide-based emitters are quenched by water molecules. Previously, we described a small luminophore, 8-methoxy-2-oxo-1,2,4,5-tetrahydrocyclopenta[de]quinoline-3-phosphonic acid (PAnt), which is capable of dynamically coordinating with Tb(III) and Eu(III), and its exchangeable behavior improved their performance in PL lifetime imaging microscopy (PLIM) compared with conventional lanthanide cryptate imaging agents. Herein, we report an in-depth photophysical and time-dependent density functional theory (TD–DFT) computational study that reveals different sensitization mechanisms for Eu(III) and Tb(III) in stable complexes formed in water. Understanding this unique behavior in aqueous media enables the exploration of different applications in bioimaging or novel emitting materials.

Short-wave Infrared Photoluminescence Lifetime Mapping of Rare-Earth Doped Nanoparticles Using All-Optical Streak Imaging.

The short-wave infrared (SWIR) photoluminescence lifetimes of rare-earth doped nanoparticles (RENPs) have found diverse applications in fundamental and applied research. Despite dazzling progress in the novel design and synthesis of RENPs with attractive optical properties, existing optical systems for SWIR photoluminescence lifetime imaging are still considerably restricted by inefficient photon detection, limited imaging speed, and low sensitivity. To overcome these challenges, SWIR photoluminescence lifetime imaging microscopy using an all-optical streak camera (PLIMASC) is developed. Synergizing scanning optics and a high-sensitivity InGaAs CMOS camera, SWIR-PLIMASC has a 1D imaging speed of up to 138.9kHz in the spectral range of 900-1700nm, which quantifies the photoluminescence lifetime of RENPs in a single shot. A 2D photoluminescence lifetime map can be acquired by 1D scanning of the sample. To showcase the power of SWIR-PLIMASC, a series of core-shell RENPs with distinct SWIR photoluminescence lifetimes is synthesized. In particular, using Er3+ -doped RENPs, SWIR-PLIMASC enables multiplexed anti-counterfeiting. Leveraging Ho3+ -doped RENPs as temperature indicators, this system is applied to SWIR photoluminescence lifetime-based thermometry. Opening up a new avenue for efficient SWIR photoluminescence lifetime mapping, this work is envisaged to contribute to advanced materials characterization, information science, and biomedicine.

Exchangeable Self-Assembled Lanthanide Antennas for PLIM Microscopy.

Lanthanides have unique photoluminescence (PL) emission properties, including very long PL lifetimes. This makes them ideal for biological imaging applications, especially using PL lifetime imaging microscopy (PLIM). PLIM is an inherently multidimensional technique with exceptional advantages for quantitative biological imaging. Unfortunately, due to the required prolonged acquisitions times, photobleaching of lanthanide PL emission currently constitutes one of the main drawbacks of PLIM. In this study, we report a small aqueous-soluble, lanthanide antenna, 8-methoxy-2-oxo-1,2,4,5-tetrahydrocyclopenta[de]quinoline-3-phosphonic acid, PAnt, specifically designed to dynamically interact with lanthanide ions, serving as exchangeable dye aimed at mitigating photobleaching in PLIM microscopy in cellulo. Thus, self-assembled lanthanide complexes that may be photobleached during image acquisition are continuously replenished by intact lanthanide antennas from a large reservoir. Remarkably, our self-assembled lanthanide complex clearly demonstrated a significant reduction of PL photobleaching when compared to well-established lanthanide cryptates, used for bioimaging. This concept of exchangeable lanthanide antennas opens new possibilities for quantitative PLIM bioimaging.

