Mapping Live Cell Viscosity with an Aggregation‐Induced Emission Fluorogen by Means of Two‐Photon Fluorescence Lifetime Imaging

Intracellular viscosity is a crucial parameter that indicates the functioning of cells. In this work, we demonstrate the utility of TPE‐Cy, a cell‐permeable dye with aggregation‐induced emission (AIE) property, in mapping the viscosity inside live cells. Owing to the AIE characteristics, both the fluorescence intensity and lifetime of this dye are increased along with an increase in viscosity. Fluorescence lifetime imaging of live cells stained with TPE‐Cy reveals that the lifetime in lipid droplets is much shorter than that from the general cytoplasmic region. The loose packing of the lipids in a lipid droplet results in low viscosity and thus shorter lifetime of TPE‐Cy in this region. It demonstrates that the AIE dye could provide good resolution in intracellular viscosity sensing. This is also the first work in which AIE molecules are applied in fluorescence lifetime imaging and intracellular viscosity sensing.     

Fluorescence ratiometry and fluorescence lifetime imaging: using a single molecular sensor for dual mode imaging of cellular viscosity

Intracellular viscosity strongly influences transportation of mass and signal, interactions between the biomacromolecules, and diffusion of reactive metabolites in live cells. Fluorescent molecular rotors are recently developed reagents used to determine the viscosity in solutions or biological fluid. Due to the complexity of live cells, it is important to carry out the viscosity determinations in multimode for high reliability and accuracy. The first molecular rotor (RY3) capable of dual mode fluorescence imaging (ratiometry imaging and fluorescence lifetime imaging) of intracellular viscosity is reported. RY3 is a pentamethine cyanine dye substituted at the central (meso-) position with an aldehyde group (CHO). In nonviscous media, rotation of the CHO group gives rise to internal conversion by a nonradiative process. The restraining of rotation in viscous or low-temperature media results in strong fluorescence (6-fold increase) and lengthens the fluorescence lifetime (from 200 to 1450 ps). The specially designed molecular sensor has two absorption maxima (λ(abs) 400 and 613 nm in ethanol) and two emission maxima (in blue, λ(em) 456 nm and red, 650 nm in ethanol). However it is only the red emission which is markedly sensitive to viscosity or temperature changes, providing a ratiometric response (12-fold) as well as a large pseudo-Stokes shift (250 nm). A mechanism is proposed, based on quantum chemical calculations and (1)H NMR spectra at low-temperature. Inside cells the viscosity changes, showing some regional differences, can be clearly observed by both ratiometry imaging and fluorescence lifetime imaging (FLIM). Although living cells are complex the correlation observed between the two imaging procedures offers the possibility of previously unavailable reliability and accuracy when determining intracellular viscosity.     

Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging

The fluorescence intensity and lifetime of the 4,4'-difluoro-4-bora-5-(p-oxoalkyl)phenyl-3a,4a-diaza-s-indacene (1) show a strong correlation with the viscosity of the medium due to the viscosity-dependent twisting of the 5-phenyl group, which gives access to the dark nonemissive excited state. We propose a sensitive and versatile method for measuring the local microviscosity in biological systems, based on the determination of the fluorescence lifetime of 1. Fluorescence lifetime imaging (FLIM) performed on live cells incubated with 1 demonstrates the distinct intracellular lifetime of the molecular rotor of 1.6 +/- 0.2 ns corresponding to the intracellular viscosity of ca. 140 cP. Time-resolved fluorescence anisotropy of 1 in cells confirms insignificant binding of the fluorophore. The viscosity value obtained in the present study is considerably higher than that of water and of cellular cytoplasm. The high viscosity of intracellular compartments is likely to play an important role in vital intracellular processes, including the rate of diffusion of reactive oxygen species, causing programmed cell destruction.     

