Albumin-seeking dyes with adjustable assemblies in situ enable programmable imaging windows and targeting tumor imaging.

Cyanine dyes are widely used organic probes for in vivo imaging due to their tunable fluorescence. They can form complexes with endogenous albumin, resulting in enhanced brightness and photostability. However, this binding is uncontrollable and irreversible, leading to considerable nonspecific background signals and unregulated circulation time. Methods: Here, we connect varying numbers of 4-(4-iodophenyl) butanoic acid (IP) as albumin-binding moieties (ABM) to the cyanine dye, enabling dynamic and controllable binding with albumin. Meanwhile, we provide a blocking method to completely release the dye from covalent capture with albumin, resulting in specific targeting fluorescence. Furthermore, we evaluate the pharmacokinetics and tumor targeting of the developed dyes. Results: The engineered dyes can dynamically and selectively bind with multiple albumins to change the in situ size of assemblies and circulation time, providing programmable regulation over the imaging time window. The nucleophilic substitution of meso-Cl with water-soluble amino acids or targeting peptides for IP-engineered dye further addresses the nonspecific signals caused by albumin, allowing for adjustable angiography time and efficient tumor targeting. Conclusion: This study rationalizes the binding modes of dyes and proteins, applicable to a wide range of near-infrared (NIR) dyes for improving their in vivo molecular imaging.

Ultrabright Renal-Clearable Cyanine-Protein Nanoprobes for High-Quality NIR-II Angiography and Lymphography.

The renal-clearable aspect of imaging agent with minimum toxicity issues and side effects is essential for clinical translation, yet clinical near-infrared-I/II (NIR-I/II) fluorophores with timely renal-clearance pathways are very limited. Herein, we rationally develop the cyanine-protein composite strategy through covalent bonding of ß-lactoglobulin (ß-LG) and chloride-cyanine dye to produce a brilliant and stable NIR-I/II fluorophore (e.g., ß-LG@IR-780). The ß-LG acts as a protecting shell with small molecular weight (18.4 kDa) and ultrasmall size (<5 nm), thus endowing the ß-LG@IR-780 with excellent biocompatibility and renal excretion. Our ß-LG@IR-780 probe enables noninvasive and precise NIR-II visualization of the physiological and pathological conditions of the vascular and lymphatic drainage system, facilitating intraoperative imaging-guided surgery and postoperative noninvasive monitoring. The minimum accumulation of our probes in the main organs improves the overall biosafety. This study provides a facile methodology for new-generation NIR-II fluorophores and largely improves the brightness and pharmacokinetics of small molecular dyes.

In vivo accurate detection of the liver tumor with pharmacokinetic parametric images from dynamic fluorescence molecular tomography.

SIGNIFICANCE: Pharmacokinetic parametric images in dynamic fluorescence molecular tomography (FMT) can describe three-dimensional (3D) physiological and pathological information inside biological tissues, potentially providing quantitative assessment tools for biological research and drug development. AIM: In vivo imaging of the liver tumor with pharmacokinetic parametric images from dynamic FMT based on the differences in metabolic properties of indocyanine green (ICG) between normal liver cells and tumor liver cells inside biological tissues. APPROACH: First, an orthotopic liver tumor mouse model was constructed. Then, with the help of the FMT/computer tomography (CT) dual-modality imaging system and the direct reconstruction algorithm, 3D imaging of liver metabolic parameters in nude mice was achieved to distinguish liver tumors from normal tissues. Finally, pharmacokinetic parametric imaging results were validated against in vitro anatomical results. RESULTS: This letter demonstrates the ability of dynamic FMT to monitor the pharmacokinetic delivery of the fluorescent dye ICG in vivo, thus, enabling the distinction between normal and tumor tissues based on the pharmacokinetic parametric images derived from dynamic FMT. CONCLUSIONS: Compared with CT structural imaging technology, dynamic FMT combined with compartmental modeling as an analytical method can obtain quantitative images of pharmacokinetic parameters, thus providing a more powerful research tool for organ function assessment, disease diagnosis and new drug development.

Adrenergic receptors control frequency-dependent switching of the exocytosis mode between "full-collapse" and "kiss-and-run" in murine motor nerve terminal.

