Molecular Probes Research Articles

Bienzyme-Locked Activatable Fluorescent Probes for Specific Imaging of Tumor-Associated Mast Cells.

Tumor-associated mast cells (TAMCs) have been recently revealed to play a multifaceted role in the tumor microenvironment. Noninvasive optical imaging of TAMCs is thus highly desired to gain insights into their functions in cancer immunotherapy. However, due to the lack of a single enzyme that is specific to mast cells, a common probe design approach based on single-enzyme activation is not applicable. Herein, we reported a bienzyme-locked molecular probe (THCMC) based on a photoinduced electron transfer-intramolecular charge-transfer hybrid strategy for in vivo imaging of TAMCs. The bienzyme-locked activation mechanism ensures that THCMC exclusively turns on near-infrared (NIR) fluorescence only in the presence of both tryptase and chymase specifically coexpressed by mast cells. Thus, THCMC effectively distinguishes mast cells from other leukocytes, including T cells, neutrophils, and macrophages, a capability lacking in single-locked probes. Such a high specificity of THCMC allows noninvasive tracking of the fluctuation of TAMCs in the tumor of living mice during cancer immunotherapy. The results reveal that the decreased intratumoral signal of THCMC after combination immunotherapy correlates well with the reduced population of TAMCs, accurately predicting the inhibition of tumor growth. Thus, this study not only presents the first NIR fluorescent probe specific for TAMCs but also proposes a generic bienzyme-locked probe design approach for in vivo cell imaging.

Engineering Cyanine- and Hemicyanine-Based Probes for Optical Imaging of Kidney Diseases.

Molecular optical probes play pivotal roles in in vivo imaging of biomarkers associated to kidney diseases. Relying on structural tunability and high fluorescence quantum yields, versatile optical probes have been constructed on cyanine or hemicyanine-based scaffold in recent years. This review summaries the recent progress on the development of optical probes for imaging of kidney diseases, particularly through near-infrared fluorescence, chemiluminescence and photoacoustic imaging modalities. The chemical design and sensing mechanisms are discussed along with applications in the detection of renal cell carcinoma and acute kidney injury. This progress provides insights and directions for the development of next generation kidney-targeted probes and for pushing their further applications in preclinical and clinical research.

Preclinical evaluation of AGTR1-Targeting molecular probe for colorectal cancer imaging in orthotopic and liver metastasis mouse models

Despite advancements in colorectal cancer (CRC) treatment, the prognosis remains unfavorable for patients with distant liver metastasis. Fluorescence molecular imaging with specific probes is increasingly used to guide CRC surgical resection in real-time and treatment planning. Here, we demonstrate the targeted imaging capacity of an MPA-PEG4-N3-Ang II probe labeled with near-infrared (NIR) fluorescent dye targeting the angiotensin II (Ang II) type 1 receptor (AGTR1) that is significantly upregulated in CRC. MPA-PEG4-N3-Ang II was highly selective and specific to in vitro tumor cells and in vivo tumors in a mouse CRC xenograft model. The favorable ex vivo imaging and in vivo biodistribution of MPA-PEG4-N3-Ang II afforded tumor-specific accumulation with low background and >10 contrast tumor-to-colorectal values in multiple subcutaneous CRC models at 8 h following injection. Biodistribution analysis confirmed the probe's high uptake in HT29 and HCT116 orthotopic and liver metastatic models of CRC with signal-to-noise ratio (SNR) values of tumor-to-colorectal and -liver fluorescence of 5.8 ± 0.6, 5.3 ± 0.7, and 2.7 ± 0.5, 2.6 ± 0.5, respectively, enabling high-contrast intraoperative tumor visualization for surgical navigation. Given its rapid tumor targeting, precise tumor boundary delineation, durable tumor retention and docking study, MPA-PEG4-N3-Ang II is a promising high-contrast imaging agent for the clinical detection of CRC.

Iridium Pyridylpyrrolides Hyperpolarised by Para-Hydrogen.

Chemical activation of the nuclear singlet state of dihydrogen, para-hydrogen, can dramatically increase the sensitivity of magnetic resonance spectroscopy and imaging. Here, we show that the highly reversible activation of para-hydrogen by an iridium pyridylpyrrolide complex is capable of producing this hyperpolarisation effect. Bound alkene ligands exhibit signal enhancement without reduction to alkanes, which is in contrast to the most widely used hyperpolarisation catalysts. The complex is recoverable due to the highly reversible binding and release of H2, and result in enhanced hydride signals in a wide range of coordinating and non-coordinating deuterated solvents. Synthetic modification of the ligand substituents and the addition of co-ligands show a strong dependence of chemical structure on reactivity, which reveals an untapped potential to exploit pyridylpyrrolides as ligands in the development of tunable para-hydrogen induced hyperpolarisation catalysts or molecular probes.