Exchangeable Self‐Assembled Lanthanide Antennas for PLIM Microscopy

AbstractLanthanides have unique photoluminescence (PL) emission properties, including very long PL lifetimes. This makes them ideal for biological imaging applications, especially using PL lifetime imaging microscopy (PLIM). PLIM is an inherently multidimensional technique with exceptional advantages for quantitative biological imaging. Unfortunately, due to the required prolonged acquisitions times, photobleaching of lanthanide PL emission currently constitutes one of the main drawbacks of PLIM. In this study, we report a small aqueous‐soluble, lanthanide antenna, 8‐methoxy‐2‐oxo‐1,2,4,5‐tetrahydrocyclopenta[de]quinoline‐3‐phosphonic acid, PAnt, specifically designed to dynamically interact with lanthanide ions, serving as exchangeable dye aimed at mitigating photobleaching in PLIM microscopy in cellulo. Thus, self‐assembled lanthanide complexes that may be photobleached during image acquisition are continuously replenished by intact lanthanide antennas from a large reservoir. Remarkably, our self‐assembled lanthanide complex clearly demonstrated a significant reduction of PL photobleaching when compared to well‐established lanthanide cryptates, used for bioimaging. This concept of exchangeable lanthanide antennas opens new possibilities for quantitative PLIM bioimaging.

Cellular imaging properties of phosphorescent iridium(III) complexes substituted with ester or amide groups.

Phosphorescent iridium(III) complexes have been extensively investigated as cellular imaging reagents and sensors. The intracellular localization of the complexes is known to be closely related to their formal charge, molecular size, lipophilicity, and bioactive pendants. Herein, we reported four phosphorescent iridium(III) complexes with the diimine ligands being modified with ester or amide groups as imaging reagents for living cells. The complexes have the same positive charge and very similar molecular size and weight. The lipophilicity of the complexes is similar ranging from 1.45 to 2.14. Upon internalization into living HeLa cells, while complexes 2-4, like most other iridium(III) complexes, were localized in the cytoplasm, complex 1 unexpectedly stained the whole cells including nuclei. The nuclear uptake of complex 1 was not observed when the cells were pretreated with chlorpromazine or nocodazole, suggesting that clathrin and microtubules mediated the nuclear uptake of complex 1. Additionally, the nuclear uptake efficiency is related to the cell division cycle. The complex was mainly concentrated in the nucleus when the cells were in mitosis, and distributed in whole cells when the cells were in the interphases. Furthermore, complex 1 exhibited a longer luminescence lifetime in the nucleus than in the cytoplasm as revealed by photoluminescence lifetime imaging microscopy (PLIM). Incubation of the cells in the hypoxia environment elongated the lifetime of the cytoplasmic complex, but hardly affected the luminescence properties of the intranuclear complex.

Conjugated Pt(II) Complexes as Luminescence-Switch-On Reporters Addressing the Microenvironment of Bacterial Biofilms.

In this work, the synthesis, structural and photophysical characterization of six phosphorescent H2O-soluble Pt(II) complexes are reported while addressing their emission maxima, photoluminescence quantum yields (ΦL), lifetimes (τ), aggregation tendency, and microenvironment sensitivity as a function of the substitution pattern on the main tridentate luminophore. Different ancillary ligands, namely, a trisulfonated phosphane and maltohexaose-conjugated pyridines (with or without amide bridges), were introduced and evaluated for the realization of switch-on-photoluminescent labels reporting on the microenvironment sensed in biofilms of Gram+ and Gram- models, namely, Staphylococcus aureus and Escherichia coli. With the aid of confocal luminescence micro(spectro)scopy, we observed that selected complexes specifically interact with the biofilms while leaving planktonic cells unlabeled. By using photoluminescence lifetime imaging microscopy, excited-state lifetimes within S. aureus biofilms were measured. The photoluminescence intensities were drastically boosted, and the excited state lifetimes were significantly prolonged upon binding to the viscous biofilm matrix, mainly due to the suppression of radiationless deactivation pathways upon shielding from physical quenching processes, such as interactions with solvent molecules and 3O2. The best performances were attained for non-aggregating complexes with maltohexaose targeting units and without amide bridges. Notably, in the absence of the maltodextrin, a hydrophobic adamantyl moiety suffices to attain a sizeable labeling capacity. Moreover, photoluminescence studies showed that selected complexes can also effectively interact with E. coli biofilms, where the bacterial cells are able to partially uptake the maltodextrin-based agents. In summary, the herein introduced concepts enable the development of specific biofilm reporters providing spatial resolution as well as lifetime- and spectrum-based readouts. Considering that most theragnostic agents reported so far mainly address metabolically active bacteria at the surface of biofilms but without reaching cells deeply immersed in the matrix, a new platform with a clear structure-property correlation is provided for the early detection of such bacterial arrays.