AIMS: Neurotransmitter release from the synaptic vesicles can occur through two modes of exocytosis: "full-collapse" or "kiss-and-run". Here we investigated how increasing the nerve activity and pharmacological stimulation of adrenoceptors can influence the mode of exocytosis in the motor nerve terminal. METHODS: Recording of endplate potentials with intracellular microelectrodes was used to estimate acetylcholine release. A fluorescent dye FM1-43 and its quenching with sulforhodamine 101 were utilized to visualize synaptic vesicle recycling. KEY FINDINGS: An increase in the frequency of stimulation led to a decrease in the rate of FM1-43 unloading despite the higher number of quanta released. High frequency activity promoted neurotransmitter release via the kiss-and-run mechanism. This was confirmed by experiments utilizing (I) FM1-43 dye quencher, that is able to pass into the synaptic vesicle via fusion pore, and (II) loading of FM1-43 by compensatory endocytosis. Noradrenaline and specific α2-adrenoreceptors agonist, dexmedetomidine, controlled the mode of synaptic vesicle recycling at high frequency activity. Their applications favored neurotransmitter release via full-collapse exocytosis rather than the kiss-and-run pathway. SIGNIFICANCE: At the diaphragm neuromuscular junctions, neuronal commands are translated into contractions necessary for respiration. During stress, an increase in discharge rate of the phrenic nerve shifts the exocytosis from the full-collapse to the kiss-and-run mode. The stress-related molecule, noradrenaline, restricts neurotransmitter release in response to a high frequency activity, and prevents the shift in the mode of exocytosis through α2-adrenoceptor activation. This may be a component of the mechanism that limits overstimulation of the respiratory system during stress.

Guest-host doped strategy for constructing ultralong-lifetime near-infrared organic phosphorescence materials for bioimaging.

Organic near-infrared room temperature phosphorescence materials have unparalleled advantages in bioimaging due to their excellent penetrability. However, limited by the energy gap law, the near-infrared phosphorescence materials (>650 nm) are very rare, moreover, the phosphorescence lifetimes of these materials are very short. In this work, we have obtained organic room temperature phosphorescence materials with long wavelengths (600/657-681/732 nm) and long lifetimes (102-324 ms) for the first time through the guest-host doped strategy. The guest molecule has sufficient conjugation to reduce the lowest triplet energy level and the host assists the guest in exciton transfer and inhibits the non-radiative transition of guest excitons. These materials exhibit good tissue penetration in bioimaging. Thanks to the characteristic of long lifetime and long wavelength emissive phosphorescence materials, the tumor imaging in living mice with a signal to background ratio value as high as 43 is successfully realized. This work provides a practical solution for the construction of organic phosphorescence materials with both long wavelengths and long lifetimes.

Photoactivatable Fluorescent Tags for Dual-Modality Positron Emission Tomography Optical Imaging.

Fluorescent protein conjugates are vital tools in a wide range of scientific disciplines from basic biochemical research to applications in clinical pathology and intraoperative surgery. We report the synthesis and characterization of photoactivatable fluorophores (PhotoTags) based on the functionalization of coumarin, fluorescein, BODIPY, rhodamine B, and cyanine dyes with a photochemically active aryl azide group. Photochemical labeling experiments using human serum albumin produced fluorescent proteins in high yields under irradiation with ultraviolet light for <15 min. We also synthesized DFO-RhodB-PEG3-ArN3─a photoactivatable compound that can be radiolabeled with 89Zr for applications in optical imaging and positron emission tomography. One-pot 89Zr-radiolabeling and light-induced protein conjugation produced [89Zr]ZrDFO-RhodB-PEG3-azepin-trastuzumab. Proof-of-concept studies in vitro and in vivo confirmed that [89Zr]ZrDFO-RhodB-PEG3-azepin-trastuzumab is a potential dual-modality agent for detecting human epidermal growth factor receptor 2 (HER2/neu) expression. Overall, the PhotoTag technology represents a rapid, synthetically versatile, and user-friendly approach for generating novel protein conjugates.

Self-assembled NIR-II Fluorophores with Ultralong Blood Circulation for Cancer Imaging and Image-guided Surgery.