Molecular Gas Tracers in Young and Old Protoplanetary Disks

Molecular emission is used to investigate both the physical and chemical properties of protoplanetary disks. Therefore, to derive disk properties accurately, we need a thorough understanding of the behavior of the molecular probes upon which we rely. Here we investigate how the molecular line emission of N2H+, HCO+, HCN, and C18O compare to other measured quantities in a set of 20 protoplanetary disks. Overall, we find positive correlations between multiple line fluxes and the disk dust mass and radius. We also generally find strong positive correlations between the line fluxes of different molecular species. However, some disks do show noticeable differences in the relative fluxes of N2H+, HCO+, HCN, and C18O. These differences occur even within a single star-forming region. This results in a potentially large range of different disk masses and chemical compositions for systems of similar age and birth environment. While we make preliminary comparisons of molecular fluxes across different star-forming regions, more complete and uniform samples are needed in the future to search for trends with birth environment or age.

Trifunctional Fe3O4@MIP nanocatalytic probe-gold nanosol SERS quantitative analysis of ultratrace dinotefuran

A new trifunctional magnetic nanosurface imprinted polymer (MMIP) nanoprobe (Fe3O4@MIP) were rapidly prepared by microwave digestion procedure, and the nanoprobe was characterized in details. It was found that the nanoprobe not only could specifically recognize dinotefuran (DNT), but also could catalyze the HAuCl4-pyruvate indication reaction to produce gold nanoparticles (AuNPs). The generated AuNPs have strong surface enhanced Raman scattering (SERS) effect using VB4r as molecular probes, resonant Rayleigh scattering (RRS) and surface plasmon resonance absorption (Abs) effect. After adding DNT, it specifically combined with Fe3O4@MIP to form Fe3O4@MIP-DNT conjugates with weak catalytic activity, and the SERS/RRS/Abs signal decreased linearly. Based on this, a new SERS/RRS/Abs trimode sensing platform was constructed for detection DNT with detection limit of 0.009, 0.01 and 0.35 nmol/L, respectively. Due to the unique magnetic properties of the nanoprobe, DNT could be easily and quickly identified, separated and enriched, the enrichment factor was up to 100 times. As a comparison, we also investigated the catalytic and specific recognition effects of MMIP nanoprobes prepared with different neonicotinoid insecticides template molecules and functional monomers.

Facile interfacial synthesis of gold micronails with adjustable length and roughness and their superior SERS properties for the detection of p-aminothiophenol

Au hierarchical architectures with special morphology and structures are strongly desired in varied applications. Herein, a simple synthesis method was developed for the one-step preparation of Au micronails (MNs) at the planar liquid-liquid interface under mild conditions. The well-defined Au MNs were grown and constructed at CHCl3–H2O interface at room temperature using aniline in CHCl3 as reducing agent and HAuCl4 in H2O as precursor and no surfactant or seed is required. The intriguing Au MNs with rough surface consist of big heads and thin rods, just like iron nails in outline. Furthermore, through simple changing the reagent concentrations, the length and surface roughness of Au MNs can be adjusted conveniently. The effects of a series of factors on the morphology and structure of the products are studied in detail. With p-aminothiophenol as a molecular probe, the as-obtained Au MNs all exhibit dramatically improved surface enhanced Raman scattering sensitivity and high reproducibility, the enhancement factor and limit of detection of Au MNs are 5.4 × 105 and 1.0 × 10−10, respectively.

Enhancing surgical outcomes: accurate identification and removal of prostate cancer with B7-H3-targeted NIR-II molecular imaging.