Cyanoarylporphyrazines with High Viscosity Sensitivity: A Step towards Dosimetry-Assisted Photodynamic Cancer Treatment

Despite the significant relevance of photodynamic therapy (PDT) as an efficient strategy for primary and adjuvant anticancer treatment, several challenges compromise its efficiency. In order to develop an “ideal photosensitizer” and the requirements applied to photosensitizers for PDT, there is still a need for new photodynamic agents with improved photophysical and photobiological properties. In this study, we performed a detailed characterization of two tetracyanotetra(aryl)porphyrazine dyes with 4-biphenyl (pz II) and 4-diethylaminophenyl (pz IV) groups in the periphery of the porphyrazine macrocycle. Photophysical properties, namely, fluorescence quantum yield and lifetime of both photosensitizers, demonstrate extremely high dependence on the viscosity of the environment, which enables them to be used as viscosity sensors. Pz II and pz IV easily enter cancer cells and efficiently induce cell death under light irradiation. Using fluorescence lifetime imaging microscopy, we demonstrated the possibility of assessing local intracellular viscosity and visualizing viscosity changes driven by PDT treatment with the compounds. Thus, pz II and pz IV combine the features of potent photodynamic agents and viscosity sensors. These data suggest that the unique properties of the compounds provide a tool for PDT dosimetry and tailoring the PDT treatment regimen to the individual characteristics of each patient.     

Emission ratiometric imaging of intracellular zinc: design of a benzoxazole fluorescent sensor and its application in two-photon microscopy

Zinc and calcium are ubiquitous intracellular metals, and while a variety of quantitative probes have been developed for measuring intracellular changes in calcium concentration, the same is not true of zinc. We describe here the design, synthesis, and properties of the benzoxazole-based, ratiometric zinc probe, Zinbo-5. This bright fluorescent reporter has a quantum yield of 0.1 in the zinc-form, exhibits a Kd for Zn2+ in the nanomolar range, and shows significant changes in both excitation and emission maxima upon zinc binding. The utility of this cell permeable probe is demonstrated in fluorescence microscopy emission ratio imaging experiments on mammalian cells. We further show that Zinbo-5 is well suited for two-photon excitation microscopy ratio imaging and can readily reveal changes in intracellular zinc concentration within optical planes of single cells. To the best of our knowledge, this is the first example of two-photon excitation microscopy applied to ratio imaging of zinc. These methods can be applied to real-time emission or excitation ratio imaging studies of zinc physiology in living cells.     

Designing a Red-Emitting Viscosity-Sensitive BODIPY Fluorophore for Intracellular Viscosity Imaging

Viscosity imaging at a microscopic scale can provide important information about biosystems, including the development of serious illnesses. Microviscosity imaging is achievable with viscosity-sensitive fluorophores, the most popular of which are based on the BODIPY group. However, most of the BODIPY probes fluoresce green light, whereas the red luminescence is desired for the imaging of biological samples. Designing a new viscosity probe with suitable spectroscopic properties is a challenging task because it is difficult to preserve viscosity sensitivity after modifying the molecular structure. Here we describe how we developed a new red-emitting, viscosity-sensitive, BODIPY fluorophore BP-PH-2M-NO₂ that is suitable for reliable intracellular viscosity imaging of lipid droplets in MCF-7 breast cancer cells. The design of BP-PH-2M-NO₂ was aided by DFT calculations that allowed a successful prediction of the viscosity sensitivity of fluorophores before synthesis. In summary, we report a new red viscosity probe possessing monoexponential fluorescence decay that makes it attractive for lifetime-based viscosity imaging.     

Photoluminescence Lifetime Imaging of Synthesized Proteins in Living Cells Using an Iridium–Alkyne Probe

Designing probes for real‐time imaging of dynamic processes in living cells is a continuous challenge. Herein, a novel near‐infrared (NIR) photoluminescence probe having a long lifetime was exploited for photoluminescence lifetime imaging (PLIM) using an iridium‐alkyne complex. This probe offers the benefits of deep‐red to NIR emission, a long Stokes shift, excellent cell penetration, low cytotoxicity, and good resistance to photobleaching. This example is the first PLIM probe applicable to the click reaction of copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) with remarkable lifetime shifts of 414 ns, before and after click reaction. The approach fully eliminates the background interference and distinguishes the reacted probes from the unreacted probes, thus enabling the wash‐free imaging of the newly synthesized proteins within single living cells. Based on the unique properties of the iridium complexes, it is anticipated to have applications for imaging other processes within living cells.     