Complete excision of the last remaining 1-2% of tumor tissue without collateral damage remains particularly challenging. Herein, we report thiophenthiadiazole (TTD)-derived fluorophores L6-PEGnk (n = 1, 2, 5) as new-generation NIR-II (1000-1700 nm) probes with exceptional nonfouling performance and significantly high fluorescence quantum yields in water. L6-PEG2k can self-assemble into vesicular micelles and exhibited minimal immunogenicity, low binding affinities, ultralong blood circulation (t1/2 = 59.5 h), and a supercontrast ratio in vivo. Most importantly, L6-PEG2k achieved excellent in vivo CT-26 and U87MG tumor targeting and accumulation (>20 d) through intraperitoneal or intravenous injection. A subcutaneous U87MG tumor and orthotopic brain glioma were successfully resected under NIR-II FIGS in our animal model via intraperitoneal injection in an extended time window (48-144 h). This study highlights the potential of using L6-PEG2K as self-assembling molecular probes with long-circulation persistence for routine preoperative tumor assessment and precise intraoperative image-guided resection.

Rational Design of Surface-State Controlled Multicolor Cross-Linked Carbon Dots with Distinct Photoluminescence and Cellular Uptake Properties.

We disclose for the first time a facile synthetic methodology for the preparation of multicolor carbon dots (CDs) from a single source barring any chromatographic separations. This was achieved via sequential intraparticle cross-linking of surface abundant carboxylic acid groups on the CDs synthesized from a precursor to control their photoluminescence (PL) spectra as well as affect their degree of cellular internalization in cancer cells. The change in PL spectra with sequential cross-linking was projected by theoretical density functional theory (DFT) studies and validated by multiple characterization tools such as X-ray photoelectron spectroscopy (XPS), PL spectroscopy, ninhydrin assay, etc. The variation in cellular internalization of these cross-linked CDs was demonstrated using inhibitor assays, confocal microscopy, and flow cytometry. We supplemented our findings with high-resolution dark-field imaging to visualize and confirm the colocalization of these CDs into distinct intracellular compartments. Finally, to prove the surface-state controlled PL mechanisms of these cross-linked CDs, we fabricated a triple-channel sensor array for the identification of different analytes including metal ions and biologically relevant proteins.

4-Azidocinnoline-Cinnoline-4-amine Pair as a New Fluorogenic and Fluorochromic Environment-Sensitive Probe.

A new type of fluorogenic and fluorochromic probe based on the reduction of weakly fluorescent 4-azido-6-(4-cyanophenyl)cinnoline to the corresponding fluorescent cinnoline-4-amine was developed. We found that the fluorescence of 6-(4-cyanophenyl)cinnoline-4-amine is strongly affected by the nature of the solvent. The fluorogenic effect for the amine was detected in polar solvents with the strongest fluorescence increase in water. The environment-sensitive fluorogenic properties of cinnoline-4-amine in water were explained as a combination of two types of fluorescence mechanisms: aggregation-induced emission (AIE) and excited state intermolecular proton transfer (ESPT). The suitability of an azide-amine pair as a fluorogenic probe was tested using a HepG2 hepatic cancer cell line with detection by fluorescent microscopy, flow cytometry, and HPLC analysis of cells lysates. The results obtained confirm the possibility of the transformation of the azide to amine in cells and the potential applicability of the discovered fluorogenic and fluorochromic probe for different analytical and biological applications in aqueous medium.

Dye-loaded mesoporous polydopamine nanoparticles for multimodal tumor theranostics with enhanced immunogenic cell death.

BACKGROUND: Tumor phototherapy especially photodynamic therapy (PDT) or photothermal therapy (PTT), has been considered as an attractive strategy to elicit significant immunogenic cell death (ICD) at an optimal tumor retention of PDT/PTT agents. Heptamethine cyanine dye (IR-780), a promising PDT/PTT agent, which can be used for near-infrared (NIR) fluorescence/photoacoustic (PA) imaging guided tumor phototherapy, however, the strong hydrophobicity, short circulation time, and potential toxicity in vivo hinder its biomedical applications. To address this challenge, we developed mesoporous polydopamine nanoparticles (MPDA) with excellent biocompatibility, PTT efficacy, and PA imaging ability, facilitating an efficient loading and protection of hydrophobic IR-780. RESULTS: The IR-780 loaded MPDA (IR-780@MPDA) exhibited high loading capacity of IR-780 (49.7 wt%), good physiological solubility and stability, and reduced toxicity. In vivo NIR fluorescence and PA imaging revealed high tumor accumulation of IR-780@MPDA. Furthermore, the combined PDT/PTT of IR-780@MPDA could induce ICD, triggered immunotherapeutic response to breast tumor by the activation of cytotoxic T cells, resulting in significant suppression of tumor growth in vivo. CONCLUSION: This study demonstrated that the as-developed compact and biocompatible platform could induce combined PDT/PTT and accelerate immune activation via excellent tumor accumulation ability, offering multimodal tumor theranostics with negligible systemic toxicity.