One of the main reasons for prostate cancer (PCa) recurrence is the difficulty in identifying and removing cancerous lesions during surgery. Accurately localizing and excising cancerous tissue remains a significant challenge. The second near-infrared window (NIR-II, 1000-1700nm) fluorescence offers enhanced resolution, a high signal-to-noise ratio, and the potential for deeper tissue penetration. However, this technology is not currently employed for intraoperative imaging of PCa. This study aims to construct a new NIR-II probe targeting B7-H3 (AbB7-H3-800CW) for accurate intraoperative identification and resection of PCa. Based on the differential expression of B7-H3 in PCa, we designed a novel imaging probe to accurately identify and guide the resection of preclinical PCa models and ex vivo human PCa tissues using NIR-II fluorescence imaging technology. Analyzing tissue samples from 60 clinical cases of PCa, along with benign prostatic hyperplasia and normal prostate tissue from 22 cases, we observed a significant difference in B7-H3 protein expression levels (P < 0.001). Subcutaneous and orthotopic mouse models of PCa were imaged using NIR-II fluorescence after AbB7-H3-800CW injection, showing promising results with successful tumor targeting and high-contrast images achieved within 24-48h post-injection. The imaging also enabled the detection of occult PCa lesions approximately 1mm in diameter. In addition, imaging analysis of human PCa and adjacent tissues using AbB7-H3-800CW incubation revealed that cancer tissues exhibited a significantly higher fluorescence intensity than adjacent tissues (P < 0.05), which was conducive to the evaluation of tumor resection margin in vitro. The findings revealed that B7-H3 was a compelling imaging target for PCa. The AbB7-H3-800CW molecular imaging probe is capable of accurately identifying PCa lesions and guiding their removal. This approach can potentially reduce the rate of surgical margins under NIR-II fluorescence guidance.

Biomarker-based human and animal sperm phenotyping: the good, the bad and the ugly†.

Conventional, brightfield-microscopic semen analysis provides important baseline information about sperm quality of an individual; however, it falls short of identifying subtle subcellular and molecular defects in cohorts of "bad," defective human and animal spermatozoa with seemingly normal phenotypes. To bridge this gap, it is desirable to increase the precision of andrological evaluation in humans and livestock animals by pursuing advanced biomarker-based imaging methods. This review, spiced up with occasional classic movie references but seriously scholastic at the same time, focuses mainly on the biomarkers of altered male germ cell proteostasis resulting in post-testicular carryovers of proteins associated with ubiquitin-proteasome system. Also addressed are sperm redox homeostasis, epididymal sperm maturation, sperm-seminal plasma interactions, and sperm surface glycosylation. Zinc ion homeostasis-associated biomarkers and sperm-borne components, including the elements of neurodegenerative pathways such as Huntington and Alzheimer disease, are discussed. Such spectrum of biomarkers, imaged by highly specific vital fluorescent molecular probes, lectins, and antibodies, reveals both obvious and subtle defects of sperm chromatin, deoxyribonucleic acid, and accessory structures of the sperm head and tail. Introduction of next-generation image-based flow cytometry into research and clinical andrology will soon enable the incorporation of machine and deep learning algorithms with the end point of developing simple, label-free methods for clinical diagnostics and high-throughput phenotyping of spermatozoa in humans and economically important livestock animals.

One electron oxidation-induced degradation of brominated flame retardants in electroactive membrane filtration system: Vital role of dichlorine radical-mediated process

Reactive chlorine species (RCS) are inevitably generated in electrochemical oxidation process for treating high-salinity industrial wastewater, thereby resulting in the competition with coexisting hydroxyl radicals (•OH) for oxidizing recalcitrant organic compounds. Due to the low redox potentials compared to •OH, the role of RCS has been often overlooked. In this work, we developed an electroactive membrane filtration (EMF) system that had a high removal efficiency (99.1 ± 0.5 %) for tetrabromobisphenol S (TBBPS) at low energy consumption (1.45 kWh m−3). Electron spin resonance spectroscopy and molecular probing tests indicated the predominance of Cl2•–, of which steady-state concentration (2.2 ×10−10 M) was extremely higher than those of ClO• (6.7 ×10−13 M), •OH (0.95 ×10−13 M), and Cl• (2.39 ×10–15 M). The density functional theory (DFT) and intermediate product analysis highlighted that Cl2•– radicals had a higher electrophilic attack efficacy than •OH radicals for inducing changes in the electron density of the carbon atoms around phenolic hydroxyl groups, thus leading to the generation of transition state intermediates and accelerating the degradation of TBBPS. Our work demonstrates the vital role of Cl2•– radicals for pollutant degradation, highlighting the potential of this technology for cost-effective removal of recalcitrant organic compounds from water and wastewater.

Development of a Fluorescent Ligand for the Intracellular Allosteric Binding Site of the Neurotensin Receptor 1.