一种红光发射的粘度荧光探针

以三苯胺和苯并噻唑盐为原料,设计合成了一种具有红光发射特性的D-π-A型荧光粘度探针N-乙酸乙酯基-2-(4-甲酸甲酯基三苯胺-4'-乙烯基)苯并噻唑六氟磷酸盐(L),运用现代分析测试手段进行了系统地表征。 研究结果表明,探针L的最大发射波长为630 nm,能有效地降低生物背景,提高生物成像的信噪比。 该探针对粘度有很好的荧光响应,其荧光强度比值(I/I₀)的对数与粘度的对数呈现很好的线性关系(R²=0.9934)。 此外,探针L对极性的敏感性小,且荧光信号不受生物分子的干扰。 生物学研究结果表明,探针L具有低的细胞毒性,可应用于细胞内微环境粘度的荧光成像。     

Dual-Phase Thermally Activated Delayed Fluorescence Luminogens: A Material for Time-Resolved Imaging Independent of Probe Pretreatment and Probe Concentration

Developing luminescent probes with long lifetime and high emission efficiency is essential for time-resolved imaging. However, the practical applications usually suffer from emission quenching of traditional luminogens in aggregated states, or from weak emission of aggregation-induced emission type luminogens in monomeric states. Herein, we overcome this dilemma by a rigid-and-flexible alternation design in donor–acceptor–donor skeletons, to achieve a thermally activated delayed fluorescence luminogen with high emission efficiency both in the monomeric state (quantum yield up to 35.3 %) and in the aggregated state (quantum yield up to 30.8 %). Such a dual-phase strong and long-lived emission allows a time-resolved luminescence imaging, with an efficiency independent of probe pretreatment and probe concentration. The findings open opportunities for developing luminescent probes with a usage in larger temporal and spatial scales.     

Dual‐Phase Thermally Activated Delayed Fluorescence Luminogens: A Material for Time‐Resolved Imaging Independent of Probe Pretreatment and Probe Concentration

Developing luminescent probes with long lifetime and high emission efficiency is essential for time‐resolved imaging. However, the practical applications usually suffer from emission quenching of traditional luminogens in aggregated states, or from weak emission of aggregation‐induced emission type luminogens in monomeric states. Herein, we overcome this dilemma by a rigid‐and‐flexible alternation design in donor–acceptor–donor skeletons, to achieve a thermally activated delayed fluorescence luminogen with high emission efficiency both in the monomeric state (quantum yield up to 35.3 %) and in the aggregated state (quantum yield up to 30.8 %). Such a dual‐phase strong and long‐lived emission allows a time‐resolved luminescence imaging, with an efficiency independent of probe pretreatment and probe concentration. The findings open opportunities for developing luminescent probes with a usage in larger temporal and spatial scales.     

Mitochondria‐Targeted Cancer Therapy Using a Light‐Up Probe with Aggregation‐Induced‐Emission Characteristics

Subcellular organelle‐specific reagents for simultaneous tumor targeting, imaging, and treatment are of enormous interest in cancer therapy. Herein, we present a mitochondria‐targeting probe (AIE‐mito‐TPP) by conjugating a triphenylphosphine (TPP) with a fluorogen which can undergo aggregation‐induced emission (AIE). Owing to the more negative mitochondrial membrane potential of cancer cells than normal cells, the AIE‐mito‐TPP probe can selectively accumulate in cancer‐cell mitochondria and light up its fluorescence. More importantly, the probe exhibits selective cytotoxicity for studied cancer cells over normal cells. The high potency of AIE‐mito‐TPP correlates with its strong ability to aggregate in mitochondria, which can efficiently decrease the mitochondria membrane potential and increase the level of intracellular reactive oxygen species (ROS) in cancer cells. The mitochondrial light‐up probe provides a unique strategy for potential image‐guided therapy of cancer cells.     