Rapidly liver-clearable rare-earth core-shell nanoprobe for dual-modal breast cancer imaging in the second near-infrared window.

BACKGROUND: Fluorescence imaging as the beacon for optical navigation has wildly developed in preclinical studies due to its prominent advantages, including noninvasiveness and superior temporal resolution. However, the traditional optical methods based on ultraviolet (UV, 200-400 nm) and visible light (Vis, 400-650 nm) limited by their low penetration, signal-to-noise ratio, and high background auto-fluorescence interference. Therefore, the development of near-infrared-II (NIR-II 1000-1700 nm) nanoprobe attracted significant attentions toward in vivo imaging. Regrettably, most of the NIR-II fluorescence probes, especially for inorganic NPs, were hardly excreted from the reticuloendothelial system (RES), yielding the anonymous long-term circulatory safety issue. RESULTS: Here, we develop a facile strategy for the fabrication of Nd3+-doped rare-earth core-shell nanoparticles (Nd-RENPs), NaGdF4:5%Nd@NaLuF4, with strong emission in the NIR-II window. What's more, the Nd-RENPs could be quickly eliminated from the hepatobiliary pathway, reducing the potential risk with the long-term retention in the RES. Further, the Nd-RENPs are successfully utilized for NIR-II in vivo imaging and magnetic resonance imaging (MRI) contrast agents, enabling the precise detection of breast cancer. CONCLUSIONS: The rationally designed Nd-RENPs nanoprobes manifest rapid-clearance property revealing the potential application toward the noninvasive preoperative imaging of tumor lesions and real-time intra-operative supervision.

A Novel NIR Fluorescent Probe for Highly Selective Detection of Nitroreductase and Hypoxic-Tumor-Cell Imaging.

Nitroreductase as a potential biomarker for aggressive tumors has received extensive attention. In this work, a novel NIR fluorescent probe for nitroreductase detection was synthesized. The probe Py-SiRh-NTR displayed excellent sensitivity and selectivity. Most importantly, the confocal fluorescence imaging demonstrated that HepG-2 cells treated with Py-SiRh-NTR under hypoxic conditions showed obvious enhanced fluorescence, which means that the NTR was overexpressed under hypoxic conditions. Moreover, the probe showed great promise that could help us to study related anticancer mechanisms research.

Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping.

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell-cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.

Cytochrome b 5 Binds Tightly to Several Human Cytochrome P450 Enzymes.

Numerous studies have been reported in the past 50-plus years regarding the stimulatory role of cytochrome b 5 (b 5) in some, but not all, microsomal cytochrome P450 (P450) reactions with drugs and steroids. A missing element in most of these studies has been a sensitive and accurate measure of binding affinities of b 5 with P450s. In the course of work with P450 17A1, we developed a fluorescent derivative of a human b 5 site-directed mutant, Alexa 488-T70C-b 5, that could be used in binding assays at sub-µM concentrations. Alexa 488-T70C-b 5 bound to human P450s 1A2, 2B6, 2C8, 2C9, 2E1, 2S1, 4A11, 3A4, and 17A1, with estimated K d values ranging from 2.5 to 61 nM. Only weak binding was detected with P450 2D6, and no fluorescence attenuation was observed with P450 2A6. All of the P450s that bound b 5 have some reported activity stimulation except for P450 2S1. The affinity of P450 3A4 for b 5 was decreased somewhat by the presence of a substrate or inhibitor. The fluorescence of a P450 3A4•Alexa 488-T70C-b 5 complex was partially restored by titration with NADPH-P450 reductase (POR) (K d,apparent 89 nM), suggesting the existence of a ternary P450 3A4-b 5-POR complex, as observed previously with P450 17A1. Gel filtration evidence was also obtained for this ternary complex with P450 3A4. Overall, the results indicated that the affinity of b 5 for many P450s is very high, and that ternary P450-b 5-POR complexes are relevant in P450 3A4 reactions as opposed to a shuttle mechanism. SIGNIFICANCE STATEMENT: High-affinity binding of cytochrome b 5 (b 5) (K d < 100 nM) was observed with many drug-metabolizing cytochrome P450 (P450) enzymes. There is some correlation of binding with reported stimulation, with several exceptions. Evidence is provided for a ternary P450 3A4-b 5-NADPH-P450 reductase complex.