The membrane protein family of G protein-coupled receptors (GPCRs) represents a major class of drug targets. Over the last years, the presence of additional intracellular binding sites besides the canonical orthosteric binding pocket has been demonstrated for an increasing number of GPCRs. Allosteric modulators harnessing these pockets may represent valuable alternatives when targeting the orthosteric pocket is not successful for drug development. Starting from SBI-553, a recently discovered intracellular allosteric modulator for neurotensin receptor subtype 1 (NTSR1), we developed the fluorescent molecular probe 14. Compound 14 binds to NTSR1 with an affinity of 0.68 μM in the presence of the agonist NT(8-13). NanoBRET-based ligand binding assays with 14 were established to derive the affinity and structure-activity relationships for allosteric NTSR1 modulators in a direct and nonisotopic manner, thereby facilitating the search for and optimization of novel allosteric NTSR1 ligands. As a consequence of cooperativity between the ligands binding to the allosteric and orthosteric pocket, compound 14 can also be used to investigate orthosteric NTSR1 agonists and antagonists. Moreover, employing 14 as a probe in a drug library screening, we identified novel chemotypes as binders for the intracellular allosteric SBI-553 binding pocket of NTSR1 with single-digit micromolar affinity. These hits may serve as interesting starting points for the development of novel intracellular allosteric ligands for NTSR1 as a highly interesting yet unexploited drug target in the fields of pain and addiction disorder therapy.

Quinone Methide Based Self-Immobilizing Molecular Fluorescent Probes for In Situ Imaging of Enzymes.

Enzymes play important roles not only in normal physiological processes but in the development of many diseases. In situ imaging of enzymes with high-resolution in living systems would helpful for clinical diagnosis and treatment. However, many molecular fluorescent probes suffer from the drawback of diffusing away from the reaction site of enzymes even out of the cells, losing the in situ information and resulting in poor imaging resolution. Quinone methide (QM) based self-immobilizing probes allow the fluorescent signal to be immobilized near the target for an extended period without deactivating the target enzymes, ensuring that it will provide amplified signals and in situ information of the target with high resolution. In this review, we summarized the recent progress of QM-based self-immobilizing probes including their design strategies, working mechanisms, classifications and applications in in situ enzyme imaging. This review calls for the development of more activatable QM-based probe with the advantages of high stability in the absence of the target but very high labeling efficiency after activation.

Introduction to the Optical Molecular Probes, Imaging and Drug Delivery 2023 feature issue

A feature issue is being presented by a team of guest editors containing papers based on studies presented at the Optical Molecular Probes, Imaging and Drug Delivery conference as part of the Optica Biophotonics Congress in Vancouver, Canada from April 24–27, 2023.

Ligustilide covalently binds to Cys703 in the pre-S1 helix of TRPA1, blocking the opening of channel and relieving pain in rats with acute soft tissue injury

Ethnopharmacological relevanceThe natural anodyne Ligustilide (Lig), derived from Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort., has been traditionally employed for its analgesic properties in the treatment of dysmenorrhea and migraine, and rheumatoid arthritis pain. Despite the existing reports on the correlation between TRP channels and the analgesic effects of Lig, a comprehensive understanding of their underlying mechanisms of action remains elusive. Aim of the studyThe objective of this study is to elucidate the mechanism of action of Lig on the analgesic target TRPA1 channel. MethodsThe therapeutic effect of Lig was evaluated in a rat acute soft tissue injury model. The analgesic target was identified through competitive inhibition of TRP channel agonists at the animal level, followed by Fluo-4/Ca2+ imaging on live cells overexpressing TRP proteins. The potential target was verified through in-gel imaging, colocalization using a Lig-derived molecular probe, and a drug affinity response target stability assay. The binding site of Lig was identified through protein spectrometry and further analyzed using molecular docking, site-specific mutation, and multidisciplinary approaches. ResultsThe administration of Lig effectively ameliorated pain and attenuated oxidative stress and inflammatory responses in rats with soft tissue injuries. Moreover, the analgesic effects of Lig were specifically attributed to TRPA1. Mechanistic studies have revealed that Lig directly activates TRPA1 by interacting with the linker domain in the pre-S1 region of TRPA1. Through metabolic transformation, 6,7-epoxyligustilide (EM-Lig) forms a covalent bond with Cys703 of TRPA1 at high concentrations and prolonged exposure time. This irreversible binding prevents endogenous electrophilic products from entering the cysteine active center of ligand-binding pocket of TRPA1, thereby inhibiting Ca2+ influx through the channel opening and ultimately relieving pain. ConclusionsLig selectively modulates the TRPA1 channel in a bimodal manner via non-electrophilic/electrophilic metabolic conversion. The epoxidized metabolic intermediate EM-Lig exerts analgesic effects by irreversibly inhibiting the activation of TRPA1 on sensory neurons. These findings not only highlight the analgesic mechanism of Lig but also offer a novel nucleophilic attack site for the development of TRPA1 antagonists in the pre-S1 region.

