Noninvasive in vivo imaging of pancreatic islet cell biology

Dimitri presented this paper on 1/22/09

Nature Medicine, 14, 2008, 574

Advanced imaging techniques have become a valuable tool in the study of complex biological processes at the cellular level in biomedical research. Here, we introduce a new technical platform for noninvasive in vivo fluorescence imaging of pancreatic islets using the anterior chamber of the eye as a natural body window. Islets transplanted into the mouse eye engrafted on the iris, became vascularized, retained cellular composition, responded to stimulation and reverted diabetes. Laser-scanning microscopy allowed repetitive in vivo imaging of islet vascularization, beta cell function and death at cellular resolution. Our results thus establish the basis for noninvasive in vivo investigations of complex cellular processes, like beta cell stimulus-response coupling, which can be performed longitudinally under both physiological and pathological conditions.

Nanoscale magnetic resonance imaging

PNAS, 2009

We have combined ultrasensitive magnetic resonance force microscopy (MRFM) with 3D image reconstruction to achieve magnetic resonance imaging (MRI) with resolution <10 nm. The image reconstruction converts measured magnetic force data into a 3D map of nuclear spin density, taking advantage of the unique characteristics of the “resonant slice” that is projected outward from a nanoscale magnetic tip. The basic principles are demonstrated by imaging the 1H spin density within individual tobacco mosaic virus particles sitting on a nanometer-thick layer of adsorbed hydrocarbons. This result, which represents a 100 million-fold improvement in volume resolution over conventional MRI, demonstrates the potential of MRFM as a tool for 3D, elementally selective imaging on the nanometer scale.

Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes

Elizabeth presented this paper on 1/29/09

Nature Medicine, 15, 2008, 104

A long-term goal of cancer diagnosis is to develop tumor imaging techniques that have sufficient specificity and sensitivity. To achieve this goal, minimizing the background signal originating from nontarget tissues is crucial. Here we achieve highly specific in vivo cancer visualization by using a newly designed targeted ‘activatable’ fluorescent imaging probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel acidic pH–activatable probes based on the boron-dipyrromethene fluorophore were synthesized and then conjugated to a cancer-targeting monoclonal antibody. As proof of concept, ex vivo and in vivo imaging of human epidermal growth factor receptor type 2–positive lung cancer cells in mice was performed. The probe was highly specific for tumors with minimal background signal. Furthermore, because the acidic pH in lysosomes is maintained by the energy-consuming proton pump, only viable cancer cells were successfully visualized. The design concept can be widely adapted to cancer-specific, cell surface–targeting molecules that result in cellular internalization.

Directed self-organization of single DNA molecules in a nanoslit via embedded nanopit arrays

PNAS, 106, 2009, 79

We show that arrays of nanopit structures etched in a nanoslit can control the positioning and conformation of single DNA molecules in nanofluidic devices. By adjusting the spacing, organization and placement of the nanopits it is possible to immobilize DNA at predetermined regions of a device without additional chemical modification and achieve a high degree of control over local DNA conformation. DNA can be extended between two nanopits and in closely spaced arrays will self-assemble into “connect-the-dots” conformations consisting of locally pinned segments joined by fluctuating linkers. These results have broad implications for nanotechnology fields that require methods for the nanoscale positioning and manipulation of DNA.

Accurate SHAPE-directed RNA structure determination

PNAS, 106, 2009, 97

Almost all RNAs can fold to form extensive base-paired secondary structures. Many of these structures then modulate numerous fundamental elements of gene expression. Deducing these structure–function relationships requires that it be possible to predict RNA secondary structures accurately. However, RNA secondary structure prediction for large RNAs, such that a single predicted structure for a single sequence reliably represents the correct structure, has remained an unsolved problem. Here, we demonstrate that quantitative, nucleotide-resolution information from a SHAPE experiment can be interpreted as a pseudo-free energy change term and used to determine RNA secondary structure with high accuracy. Free energy minimization, by using SHAPE pseudo-free energies, in conjunction with nearest neighbor parameters, predicts the secondary structure of deproteinized Escherichia coli 16S rRNA (>1,300 nt) and a set of smaller RNAs (75–155 nt) with accuracies of up to 96–100%, which are comparable to the best accuracies achievable by comparative sequence analysis.