Cellular binding and uptake of fluorescent glucose analogs 2-NBDG and 6-NBDG occurs independent of membrane glucose transporters.

The classical methods for determining glucose uptake rates in living cells involve the use of isotopically labeled 2-deoxy-d-glucose or 3-O-methyl-d-glucose, which enter cells via well-characterized membrane transporters of the SLC2A and SLC5A families, respectively. These classical methods, however, are increasingly being displaced by high-throughput assays that utilize fluorescent analogs of glucose. Among the most commonly used of these analogs are 2-NBDG and 6-NBDG, which contain a bulky 7-nitro-2,1,3-benzoxadiazol-4-yl-amino moiety in place of a hydroxy group on d-glucose. This fluorescent group significantly alters both the size and shape of these molecules compared to glucose, calling into question whether they actually enter cells by the same transport mechanisms. In this study, we took advantage of the well-defined glucose uptake mechanism of L929 murine fibroblasts, which rely exclusively on the Glut1/Slc2a1 membrane transporter. We demonstrate that neither pharmacologic inhibition of Glut1 nor genetic manipulation of its expression has a significant impact on the binding or uptake of 2-NBDG or 6-NBDG by L929 cells, though both approaches significantly impact [3H]-2-deoxyglucose uptake rates. Together these data indicate that 2-NBDG and 6-NBDG can bind and enter mammalian cells by transporter-independent mechanisms, which calls into question their utility as an accurate proxy for glucose transport.

Study on the pharmacokinetics of mulberry fruit polysaccharides through fluorescence labeling.

A sensitive and efficient fluorescence labeling method was developed and validated for the microanalysis and detection of polysaccharides. Fluorescein isothiocyanate (FITC) was successfully labeled on mulberry fruit polysaccharides (MFP) through a reductive amination reaction with the assistant of tyramine. The fluorescent labeled polysaccharides (FMFP) was identified by fluorescence, UV-visible, flourier transform infrared (FT-IR) and 1H NMR spectrum. Results demonstrated that the labeling efficiency of FMFP was 0.32%, and the FMFP was stable in simulated digestion fluid without cytotoxicity. The pharmacokinetics and biodistribution after administration were analyzed in rats, which indicated that the FMFP obtained could be absorbed in a short time (tmax 0.50 h) but eliminated slowly (t1/2 8.77 ± 1.38 h). At 24 h after administration, the polysaccharide could be tested mainly in intestine, stomach, liver and kidney. The FITC labeling method lays a foundation for investigating the absorption and metabolism of MFP, and provides references for the microanalysis research of bioactive polysaccharides.

Tetrahydrocannabinol and Its Major Metabolites Are Not (or Are Poor) Substrates or Inhibitors of Human P-Glycoprotein [ATP-Binding Cassette (ABC) B1] and Breast Cancer Resistance Protein (ABCG2).

(-)-Δ9-Tetrahydrocannabinol (THC) is the primary psychoactive constituent of cannabis. In humans, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH) are psychoactive and nonpsychoactive circulating metabolites of THC, respectively. Whether these cannabinoids are substrates or inhibitors of human P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP) is unknown. Previous animal studies suggest that THC and its metabolites could be substrates of these transporters. Therefore, we performed Transwell, cellular accumulation, and vesicular transport assays, at pharmacologically relevant concentrations of these cannabinoids, using Madin-Darby canine kidney (MDCK) II cells or plasma membrane vesicles overexpressing human P-gp or BCRP. Neither THC nor 11-OH-THC was found to be a substrate or inhibitor of P-gp or BCRP. The efflux ratio of THC-COOH in MDCKII-BCRP cells was 1.6, which was significantly decreased to 1.0 by the BCRP inhibitor Ko143. Likewise, cellular accumulation of THC-COOH was significantly increased 1.6-fold in the presence versus absence of Ko143. THC-COOH also significantly inhibited BCRP-mediated transport of Lucifer yellow, a BCRP substrate; however, THC-COOH was neither a substrate nor an inhibitor of P-gp. Collectively, these results indicate that THC and 11-OH-THC are not substrates or inhibitors (at pharmacologically relevant concentrations) of either P-gp or BCRP. THC-COOH is a weak substrate and inhibitor of BCRP, but not of P-gp. Accordingly, we predict that P-gp/BCRP will not modulate the disposition of these cannabinoids in humans. In addition, use of these cannabinoids will not result in P-gp- or BCRP-based drug interactions. SIGNIFICANCE STATEMENT: This study systematically investigated whether Δ9-tetrahydrocannabinol (THC) and its major metabolites, 11-hydroxy-THC and 11-nor-9-carboxy-THC, are substrates and/or inhibitors of human P-gp and BCRP at pharmacologically relevant concentrations. The results obtained are highly valuable for mechanistic understanding and prediction of the roles of P-gp and BCRP in determining the human pharmacokinetics, tissue distribution, and drug interactions of cannabinoids.