The 5HT2b Receptor in Alzheimer's Disease: Increased Levels in Patient Brains and Antagonist Attenuation of Amyloid and Tau Induced Dysfunction.

Background: Neurodegenerative diseases manifest behavioral dysfunction with disease progression. Intervention with neuropsychiatric drugs is part of most multi-drug treatment paradigms. However, only a fraction of patients responds to the treatments and those responding must deal with drug-drug interactions and tolerance issues generally attributed to off-target activities. Recent efforts have focused on the identification of underexplored targets and exploration of improved outcomes by treatment with selective molecular probes. As part of ongoing efforts to identify and validate additional targets amenable to therapeutic intervention, we examined levels of the serotonin 5-HT2b receptor (5-HT2bR) in Alzheimer's disease (AD) brains and the potential of a selective 5-HT2bR antagonist to counteract synaptic plasticity and memory damage induced by AD-related proteins, amyloid-β, and tau. This work used a combination of biochemical, chemical biology, electrophysiological, and behavioral techniques. Biochemical methods included analysis of protein levels. Chemical biology methods included the use of an in vivo molecular probe MW071, a selective antagonist for the 5HT2bR. Electrophysiological methods included assessment of long-term potentiation (LTP), a type of synaptic plasticity thought to underlie memory formation. Behavioral studies investigated spatial memory and associative memory. 5HT2bR levels are increased in brain specimens of AD patients compared to controls. 5HT2bR antagonist treatment rescued amyloid-β and tau oligomer-induced impairment of synaptic plasticity and memory. The increased levels of 5HT-2bR in AD patient brains and the attenuation of disease-related synaptic and behavioral dysfunctions by MW071 treatment suggest that the 5HT-2bR is a molecular target worth pursuing as a potential therapeutic target.

Graphene oxide functionalized organosilanes for electrochemical detection of uric acid in urine and water

The development of a sensitive molecular probe for recognition of uric acid is imperative because of diseases caused from change in its normal level. In the present article, new nanocomposites are developed based on graphene oxide (GO) functionalized organosilanes and applied for electrochemical detection of uric acid using GO-Silane/Glassy Carbon Electrode (GCE). Firstly, Isoindolone tethered organosilanes are synthesized by inserting amino phenol into pthallic anhydride followed by azide-alkyne click reaction catalyzed by [CuBr (PPh3)3]. The presence of silane unit helped in the extension of this molecular system in the material field and the prepared organosilanes have been grafted over the graphene oxide surface to get the desired probe for uric acid. The newly synthesized compounds have been well characterized by Infrared Spectroscopy (IR), Nuclear Magnetic Resonance (1H and 13C NMR), X-ray Diffraction (XRD), Field Emission Scanning Electron Microscope (FE-SEM) and mass spectral analysis. Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) are used to evaluate the electrochemical signal of the prepared GO-Silane/GCE electrode. The results obtained shows excellent selectivity for uric acid against different species with a low detection limit of 65.7 nM in the linear range 2.5 μM–80.0 μM. Further, the outcome of the work suggests high specificity of the current GO-silane based receptor for uric acid in the presence of different interfering ions. Moreover, the real sample analysis of the present organosilane-based receptor in urine and water pave the way for its practical applicability.

Assessing the role of surface layer and molecular probe size in diffusion within meniscus tissue.

Diffusion within extracellular matrix is essential to deliver nutrients and larger metabolites to the avascular region of the meniscus. It is well known that both structure and composition of the meniscus vary across its regions; therefore, it is crucial to fully understand how the heterogenous meniscal architecture affects its diffusive properties. The objective of this study was to investigate the effect of meniscal region (core tissue, femoral, and tibial surface layers) and molecular weight on the diffusivity of several molecules in porcine meniscus. Tissue samples were harvested from the central area of porcine lateral menisci. Diffusivity of fluorescein (MW 332 Da) and three fluorescence-labeled dextrans (MW 3k, 40k, and 150k Da) was measured via fluorescence recovery after photobleaching. Diffusivity was affected by molecular size, decreasing as the Stokes' radius of the solute increased. There was no significant effect of meniscal region on diffusivity for fluorescein, 3k and 40k dextrans (p>0.05). However, region did significantly affect the diffusivity of 150k Dextran, with that in the tibial surface layer being larger than in the core region (p = 0.001). Our findings contribute novel knowledge concerning the transport properties of the meniscus fibrocartilage. This data can be used to advance the understanding of tissue pathophysiology and explore effective approaches for tissue restoration.