Multifunctional Chiral π‐Conjugated Polymer Microspheres: Production and Confinement of NLO signal, Detection of Circularly Polarized Light, and Display of Laser‐Triggered NLO Emission Shifts

AbstractTwo chiral π‐conjugated polymers with optical nonlinearity (NLO), abbreviated as R‐ and S‐BP, are prepared by copolymerization of R‐ and S‐6,6'‐dibromo‐2,2'‐diethoxy‐1,1'‐binaphthyl with 1,4‐diethynyl‐2,5‐bis(octyloxy)benzene by Sonogashira coupling. The R‐ and S‐BP copolymers self‐assemble in a tetrahydrofuran/H2O/ethanol mixture producing microspherical particles. Remarkably, the obtained polymer microspheres support excitation of whispering gallery mode resonances (500–720 nm) in one‐ and two‐photon excited luminescence or two‐photon luminescence (TPL) with a quality factor (Q) of up to ≈700. As a result of a high Q value, a single (R‐ or S‐BP) microsphere displays a circular dichroism (CD) effect in the TPL signal. At high laser pump‐power (≥10 mW), the TPL spectral features of microspheres change significantly, suggesting alterations of the polymer packing. Similar spectral variations are observed for microspheres heated thermally or with high power continuous‐wave laser. Photoluminescence lifetime imaging microscopy down to the single microsphere level corroborate transformations of polymer packing from a J type to a mixture of J, H, and H‐like (with disordered side chains) types. This work demonstrates the tremendous potential of tailor‐made chiral polymer microspheres exhibiting multifunctional properties, such as i) light‐confinement, ii) NLO‐CD, and iii) laser‐driven optical emission shifts, useful for many optoelectronic and photonic device applications.

Bioorthogonal "Labeling after Recognition" Affording an FRET-Based Luminescent Probe for Detecting and Imaging Caspase-3 via Photoluminescence Lifetime Imaging.

Bis-labeling with a luminescent energy donor/acceptor pair onto biological substrates affords probes which give FRET readouts for the detection of interaction partners. However, the covalently bound luminophores bring about steric hindrance and nonspecific interaction, which probably perturb the biological recognition. Herein, we designed a highly sensitive and specific "labeling after recognition" sensing approach, where luminophore labeling occurred after the biological recognition. Taking the cutting enzyme caspase-3 as an example, we demonstrated the detection of its catalytic activity in solution and apoptotic cells using the tetrapeptide motif Asp-Glu-Val-Asp (DEVD) as the cleavable substrate, and an iridium(III) complex and a rhodamine derivative as the energy donor/acceptor pair. The DEVD tetrapeptide was modified with an azide and a GK-norbornylene groups at the amino and carboxyl terminuses, respectively, which allowed donor/acceptor bis-labeling via two independent catalysis-free bioorthogonal reactions. The phosphorescence lifetime of the iridium(III) complex was quenched upon bis-labeling owing to the intracellular FRET to the rhodamine derivative, and significantly elongated upon the peptide being catalytically cleaved by caspase-3. Interestingly, the sensitivity and efficiency of the lifetime responses were much higher in the "labeling after recognition" sensing approach. Molecular docking analysis showed that the steric hindrance and nonspecific interactions partially inhibited the biological recognition of the DEVD substrate by caspase-3. The imaging of the catalytic activity of caspase-3 in apoptotic cells was demonstrated via photoluminescence lifetime imaging microscopy. Lifetime analysis not only confirmed the occurrence of intracellular bioorthogonal bis-labeling and catalytic cleavage, but also showed the extent to which the two dynamic processes occurred.

Monocycloplatinated Solvento Complex Displays Turn-on Ratiometric Phosphorescence Responses to Histamine.