Imaging-Guided Chemo-Photothermal Polydopamine Carbon Dots for EpCAM-Targeted Delivery toward Liver Tumor.

We demonstrate a versatile nanoparticle with imaging-guided chemo-photothermal synergistic therapy and EpCAM-targeted delivery of liver tumor cells. EpCAM antibody (anti-EpCAM) and Pt(IV) were grafted onto the polydopamine carbon dots (PDA-CDs) by the amidation reaction. The EpCAM antibody of particles enables the targeted interaction with liver progenitor cells due to their overexpressed EpCAM protein. The tetravalent platinum prodrug [Pt(IV)] induces apoptosis with minimum toxic side effects through the interaction between cisplatin and tumor cell DNA. The nanoparticles displayed stable photothermal property and considerable anti-tumor therapeutic effect in vivo. Coupling with cellular imaging due to their fluorescence property, anti-EpCAM@PDA-CDs@Pt(IV) offers a convenient and effective platform for imaging-guided chemo-photothermal synergistic therapy toward liver cancers in the near future.

Diffusion MRI and anatomic tracing in the same brain reveal common failure modes of tractography.

Anatomic tracing is recognized as a critical source of knowledge on brain circuitry that can be used to assess the accuracy of diffusion MRI (dMRI) tractography. However, most prior studies that have performed such assessments have used dMRI and tracer data from different brains and/or have been limited in the scope of dMRI analysis methods allowed by the data. In this work, we perform a quantitative, voxel-wise comparison of dMRI tractography and anatomic tracing data in the same macaque brain. An ex vivo dMRI acquisition with high angular resolution and high maximum b-value allows us to compare a range of q-space sampling, orientation reconstruction, and tractography strategies. The availability of tracing in the same brain allows us to localize the sources of tractography errors and to identify axonal configurations that lead to such errors consistently, across dMRI acquisition and analysis strategies. We find that these common failure modes involve geometries such as branching or turning, which cannot be modeled well by crossing fibers. We also find that the default thresholds that are commonly used in tractography correspond to rather conservative, low-sensitivity operating points. While deterministic tractography tends to have higher sensitivity than probabilistic tractography in that very conservative threshold regime, the latter outperforms the former as the threshold is relaxed to avoid missing true anatomical connections. On the other hand, the q-space sampling scheme and maximum b-value have less of an impact on accuracy. Finally, using scans from a set of additional macaque brains, we show that there is enough inter-individual variability to warrant caution when dMRI and tracer data come from different animals, as is often the case in the tractography validation literature. Taken together, our results provide insights on the limitations of current tractography methods and on the critical role that anatomic tracing can play in identifying potential avenues for improvement.

Spatiotemporal imaging and pharmacokinetics of fluorescent compounds in zebrafish eleuthero-embryos after different routes of administration.

Zebrafish (Danio rerio) is increasingly used to assess the pharmacological activity and toxicity of compounds. The spatiotemporal distribution of seven fluorescent alkyne compounds was examined during 48 h after immersion (10 µM) or microinjection (2 mg/kg) in the pericardial cavity (PC), intraperitoneally (IP) and yolk sac (IY) of 3 dpf zebrafish eleuthero-embryos. By modelling the fluorescence of whole-body contours present in fluorescence images, the main pharmacokinetic (PK) parameter values of the compounds were determined. It was demonstrated that especially in case of short incubations (1-3 h) immersion can result in limited intrabody exposure to compounds. In this case, PC and IP microinjections represent excellent alternatives. Significantly, IY microinjections did not result in a suitable intrabody distribution of the compounds. Performing a QSPkR (quantitative structure-pharmacokinetic relationship) analysis, LogD was identified as the only molecular descriptor that explains the final uptake of the selected compounds. It was also shown that combined administration of compounds (immersion and microinjection) provides a more stable intrabody exposure, at least in case of a prolonged immersion and compounds with LogD value > 1. These results will help reduce the risk of false negative results and can offer an invaluable input for future translational research and safety assessment applications.