Organelle-targeted Probes Research Articles

Modular development of organelle-targeting fluorescent probes for imaging formaldehyde in live cells.

Formaldehyde (FA) is endogenously generated via fundamental biological processes in living systems. Aberrant FA homeostasis in subcellular microenvironments is implicated in numerous pathological conditions. Fluorescent probes for detecting FA in specific organelles are thus of great research interest. Herein, we present a modular strategy to construct diverse organelle-targeting FA probes by incorporating selective organelle-targeting moieties into the scaffold of a 1,8-naphthalimide-derived FA fluorescent probe. These probes react with FA through the 2-aza-Cope arrangement and exhibit highly selective fluorescence increases for detecting FA in aqueous solutions. Moreover, these organelle-targeting probes, i.e., FFP551-Nuc, FFP551-ER, FFP551-Mito, and FFP551-Lyso, allow selective localization and imaging of FA in the nucleus, endoplasmic reticulum, mitochondria, and lysosomes of live mammalian cells, respectively. Furthermore, FFP551-Nuc has been successfully employed to monitor changes of endogenous FA levels in the nucleus of live mammalian cells. Overall, these probes should represent new imaging tools for studying the biology and pathology associated with FA in different intracellular compartments.

An acetylpyrene-based fluorescent probe for selective detection of cysteine in vitro and in lipid droplets

The development of organelle-targeted probes that enable specific recognition and detection Cysteine (Cys) may provide an effective way to explore physiological processes and pathological mechanisms associated with the related diseases. In this work, an acetylpyrene-based fluorescent probe CP-1 was designed and synthesized for detecting Cys in vitro and in vivo. The probe exhibited a noticeable fluorescence blue shift upon the addition of Cys under UV light, attributed to the mechanism of the nucleophilic addition reaction. The response mechanism for Cys was studied by time-dependent density functional theory (TD-DFT) calculations and mass spectra (MS). CP-1 can not only specifically recognize Cys in the presence of other amino acids and biothiols, but also exhibit the property of targeting lipid droplets (LDs). Due to the good cell biocompatibility, CP-1 has been successfully employed for real-time tracking Cys in LDs of living cells. The ability to target LDs and the favorable biocompatibility make the probe valuable for investigating Cys-related diseases in living cell systems.

The rational utilization of organelle microenvironment for imaging of lysosomal SO2 with high fidelity

Nowadays, more and more studies have linked the abnormal expression of active molecules in organelles with the occurrence of diseases, so there is an urgent need to develop tools for detecting active molecules in specific organelles. However, the recognition receptors of most organelle-targeting probes currently developed always remain active, which easily causes them to react with the analyte in the cytoplasm, thus misjudging the role of the analyte in the physiological and pathological processes. Therefore, it is of great significance to develop a new strategy for the design of probes capable of high-fidelity imaging of the analyte in specific organelles. Herein, we propose a new strategy that the activation of recognition receptors that can be triggered by the microenvironment of targeting organelles. Based on this strategy, we develop a novel lysosome-targeting fluorescent probe (Lyso-SO2) for imaging of sulfur dioxide (SO2) with high-fidelity in lysosomes. The inert probe is activated by the acidic environment in the lysosome and then responds quickly (<2 s) and sensitively (LOD = 0.34 μM) to SO2. This paradigm by taking full advantage of the features of the organelle microenvironment provides a promising methodology for developing organelle-targeting probes for high-fidelity imaging.

Electrostimulus-triggered reactive oxygen species level in organelles revealed by organelle-targeting SERS nanoprobes.

Electrostimulation (ES) is an important therapeutic method for diseases caused by abnormal intracellular electrical activity. Also, it can induce apoptosis of cells, which is a potential tumor treatment method. At present, there are no relevant studies on changes in intracellular reactive oxygen species (ROS) levels produced in the process of ES, or on the effects of simultaneous implementation of conventional antioxidant inhibitor drugs and ES therapy. To reveal these, two organelle-targeting core-shell plasmonic probes were designed for measuring ROS produced during ES. The probes were delivered into target organelles (nucleus and mitochondrion) before the cells were electrically stimulated for different periods of time. Surface-enhanced Raman scattering (SERS) signals were detected in situ, and the sensing mechanism for the quantitative analysis of ROS is based on the signal reduction of SERS caused by the ROS-etching effect on the silver shell. The detection results revealed that ES could trigger ROS generation in cells, and the ROS levels localized around organelles were assessed by SERS. This study has great potential for exploring abnormal organelle microenvironments via organelle-targeting probes combined with SERS technology.