The study of biological histamine (HA) requires probes capable of ratiometric photoluminescence detection of HA. We discovered that a monocycloplatinated complex having two solvento ligands ([Pt(2-(2-naphthyl)quinolinate)(NCCH3)2]ClO4) could produce ratiometric phosphorescence responses to HA in aerated aqueous solutions buffered to pH 7.4. The HA response was characterized with a hypsochromic shift of an emission peak wavelength from 635 to 567 nm. The corresponding phosphorescence intensity ratio (i.e., I567 nm/ I635 nm) increased from 0.26 to 1.90. Spectroscopic and spectrometric investigations indicated an occurrence of spontaneous displacement of the labile CH3CN ligands with HA. An independently prepared HA adduct supported this notion. The ratiometric phosphorescence responses to HA were highly tolerant to other biological stimuli, including changes in pH and the presence of biometals and biological Lewis bases such as amino acids, nucleosides, biothiols, neurotransmitters, and small molecular metabolites. Of note was the high selectivity toward HA over common biological ligands, including histidine, cysteine, and homocysteine, which was ascribed to tighter HA binding. Our phosphorescence measurements employing Boc-protected derivatives of HA suggested that the bis-chelate motif involving imidazolyl and terminal amino groups was crucial for eliciting the ratiometric phosphorescence signaling. Finally, the bioimaging utility of the HA probe was validated using RAW 264.7 macrophages that were exogenously supplemented with HA or stimulated with thapsigargin to enrich intracellular HA. Ratiometric phosphorescence imaging microscopy experiments demonstrated the ability of the probe for monitoring intracellular HA uptake. In addition, photoluminescence lifetime imaging microscopy techniques could be applied for visualization of HA within the RAW 264.7 cells, because the HA binding elongated the photoluminescence lifetime. Our study demonstrated the promising utility of inner-sphere interactions of phosphorescent Pt(II) complexes for detection of biological HA.

Rational design of a luminescent nanoprobe for hypoxia imaging in vivo via ratiometric and photoluminescence lifetime imaging microscopy.

A luminescent nanoprobe has been designed for detection of oxygen. The nanoprobe exhibits high sensitivity, selectivity and excellent reversibility, and has been employed for hypoxia imaging in vitro and in vivo by ratiometric and photoluminescence lifetime imaging techniques.

Phosphorescent soft salt for ratiometric and lifetime imaging of intracellular pH variations.

In contrast to traditional short-lived fluorescent probes, long-lived phosphorescent probes based on transition-metal complexes can effectively eliminate unwanted background interference by using time-resolved luminescence imaging techniques, such as photoluminescence lifetime imaging microscopy. Hence, phosphorescent probes have become one of the most attractive candidates for investigating biological events in living systems. However, most of them are based on single emission intensity changes, which might be affected by a variety of intracellular environmental factors. Ratiometric measurement allows simultaneous recording of two separated wavelengths instead of measuring mere intensity changes and thus offers built-in correction for environmental effects. Herein, for the first time, a soft salt based phosphorescent probe has been developed for ratiometric and lifetime imaging of intracellular pH variations in real time. Specifically, a pH sensitive cationic complex (C1) and a pH insensitive anionic complex (A1) are directly connected through electrostatic interaction to form a soft salt based probe (S1), which exhibits a ratiometric phosphorescent response to pH with two well-resolved emission peaks separated by about 150 nm (from 475 to 625 nm). This novel probe was then successfully applied for ratiometric and lifetime imaging of intracellular pH variations. Moreover, quantitative measurements of intracellular pH fluctuations caused by oxidative stress have been performed for S1 based on the pH-dependent calibration curve.

Dual-emissive Polymer Dots for Rapid Detection of Fluoride in Pure Water and Biological Systems with Improved Reliability and Accuracy.

It is of paramount importance to develop new probes that can selectively, sensitively, accurately and rapidly detect fluoride in aqueous media and biological systems, because F- is found to be closely related to many health and environmental concerns. Herein, a dual-emissive conjugated polyelectrolyte P1 containing phosphorescent iridium(III) complex was designed and synthesized, which can form ultrasmall polymer dots (Pdots) in aqueous media. The F--responsive tert-butyldiphenylsilyl moiety was introduced into iridium(III) complex as the signaling unit for sensing F− with the quenched phosphorescence. Thus, the dual-emissive Pdots can rapidly and accurately detect F− in aqueous media and live cells as a ratiometric probe by measuring the change in the ratio of the F−-sensitive red phosphorescence from iridium(III) complex to the F−-insensitive blue fluorescence from polyfluorene. Moreover, the interaction of Pdots with F− also changes its emission lifetime, and the lifetime-based detection of F− in live cells has been realized through photoluminescence lifetime imaging microscopy for the first time. Both the ratiometric luminescence and lifetime imaging have been demonstrated to be resistant to external influences, such as the probe’s concentration and excitation power. This study provides a new perspective for the design of promising Pdots-based probes for biological applications.