Novel cutting edge nano-strategies to address old long-standing complications in cardiovascular diseases. A comprehensive review.

Cardiovascular diseases (CVD) impact a substantial portion of the global population and represent a significant threat to experiencing life-threatening outcomes, such as atherosclerosis, myocardial infarction, stroke and heart failure. Despite remarkable progress in pharmacology and medical interventions, CVD persists as a major public health concern, and now ranks as the primary global cause of death and the highest consumer of global budgets. Ongoing research endeavours persist in seeking novel therapeutic avenues and interventions to deepen our understanding of CVD, enhance prevention measures, and refine treatment strategies. Nanotechnology applied to the development of new molecular probes with diagnostic and theranostic properties represents one of the greatest technological challenges in preclinical and clinical research. The application of nanotechnology in cardiovascular medicine holds great promise for advancing our understanding of CVDs and revolutionizing their diagnosis and treatment strategies, ultimately improving patient care and outcomes. In addition, the capacity of drug encapsulation in nanoparticles has significantly bolstered their biological safety, bioavailability and solubility. In combination with imaging technologies, molecular imaging has emerged as a pivotal therapeutic tool, offering insight into the molecular events underlying disease and facilitating targeted treatment approaches. Here, we present a comprehensive overview of the recent advancements in targeted nanoparticle approaches for diagnosing CVDs, encompassing molecular imaging techniques, underscoring the significant progress in theranostic, as a novel and promising therapeutic strategy.

An Innovative Methodology to Characterize, at the Molecular Scale, Interactions in Polysaccharide Aqueous Solutions.

Characterizing molecular interactions at the microscopic level remains difficult and, therefore, represents a key target to better understand macromolecule and biomacromolecule behaviors in solution, alone, or in mixtures with others. Therefore, accurate characterization in liquid media, especially in aqueous solutions, without causing any perturbation of the system in which they are studied, is quite difficult. To this purpose, the present paper describes an innovative methodology based on fluorescence spectrophotometry. Two molecular fluorescent probes, namely 8-anilino-1-naphtalenesulfonic acid (ANS) and 2-benzofuryl-3-hydroxy-4(1H)-quinolone (3HQ-Bf), were selected to characterize, respectively, the dipole-dipole interactions and hydrophobic micro-domains, for the first one, and hydrogen bonding, for the second. As a support to study molecular interactions, xanthan, galactomannan, and corresponding mixtures of these substances which are well known to exhibit a synergy of interactions in well-defined mixture conditions were chosen. Once the methodology was set up, the existence of the three types of interactions in these systems was demonstrated, thus allowing the elucidation of the mechanisms of interactions at the molecular scale.

Synthesis of Monofluorinated 7-Hydroxycoumarin-3-Carboxamides as Cell-Permeable Fluorescent Molecular Probes.

To facilitate studies of engagement of protein targets by small molecules in living cells, we synthesized fluorinated derivatives of the fluorophore 7-hydroxycoumarin-3-carboxylic acid (7OHCCA). Compared to the related difluorinated coumarin Pacific Blue (PB), amide derivatives of 6-fluoro-7-hydroxycoumarin-3-carboxylic acid (6FC) exhibited substantially brighter fluorescence. When linked to the anticancer drug paclitaxel (Taxol) via gamma-aminobutyric acid (GABA), the acidity of the phenol of these coumarins profoundly affected cellular efflux and binding to microtubules in living cells. In contrast to the known fluorescent taxoid PB-GABA-Taxol, the less acidic 6FC-GABA-Taxol was more cell-permeable due to a lower susceptibility to active efflux. In living cells, this facilitated the imaging of microtubules by confocal microscopy and enabled quantification of binding to microtubules by flow cytometry without added efflux inhibitors. The photophysical, chemical, and biological properties of 6FC derivatives make these compounds particularly attractive for the construction of fluorescent molecular probes suitable for quantitative analysis of intracellular small molecule-protein interactions.