Chromene-based fluorescent probes for sensing and bioimaging

Chromene is an important motif in natural products and is the backbone for a range of fluorescent molecules. Chromene derivatives are particularly important for biological applications because of their distinctive photophysical and chemical properties. Recent research efforts have been focused on utilizing chromene-based probes motif for biological sensing applications. Most notably, the “thiol-chromene” click reaction realized the selective and rapid recognition of thiols. This Review presents the various chromene-based probes for a range of biologically relevant analytes. This includes examples of sensors for thiols, amino acids, hydrogen peroxide, and nitroreductase, as well as subcellular organelle-targeted probes.

Rational engineering of biomimetic flavylium fluorophores for regulating the lysosomal and mitochondrial localization behavior by pH-induced structure switch and application to fluorescence imaging.

Mitochondria and lysosomes, as the important subcellular organelles, play vital roles in cell metabolism and physiopathology. However, there is still no general method to precisely regulate the lysosomal and mitochondrial localization behavior of fluorescent probes except by selecting specific targeting groups. Herein, we proposed a pH-induced structure switch (pHISS) strategy to solve this tricky puzzle. For the proof-of-concept, we have rationally designed and synthesized a series of cationic flavylium derivatives FL-1-9 with tunable pH-induced structure switch through adjusting the electron-donating ability of the substituents. As expected, the co-localization imaging experiments revealed that the lysosomal and mitochondrial localization behavior of FL-1-9 dyes is closely related to their pHISS ability. It is noteworthy that FL cationic dyes with strong electron-donors are not prone to pHISS and can be well enriched in mitochondria, while FL cationic dyes with weak electron-donors are highly susceptible to pHISS and display an unusual lysosome-targeting capability. This also provided a feasible strategy for lysosomal localization without basic groups and presented new application options for some flavylium dyes previously thought to be less stable. Furthermore, FL cationic dyes with medium electron-donor exhibit certain localization abilities both in mitochondria and lysosomes. Finally, through a detailed study of pH-induced structure switch and exploiting the pH inertia brought by the strong electron-donors, a novel NIR ratiometric fluorescent probe with large wavelength-shift was constructed for monitoring mitochondrial H2S in living cells, tumor tissues and living mice, highlighting the value of the pHISS strategy in precisely regulating organelle targeting and constructing corresponding organelle targeting probes.

Multicolor Photoactivatable Raman Probes for Subcellular Imaging and Tracking by Cyclopropenone Caging.

Photoactivatable probes, with high-precision spatial and temporal control, have largely advanced bioimaging applications, particularly for fluorescence microscopy. While emerging Raman probes have recently pushed the frontiers of Raman microscopy for noninvasive small-molecule imaging and supermultiplex optical imaging with superb sensitivity and specificity, photoactivatable Raman probes remain less explored. Here, we report the first general design of multicolor photoactivatable alkyne Raman probes based on cyclopropenone caging for live-cell imaging and tracking. The fast photochemically generated alkynes from cyclopropenones enable background-free Raman imaging with desired photocontrollable features. We first synthesized and spectroscopically characterized a series of model cyclopropenones and identified the suitable light-activating scaffold. We further engineered the scaffold for enhanced chemical stability in a live-cell environment and improved Raman sensitivity. Organelle-targeting probes were then generated to achieve targeted imaging of mitochondria, lipid droplets, endoplasmic reticulum, and lysosomes. Multiplexed photoactivated imaging and tracking at both subcellular and single-cell levels was next demonstrated to monitor the dynamic migration and interactions of the cellular contents. We envision that this general design of multicolor photoactivatable Raman probes would open up new ways for spatial-temporal controlled profiling and interrogations in complex biological systems with high information throughput.

Recent Advances in Organelle-Targeted Fluorescent Probes.

Recent advances in fluorescence imaging techniques and super-resolution microscopy have extended the applications of fluorescent probes in studying various cellular processes at the molecular level. Specifically, organelle-targeted probes have been commonly used to detect cellular metabolites and transient chemical messengers with high precision and have become invaluable tools to study biochemical pathways. Moreover, several recent studies reported various labeling strategies and novel chemical scaffolds to enhance target specificity and responsiveness. In this review, we will survey the most recent reports of organelle-targeted fluorescent probes and assess their general strategies and structural features on the basis of their target organelles. We will discuss the advantages of the currently used probes and the potential challenges in their application as well as future directions.