Dual-emissive nanohybrid for ratiometric luminescence and lifetime imaging of intracellular hydrogen sulfide.

We design a nanohybrid for the detection of hydrogen sulfide (H2S) based on mesoporous silica nanoparticles (MSNs). A phosphorescent iridium(III) complex and a specific H2S-sensitive merocyanine derivative are embedded into the nanohybrid. It exhibits a unique dual emission that is ascribed to the iridium(III) complex and the merocyanine derivative, respectively. Upon addition of sodium hydrogen sulfide (NaHS), the emission from the merocyanine derivative is quenched, while the emission from the iridium(III) complex is almost unchanged, which enables the ratiometric detection of H2S. Additionally, the nanohybrid has a long luminescence lifetime and displays a significant change in luminescence lifetime in response to H2S. Intracellular detection of H2S is performed via ratiometric imaging and photoluminescence lifetime imaging microscopy. Compared with the intensity-based method, the lifetime-based detection is independent of the probe concentration and can efficiently distinguish the signals of the probe from the autofluorescence in complex biological samples.

Core-shell structured phosphorescent nanoparticles for detection of exogenous and endogenous hypochlorite in live cells via ratiometric imaging and photoluminescence lifetime imaging microscopy.

We report a ratiometric phosphorescence sensory system for hypochlorite (ClO-) based on core-shell structured silica nanoparticles. Two phosphorescent iridium(iii) complexes were immobilised in the inner solid core and outer mesoporous layer of the nanoparticles, respectively. The former is insensitive to ClO- and thus serves as an internal standard to increase the accuracy and precision, while the latter exhibits a specific and significant luminogenic response to ClO-, providing high selectivity and sensitivity. Upon exposure to ClO-, the nanoparticles display a sharp luminescence colour change from blue to red. Additionally, intracellular detection of exogenous and endogenous ClO- has been demonstrated via ratiometric imaging and photoluminescence lifetime imaging microscopy. Compared to intensity-based sensing, ratiometric and lifetime-based measurements are independent of the probe concentration and are thus less affected by external influences, especially in intracellular applications.

Cyclometalated Iridium(III) Complexes for Phosphorescence Sensing of Biological Metal Ions

Phosphorescence signaling provides a valuable alternative to conventional bioimaging based on fluorescence. The benefits of using phosphorescent molecules include improved sensitivity and capabilities for effective elimination of background signals by time-gated acquisition. Cyclometalated Ir(III) complexes are promising candidates for facilitating phosphorescent bioimaging because they provide synthetic versatility and excellent phosphorescence properties. In this Forum Article, we present our recent studies on the development of phosphorescence sensors for the detection of metal ions based on cyclometalated iridium(III) complexes. The constructs contained cyclometalating (C^N) ligands with the electron densities and band-gap energies of the C^N ligand structures systematically varied. Receptors that chelated zinc, cupric, and chromium ions were tethered to the ligands to create phosphorescence sensors. The alterations in the C^N ligand structures had a profound influence on the phosphorescence responses to metal ions. Mechanistic studies suggested that the phosphorescence responses could be explained on the basis of the modulation of photoinduced electron transfer (PeT) from the receptor to the photoexcited iridium species. The PeT behaviors strictly adhered to the Rehm-Weller principle, and the occurrence of PeT was located in the Marcus-normal region. It is thus anticipated that improved responses will be obtainable by increasing the excited-state reduction potential of the iridium(III) complexes. Femtosecond transient absorption experiments provided evidence for the presence of an additional photophysical mechanism that involved metal-ion-induced alteration of the intraligand charge-transfer (ILCT) transition state. Utility of the mechanism by PeT and ILCT has been demonstrated for the phosphorescence sensing of biologically important transition-metal ions. In particular, the phosphorescence zinc sensor could report the presence of intracellular zinc pools by using confocal laser scanning microscopy and photoluminescence lifetime imaging microscopy techniques. We hope that the significant knowledge gained from our studies will be of great help in the design of new molecules as phosphorescence sensors.