Microfluorometric study of oxygen dependence of (1"-pyrene butyl)-2-rhodamine ester probe in mitochondria of living cells

The access to oxygen concentration is of importance in various organelles of living cells, especially in mitochondria. A new probe, (1"-pyrene butyl)-2-rhodamine ester, was designed to target this organelle. We present here the properties of the probe in such an environment. Microspectrofluorometry confirms the localization of the probe in the mitochondrial environment at low doses (1 microM). At these doses, the cell toxicity experiments show no effect on the cell growth. The emission spectrum measured at an excitation wavelength of 340 nm (pyrene centered) indicates energy transfer from the pyrene to the rhodamine chromophore, as also observed in an ethanol solution. With excitation at 337 nm, the excited state decays biexponentially with lifetime decays of 6-9 ns and 90 ns. The first corresponds to the intrinsic fluorescence of the cell and the latter corresponds to the pyrene chromophore. In degassed conditions the pyrene lifetime decay increases up to 130 ns. Under an oxygen atmosphere the lifetime decays decrease to 62 ns. The lifetime changes with the oxygen concentration were compared with the range obtained during our previous study in ethanol solution (5-220 ns). The observed differences were interpreted by studying the lifetime of the probe in simplified environments, liposome suspensions and protein solutions. In this paper we show that the new probe can be used to measure the fluctuation of oxygen concentration in the surroundings of mitochondria.     

The Number of Accumulated Photons and the Quality of Stimulated Emission Depletion Lifetime Images

Time binning is used to increase the number of photon counts in the peak channel of stimulated emission depletion fluorescence lifetime decay curves to determine how it affects the resulting lifetime image. The fluorescence lifetime of the fluorophore, Alexa Fluor 594 phalloidin, bound to F‐actin is probed in cultured S2 cells at a spatial resolution of ~40 nm. This corresponds to a 10‐fold smaller probe volume compared to confocal imaging, and a reduced number of photons contributing to the signal. Pixel‐by‐pixel fluorescence lifetime measurements and error analysis show that an average of 40 ± 30 photon counts in the peak channel with a signal‐to‐noise ratio of 20 is enough to calculate a reliable fluorescence lifetime from a single exponential fluorescence decay. No heterogeneity in the actin cytoskeleton in different regions of the cultured cells was measured in the 40–400 nm spatial regime.     

Ultra-Small Air-Stable Triplet-Triplet Annihilation Upconversion Nanoparticles for Anti-Stokes Time-Resolved Imaging

Image contrast is often limited by background autofluorescence in steady-state bioimaging microscopy. Upconversion bioimaging can overcome this by shifting the emission lifetime and wavelength beyond the autofluorescence window. Here we demonstrate the first example of triplet-triplet annihilation upconversion (TTA-UC) based lifetime imaging microscopy. A new class of ultra-small nanoparticle (NP) probes based on TTA-UC chromophores encapsulated in an organic–inorganic host has been synthesised. The NPs exhibit bright UC emission (400–500 nm) in aerated aqueous media with a UC lifetime of ≈1 μs, excellent colloidal stability and little cytotoxicity. Proof-of-concept demonstration of TTA-UC lifetime imaging using these NPs shows that the long-lived anti-Stokes emission is easily discriminable from typical autofluorescence. Moreover, fluctuations in the UC lifetime can be used to map local oxygen diffusion across the subcellular structure. Our TTA-UC NPs are highly promising stains for lifetime imaging microscopy, affording excellent image contrast and potential for oxygen mapping that is ripe for further exploitation.     

Direct Visualization of Amlodipine Intervention into Living Cells by Means of Fluorescence Microscopy

Amlodipine, a unique long-lasting calcium channel antagonist and antihypertensive drug, has weak fluorescence in aqueous solutions. In the current paper, we show that direct visualization of amlodipine in live cells is possible due to the enhanced emission in cellular environment. We examined the impact of pH, polarity and viscosity of the environment as well as protein binding on the spectral properties of amlodipine in vitro, and used quantum chemical calculations for assessing the mechanism of fluorescence quenching in aqueous solutions. The confocal fluorescence microscopy shows that the drug readily penetrates the plasma membrane and accumulates in the intracellular vesicles. Visible emission and photostability of amlodipine allow confocal time-lapse imaging and the drug uptake monitoring.     