Fluorescent Probe for Transmembrane Dynamics during Osmotic Effects.

Membrane tension pores determine organelle dynamics and functions, giving rise to physical observables during the cell death process. While fluorescent organelle-targeted probes for specific chemical analytes are increasingly available, subcellular dynamic processes involving not only chemical parameters but also physicochemical and physical parameters are uncommon. Here, we report a mitochondrial chemical probe, named RCN, rationally designed to monitor osmotic effects during transmembrane tension pore formation by using local mitochondrial polarity and a subcellular localization redistribution property of the probe. Utilizing fluorescence spectroscopy, high-resolution confocal imaging, and spectrally resolved confocal microscopy, we provide a new correlation between mitochondrial dynamics and bleb vesicle formation using osmotic pressure stimuli in the cell, where the mitochondrial local polarity was found to drastically increase. The RCN provides a reliable protocol to assess transmembrane pore formation driven by osmotic pressure increments through local polarity variations and is a more robust physicochemical parameter allowing the health and decease status of the cell to be measured.

BODIPY-Based Fluorescent Probes for Biothiols.

Fluorescent probes for biothiols have aroused increasing interest owing to their potential to enable better understanding of the diverse physiological and pathological processes related to the biothiol species. BODIPY fluorophores exhibit excellent optical properties, which can be readily tailored by introducing diverse functional units at various positions of the BODIPY core. In the present review, the development of fluorescent probes based on BODIPYs for the detection of biothiols are systematically summarized, with emphasis on the preferable detection of individual biothiols, as well as simultaneous discrimination among cysteine (Cys), homocysteine (Hcy), reduced glutathione (GSH). In addition, organelle-targeting probes for biothiols are also highlighted. The general design principles, various recognition mechanisms, and biological applications are elaboratively discussed, which could provide a useful reference to researchers worldwide interested in this area.

Future potential of osmium complexes as anticancer drug candidates, photosensitizers and organelle-targeted probes.

Recent work has shown that the chemical reactivity of osmium complexes can be finely tuned by the choice of osmium oxidation state, the ligands (including C- and N-bound), and the coordination geometry and stereochemistry, so opening up a wide range of new potential biological and medical applications. Osmium cancer therapeutics can have diverse modes of action and targets, including DNA interactions, redox modulation and protein inhibition. Polypyridyl Os(ii) compounds have potential advantages for luminescent cell imaging and photodynamic therapy due to their favourable photophysical and photochemical properties, such as long-wavelength metal-ligand charge transfer (MLCT) absorptions, high photostability and useful near-infrared (NIR) emission. Here we summarize recent progress in the design and application of innovative organo-osmium compounds as anticancer agents with novel mechanisms and as organelle-targeted imaging probes.

Design of a simultaneous target and location-activatable fluorescent probe for visualizing hydrogen sulfide in lysosomes.

Molecular tools capable of providing information on a target analyte in an organelle of interest are especially appreciated. Traditionally, organelle-targetable probes are designed by incorporating an organelle-specific guiding unit to target the probe molecules into the organelle. The imperfect targeting function of the guiding unit and nonspecific distribution of the analyte in cytosol and each organelle would lead to low spatiotemporal resolution and limited sensitivity. To solve this problem, we report herein a new approach for detection of a target analyte in a specific organelle by engineering a target and location dual-controlled molecular switch. For this proof-of-concept study, fluorescent detection of H2S in lysosomes was performed with a simultaneous H2S and proton-activatable probe based on the acidic environment of lysosomes. The new synthesized fluorescent sensor, "SulpHensor", which contains a spirolactam moiety to bind hydrogen protons and an azide group to react with H2S, displays highly sensitive and selective fluorescence response to H2S under lysosomal pH environment but is out of operation in neutral cytosol and other organelles. Fluorescence imaging shows that SulpHensor is membrane-permeable and suitable for visualization of both the exogenous and endogenous H2S in lysosomes of living cells. The good performance of our proposed approach for H2S sensing demonstrates that this strategy might open up new opportunities for the development of efficient subcellular molecular tools for bioanalytical and biomedical applications.

Two-Photon Fluorescent Probes for Metal Ions in Live Tissues

Two-photon microscopy (TPM) is a new imaging tool that can detect biological targets deep inside a live tissue. To faciltate the use of TPM in biomedical research, a variety of two-photon (TP) probes for specific applications are needed. In this Forum Article, we describe the design strategy, photophysical properties, and biological imaging applications of a selection of our recent studies in the development of TP probes for metal ions. Small-molecule TP turn-on probes, organelle-targeted probes, and multicolor emissive probes for dual-color imaging are briefly reviewed.

