A Selective Luminescent Probe for the Direct Time-Gated Detection of Adenosine Triphosphate

A molecular probe for the luminescent detection of adenosine nucleotides is presented. The probe, Tb-DOTAm-Phen, readily distinguishes among the three adenosine nucleotides in buffered aqueous conditions at neutral pH, a requirement for the direct monitoring of enzymatic reactions converting adenosine triphosphate (ATP) to adenosine diphosphate or adenosine monophosphate. The probe is most efficient under millimolar concentrations of ATP which are relevant to intracellular conditions. Moreover, the long luminescence lifetime of the probe readily enables time-gating experiments

Fe- and Ln-DOTAm-F12 Are Effective Paramagnetic Fluorine Contrast Agents for MRI in Water and Blood

A series of fluorinated macrocyclic complexes, M-DOTAm-F12, where M is La^III, Eu^III, Gd^III, Tb^III, Dy^III, Ho^III, Er^III, Tm^III, Yb^III, and Fe^II, was synthesized, and their potential as fluorine magnetic resonance imaging (MRI) contrast agents was evaluated. The high water solubility of these complexes and the presence of a single fluorine NMR signal, two necessary parameters for in vivo MRI, are substantial advantages over currently used organic polyfluorocarbons and other reported paramagnetic ¹⁹F probes. Importantly, the sensitivity of the paramagnetic probes on a per fluorine basis is at least 1 order of magnitude higher than that of diamagnetic organic probes. This increased sensitivity is due to a substantialup to 100-folddecrease in the longitudinal relaxation time (<i>T</i>₁) of the fluorine nuclei. The shorter <i>T</i>₁ allows for a greater number of scans to be obtained in an equivalent time frame. The sensitivity of the fluorine probes is proportional to the <i>T</i>₂/<i>T</i>₁ ratio. In water, the optimal metal complexes for imaging applications are those containing Ho^III and Fe^II, and to a lesser extent Tm^III and Yb^III. Whereas <i>T</i>₁ of the lanthanide complexes are little affected by blood, the <i>T</i>₂ are notably shorter in blood than in water. The sensitivity of Ln-DOTAm-F12 complexes is lower in blood than in water, such that the most sensitive complex in water, Ho^III-DOTAm-F12, could not be detected in blood. Tm^III yielded the most sensitive lanthanide fluorine probe in blood. Notably, the relaxation times of the fluorine nuclei of Fe^II-DOTAm-F12 are similar in water and in blood. That complex has the highest <i>T</i>₂/<i>T</i>₁ ratio (0.57) and the lowest limit of detection (300 μM) in blood. The combination of high water solubility, single fluorine signal, and high <i>T</i>₂/<i>T</i>₁ of M-DOTAm-F12 facilitates the acquisition of three-dimensional magnetic resonance images

Fe- and Ln-DOTAm-F12 Are Effective Paramagnetic Fluorine Contrast Agents for MRI in Water and Blood

A series of fluorinated macrocyclic complexes, M-DOTAm-F12, where M is La^III, Eu^III, Gd^III, Tb^III, Dy^III, Ho^III, Er^III, Tm^III, Yb^III, and Fe^II, was synthesized, and their potential as fluorine magnetic resonance imaging (MRI) contrast agents was evaluated. The high water solubility of these complexes and the presence of a single fluorine NMR signal, two necessary parameters for in vivo MRI, are substantial advantages over currently used organic polyfluorocarbons and other reported paramagnetic ¹⁹F probes. Importantly, the sensitivity of the paramagnetic probes on a per fluorine basis is at least 1 order of magnitude higher than that of diamagnetic organic probes. This increased sensitivity is due to a substantialup to 100-folddecrease in the longitudinal relaxation time (<i>T</i>₁) of the fluorine nuclei. The shorter <i>T</i>₁ allows for a greater number of scans to be obtained in an equivalent time frame. The sensitivity of the fluorine probes is proportional to the <i>T</i>₂/<i>T</i>₁ ratio. In water, the optimal metal complexes for imaging applications are those containing Ho^III and Fe^II, and to a lesser extent Tm^III and Yb^III. Whereas <i>T</i>₁ of the lanthanide complexes are little affected by blood, the <i>T</i>₂ are notably shorter in blood than in water. The sensitivity of Ln-DOTAm-F12 complexes is lower in blood than in water, such that the most sensitive complex in water, Ho^III-DOTAm-F12, could not be detected in blood. Tm^III yielded the most sensitive lanthanide fluorine probe in blood. Notably, the relaxation times of the fluorine nuclei of Fe^II-DOTAm-F12 are similar in water and in blood. That complex has the highest <i>T</i>₂/<i>T</i>₁ ratio (0.57) and the lowest limit of detection (300 μM) in blood. The combination of high water solubility, single fluorine signal, and high <i>T</i>₂/<i>T</i>₁ of M-DOTAm-F12 facilitates the acquisition of three-dimensional magnetic resonance images

Effect of Lanthanide Complex Structure on Cell Viability and Association

A systematic study of the effect of hydrophobicity and charge on the cell viability and cell association of lanthanide metal complexes is presented. The terbium luminescent probes feature a macrocyclic polyaminocarboxylate ligand (DOTA) in which the hydrophobicity of the antenna and that of the carboxyamide pendant arms are independently varied. Three sensitizing antennas were investigated in terms of their function in vitro: 2-methoxy­isophthalamide (IAM­(OMe)), 2-hydroxyisophthalamide (IAM), and 6-methylphenanthridine (Phen). Of these complexes, Tb-DOTA-IAM exhibited the highest quantum yield, although the higher cell viability and more facile synthesis of the structurally related Tb-DOTA-IAM­(OMe) platform renders it more attractive. Further modification of this latter core structure with carboxyamide arms featuring hydrophobic benzyl, hexyl, and trifluoro groups as well as hydrophilic amino acid based moieties generated a family of complexes that exhibit high cell viability (ED₅₀ > 300 μM) regardless of the lipophilicity or the overall complex charge. Only the hexyl-substituted complex reduced cell viability to 60% in the presence of 100 μM complex. Additionally, cellular association was investigated by ICP-MS and fluorescence microscopy. Surprisingly, the hydrophobic moieties did not increase cell association in comparison to the hydrophilic amino acid derivatives. It is thus postulated that the hydrophilic nature of the 2-methoxyisophthalamide antenna (IAM­(OMe)) disfavors the cellular association of these complexes. As such, responsive luminescent probes based on this scaffold would be appropriate for the detection of extracellular species

Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets

Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell's RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts' cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. VIDEO ABSTRACT

Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets

Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell’s RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts’ cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution.National Human Genome Research Institute (U.S.) (P50 HG006193