Ratiometric Fluorescence Imaging of Cellular Polarity: Decrease in Mitochondrial Polarity in Cancer Cells

Mitochondrial polarity strongly influences the intracellular transportation of proteins and interactions between biomacromolecules. The first fluorescent probe capable of the ratiometric imaging of mitochondrial polarity is reported. The probe, termed BOB, has two absorption maxima (λ_abs=426 and 561 nm) and two emission maxima—a strong green emission (λ_em=467 nm) and a weak red emission (642 nm in methanol)—when excited at 405 nm. However, only the green emission is markedly sensitive to polarity changes, thus providing a ratiometric fluorescence response with a good linear relationship in both extensive and narrow ranges of solution polarity. BOB possesses high specificity to mitochondria (R_r=0.96) that is independent of the mitochondrial membrane potential. The mitochondrial polarity in cancer cells was found to be lower than that of normal cells by ratiometric fluorescence imaging with BOB. The difference in mitochondrial polarity might be used to distinguish cancer cells from normal cells.     

Integrating Time-Resolved Imaging Information by Single-Luminophore Dual Thermally Activated Delayed Fluorescence

The fact that the lifetime of photoluminescence is often difficult to access because of the weakness of the emission signals, seriously limits the possibility to gain local bioimaging information in time-resolved luminescence probing. We aim to provide a solution to this problem by creating a general photophysical strategy based on the use of molecular probes designed for single-luminophore dual thermally activated delayed fluorescence (TADF). The structural and conformational design makes the dual TADF strong in both diluted solution and in an aggregated state, thereby reducing sensitivity to oxygen quenching and enabling a unique dual-channel time-resolved imaging capability. As the two TADF signals show mutual complementarity during probing, a dual-channel means that lifetime mapping is established to reduce the time-resolved imaging distortion by 30–40 %. Consequently, the leading intracellular local imaging information is serialized and integrated, which allows comparison to any single time-resolved signal, and leads to a significant improvement of the probing capacity.     

Integrating Time‐Resolved Imaging Information by Single‐Luminophore Dual Thermally Activated Delayed Fluorescence

The fact that the lifetime of photoluminescence is often difficult to access because of the weakness of the emission signals, seriously limits the possibility to gain local bioimaging information in time‐resolved luminescence probing. We aim to provide a solution to this problem by creating a general photophysical strategy based on the use of molecular probes designed for single‐luminophore dual thermally activated delayed fluorescence (TADF). The structural and conformational design makes the dual TADF strong in both diluted solution and in an aggregated state, thereby reducing sensitivity to oxygen quenching and enabling a unique dual‐channel time‐resolved imaging capability. As the two TADF signals show mutual complementarity during probing, a dual‐channel means that lifetime mapping is established to reduce the time‐resolved imaging distortion by 30–40 %. Consequently, the leading intracellular local imaging information is serialized and integrated, which allows comparison to any single time‐resolved signal, and leads to a significant improvement of the probing capacity.     

The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging

pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow fluorescent proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced cyan fluorescent protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.     

Mitochondria-targeted fluorescent probe based on vibration-induced emission for real-time monitoring mitophagy-specific viscosity dynamic

Directly monitoring mitophagy-specific viscosity dynamic in living cells is of great significance but remains challenging. Herein, this study reported a novel mitochondria-targeted fluorescent probe DPAC-DY based on vibration-induced emission (VIE) for monitoring viscosity changes during mitochondrial autophagy. This probe contained N,N'-diphenyl- dihydrodibenzo[a,c]phenazine (DPAC) as the VIE core and two positively charged pyridinium moieties for mitochondria anchoring. As the ambient viscosity increased, the vibration of DPAC-DY could be hindered, and subsequently resulting in the enhancement of fluorescence emission. In vitro and intracellular experiments indicated that the probe DPAC-DY showed highly sensitive response to viscosity due to VIE mechanism. Importantly, by virtue of this probe, in situ and real-time visualization of the specific viscosity dynamics during the mitochondrial autophagy process was achieved. Thus, this work provides a novel strategy for VIE-based viscosity response sensors applied to specific organelles and offers a platform for in-depth study of mitochondrial viscosity-related diseases.