CYTO 2010—Overview of the XXV ISAC Congress Proceedings Issue of Cytometry Part A

issue of Cytometry Part A presents the second occasionon which a call has been issued to encourage authors to sub-mit their best work to the Journal for publication in a specialCongress Proceedings Issue of Cytometry. The incentive forsubmission was that accepted papers would be guaranteed anoral presentation at the XXV ISAC Congress in Seattle—CYTO 2010. The manuscripts submitted for the call werereviewed via the normal Cytometry Part A review process andthen by a second level of review by the Congress OrganizingCommittee. The outcome of this strict peer-review processwas that nine papers were accepted for the Proceedings Issueof Cytometry Part A. The research articles provide excellentexamples of some of the latest trends in the field of cytometryand we highlight them here in this editorial review. The topicsof the accepted papers range from application of 12-color flowcytometry through characterization of new probes and investi-gation of cytoskeleton elements to analysis of nuclear structurefrom histopathology images.Modern polychromatic flow cytometry can providehigher resolution approaches for the dissection and identifica-tion of cell subpopulations (1,2). In this issue, the paper byAutissier et al. (page 410) describes how 12-color flow cytome-try can be used for studying the phenotypes of lymphocyte,monocyte, and dendritic cell subsets. The authors developed asingle flow cytometry panel that allows for simultaneousdetection of the lymphocyte and monocyte cell populationsand all known DC subsets. Studying these crucial subpopula-tions of the immune system in one single panel may give abroader view of the immune response during HIV infectionand can be used for monitoring AIDS pathogenesis.Polychromatic cytometry has become increasingly impor-tant in slide-based applications with the drive to increase thenumber of measurable fluorochromes for multiparametricanalysis. New fluorochromes have been introduced where thefluorescent dyes possess different optical characteristicsincluding spectral range, lifetime signatures, and improvedphotostability (3–5). The special feature of slide-based cyto-metry is that analyzed fields can be revisited, providing a basisfor the application of sequential photobleaching of fluoro-chromes next to very photostable dyes to increase the numberof parameters captured. In this issue, Wessels et al. (page 420)analyzed the newly available NorthernLights secondary anti-bodies for use in slide-based cytometry and microscopy. Theypointed out that since the NorthernLights are bright, resistantto photobleaching, stable in alcohols and xylene, these dyesare promising candidates for use with most laser- and HBO/XBA-based fluorescence microscopy techniques. It is antici-pated that combination of NorthernLights with laser scanningcytometry could provide a better methodology for analysis oftumor dissemination to bone marrow and lymph node (6).The other group of fluorescent probes aims to stain cellorganelles with high specificity. Development of fluorescent,organelle-targeted probes has been driven by an interest in dis-covering new probes that excite and emit in the visible spec-

Organelle‐targeting, red‐emitting fluorescent probes with superior photo‐physical properties for multiplexing with green fluorescent protein (GFP) in live cells

Cell‐based assays are increasingly gaining in popularity in the biotechnology and pharmaceutical industry due to their high physiological relevance. As trends advance toward analysis of living biological systems, fluorescent proteins (such as GFPs) have become indispensable tools for live‐cell analysis. One important application of GFPs is fluorescence co‐localization imaging, a powerful approach for exploring the targeting of molecules to intracellular compartments and for screening of their associations and interactions. To date, however, concerted efforts in developing fluorescent organelle‐targeting probes, specifically tailored to working in concert with GFPs, has been limited. Photo‐conversion of red‐fluorescent dyes to fluoresce green and metachromatic artifacts wherein fluorescent dyes emit both red and green lead to spurious results in GFP co‐localization experiments. Additionally, many organelle‐targeting probes photo‐bleach rapidly, are subject to quenching upon concentration in organelles or only transiently associate with the target organelle, requiring imaging within a minute of dye addition. Consequently, a new family of red‐emitting, cell‐permeable probes that spontaneously localize to specific subcellular organelles was developed. It is demonstrated that the probes can be readily used in combination with other common UV‐ and visible‐light excitable organic fluorescent dyes and various fluorescent proteins in multi‐color imaging and detection applications. It is further shown that the probes emit in the Texas Red region of the visible spectrum, and are highly resistant to photo‐bleaching, concentration quenching and photo‐conversion.