Single-molecule DNA detection with an engineered MspA protein nanopore

PNAS, 105, 2008, 20647

Nanopores hold great promise as single-molecule analytical devices and biophysical model systems because the ionic current blockades they produce contain information about the identity, concentration, structure, and dynamics of target molecules. The porin MspA of Mycobacterium smegmatis has remarkable stability against environmental stresses and can be rationally modified based on its crystal structure. Further, MspA has a short and narrow channel constriction that is promising for DNA sequencing because it may enable improved characterization of short segments of a ssDNA molecule that is threaded through the pore. By eliminating the negative charge in the channel constriction, we designed and constructed an MspA mutant capable of electronically detecting and characterizing single molecules of ssDNA as they are electrophoretically driven through the pore. A second mutant with additional exchanges of negatively-charged residues for positively-charged residues in the vestibule region exhibited a factor of ≈20 higher interaction rates, required only half as much voltage to observe interaction, and allowed ssDNA to reside in the vestibule ≈100 times longer than the first mutant. Our results introduce MspA as a nanopore for nucleic acid analysis and highlight its potential as an engineerable platform for single-molecule detection and characterization applications.

Carbon nanotubes as photoacoustic molecular imaging agents in living mice

Nature Nanotechnology, 3, 2008, 557

Photoacoustic imaging of living subjects offers higher spatial resolution and allows deeper tissues to be imaged compared with most optical imaging techniques1, 2, 3, 4, 5, 6, 7. As many diseases do not exhibit a natural photoacoustic contrast, especially in their early stages, it is necessary to administer a photoacoustic contrast agent. A number of contrast agents for photoacoustic imaging have been suggested previously8, 9, 10, 11, 12, 13, 14, 15, but most were not shown to target a diseased site in living subjects. Here we show that single-walled carbon nanotubes conjugated with cyclic Arg-Gly-Asp (RGD) peptides can be used as a contrast agent for photoacoustic imaging of tumours. Intravenous administration of these targeted nanotubes to mice bearing tumours showed eight times greater photoacoustic signal in the tumour than mice injected with non-targeted nanotubes. These results were verified ex vivo using Raman microscopy. Photoacoustic imaging of targeted single-walled carbon nanotubes may contribute to non-invasive cancer imaging and monitoring of nanotherapeutics in living subjects16.

Coupling ion channels to receptors for biomolecule sensing

Nature Nanotechnology, 3, 2008, 620

Nanoscale electrical biosensors are promising tools for diagnostics and high-throughput screening systems. The electrical signal allows label-free assays with a high signal-to-noise ratio and fast real-time measurements. The challenge in developing such biosensors lies in functionally connecting a molecule detector to an electrical switch. Advances in this field have relied on synthetic ion-conducting pores1 and modified ion channels2, 3, 4 that are not yet suitable for biomolecule screening. Here we report the design and characterization of a novel bioelectric-sensing platform engineered by coupling an ion channel, which serves as the electrical probe, to G-protein-coupled receptors (GPCRs), a family of receptors that detect molecules outside the cell. These ion-channel-coupled receptors may potentially detect a wide range of ligands recognized by natural or altered GPCRs5, 6, which are known to be major pharmaceutical targets7. This could form a unique platform for label-free drug screening8.

Carbon nanotubes as templates for polymerized lipid assemblies

Nature Nanotechnology, 3, 2008, 743

Amphiphilic molecules—molecules that have both hydrophobic and hydrophilic properties—can self-assemble in water to form diverse structures such as micelles, vesicles and tubes1, 2, 3, and these nanostructures can be used for delivering drugs4, 5, stabilizing membrane proteins6 or as nanoreactors7. We have previously shown that lipids can self-organize on the surface of single-walled carbon nanotubes into regular ring-shaped assemblies8. Here we show that these lipid assemblies can be polymerized and isolated from the nanotube template by application of an electric field. We also demonstrate that these assemblies are monodispersed, water-soluble, and can dissolve various hydrophobic rylene dyes, fullerenes and membrane proteins. The stability of these constructs and their diverse applications will be useful in the fields of cosmetics, medicine and material sciences.

Direct multiplexed measurement of gene expression with color-coded probe pairs

Nature Biotechnology, 26, 2008, 317

We describe a technology, the NanoString nCounter gene expression system, which captures and counts individual mRNA transcripts. Advantages over existing platforms include direct measurement of mRNA expression levels without enzymatic reactions or bias, sensitivity coupled with high multiplex capability, and digital readout. Experiments performed on 509 human genes yielded a replicate correlation coefficient of 0.999, a detection limit between 0.1 fM and 0.5 fM, and a linear dynamic range of over 500-fold. Comparison of the NanoString nCounter gene expression system with microarrays and TaqMan PCR demonstrated that the nCounter system is more sensitive than microarrays and similar in sensitivity to real-time PCR. Finally, a comparison of transcript levels for 21 genes across seven samples measured by the nCounter system and SYBR Green real-time PCR demonstrated similar patterns of gene expression at all transcript levels.