Robert C. Dunn

Probing single molecule dynamics

The room temperature dynamics of single sulforhodamine 101 molecules dispersed on a glass surface are investigated on two different time scales with near-field optics. On the 10-2- to 102-second time scale, intensity fluctuations in the emission from single molecules are examined with polarization measurements, providing insight into their spectroscopic properties. On the nanosecond time scale, the fluorescence lifetimes of single molecules are measured, and their excited-state energy transfer to the aluminum coating of the near-field probe is characterized. A movie of the time-resolved emission demonstrates the feasibility of fluorescence lifetime imaging with single molecule sensitivity, picosecond temporal resolution, and a spatial resolving power beyond the diffraction limit.

Scanning Near-Field Fluorescence Resonance Energy Transfer Microscopy

A new microscopic technique is demonstrated that combines attributes from both near-field scanning optical microscopy (NSOM) and fluorescence resonance energy transfer (FRET). The method relies on attaching the acceptor dye of a FRET pair to the end of a near-field fiber optic probe. Light exiting the NSOM probe, which is nonresonant with the acceptor dye, excites the donor dye introduced into a sample. As the tip approaches the sample containing the donor dye, energy transfer from the excited donor to the tip-bound acceptor produces a red-shifted fluorescence. By monitoring this red-shifted acceptor emission, a dramatic reduction in the sample volume probed by the uncoated NSOM tip is observed. This technique is demonstrated by imaging the fluorescence from a multilayer film created using the Langmuir-Blodgett (LB) technique. The film consists of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers containing the donor dye, fluorescein, separated by a spacer group of three arachidic acid layers. A DPPC monolayer containing the acceptor dye, rhodamine, was also transferred onto an NSOM tip using the LB technique. Using this modified probe, fluorescence images of the multilayer film reveal distinct differences between images collected monitoring either the donor or acceptor emission. The latter results from energy transfer from the sample to the NSOM probe. This method is shown to provide enhanced depth sensitivity in fluorescence measurements, which may be particularly informative in studies on thick specimens such as cells. The technique also provides a mechanism for obtaining high spatial resolution without the need for a metal coating around the NSOM probe and should work equally well with nonwaveguide probes such as atomic force microscopy tips. This may lead to dramatically improved spatial resolution in fluorescence imaging.

High resolution fluorescence imaging with cantilevered near‐field fiber optic probes

High resolution near‐field fluorescence and topology images of intact neurons are reported using a cantilevered near‐field fiber optic probe. A bend is introduced into a normal geometry near‐field tip, allowing the probe to be used in a tapping‐mode arrangement, similar to tapping‐mode atomic force microscopy. Features with a full width at half‐maximum of 140 nm are observed in the near‐field fluorescence image, demonstrating the subdiffraction limit spatial resolution possible with the cantilevered near‐field probe design. Characteristics of the cantilevered tips include resonances between 30 and 60 kHz, Q factors greater than 100, and measured spring constants of 300 to 400 N/m.

Calcium regulation of nuclear pore permeability

The nuclear envelope is an integral part of the structural framework of the nucleus, and is involved in organizing intranuclear events. It serves as a selective barrier, actively transporting proteins required for normal nuclear function and exporting RNA. The movement of molecules across the nuclear envelope is critical for cellular homeostasis, and it allows cells to respond to external events. The only known pathway for direct communication between the cytoplasm and the nucleoplasm of a cell is through the nuclear pore complex. In the past decade, rapid advances have been made in elucidating the structure and function of the nuclear pore complex. Yet, researchers are just beginning to identify some of the regulatory mechanisms controlling transport through the pore complex. The nucleus is surrounded by a Ca2+ storage compartment, which sequesters and releases Ca2+ in response to intracellular second messengers, Recent evidence suggests that the nuclear Ca2+ store may indirectly regulate passive diffusion through the nuclear pore complex. The evidence for Ca2+ regulation of the nuclear pore complex will be discussed, along with the introduction of the simplest, testable model to describe the observations.

Combining AFM and FRET for high resolution fluorescence microscopy

Here we demonstrate a new microscopic method that combines atomic force microscopy (AFM) with fluorescence resonance energy transfer (FRET). This method takes advantage of the strong distance dependence in Förster energy transfer between dyes with the appropriate donor/acceptor properties to couple an optical dimension with conventional AFM. This is achieved by attaching an acceptor dye to the end of an AFM tip and exciting a sample bound donor dye through far‐field illumination. Energy transfer from the excited donor to the tip immobilized acceptor dye leads to emission in the red whenever there is sufficient overlap between the two dyes. Because of the highly exponential distance dependence in this process, only those dyes located at the apex of the AFM tip, nearest the sample, interact strongly. This limited and highly specific interaction provides a mechanism for obtaining fluorescence contrast with high spatial resolution. Initial results in which 400 nm resolution is obtained through this AFM/FRET imaging technique are reported. Future modifications in the probe design are discussed to further improve both the fluorescence resolution and imaging capabilities of this new technique.

Single molecule detection and underwater fluorescence imaging with cantilevered near-field fiber optic probes

Tapping-mode near-field scanning optical microscopy (NSOM) employing a cantilevered fiber optic probe is utilized to image the fluorescence from single molecules and samples in aqueous environments. The single molecule fluorescence images demonstrate both the subdiffraction limit spatial resolution and low detection limit capabilities of the cantilevered probe design. Images taken as a function of tip oscillation drive amplitude reveal a degradation in the resolution as the amplitude is increased. With all cantilevered probes studied, however, a minimum plateau region in the resolution is reached as the drive amplitude is decreased, indicating that the tapping mode of operation does not reduce the optical resolution. Images of fluorescently doped lipid films illustrate the ability of the probe to track small height changes (<1.5 nm) in ambient and aqueous environments, while maintaining high resolution in the fluorescence image. When the tip is immersed in water (1.3 mm), the cantilevered NSOM tip resonan...

Regulation of Nuclear Pore Complex Conformation by IP3 Receptor Activation

In recent years, both the molecular architecture and functional dynamics of nuclear pore complexes (NPCs) have been revealed with increasing detail. These large, supramolecular assemblages of proteins form channels that span the nuclear envelope of cells, acting as crucial regulators of nuclear import and export. From the cytoplasmic face of the nuclear envelope, nuclear pore complexes exhibit an eightfold symmetric ring structure encompassing a central lumen. The lumen often appears occupied by an additional structure alternatively referred to as the central granule, nuclear transport complex, or nuclear plug. Previous studies have suggested that the central granule may play a role in mediating calcium-dependent regulation of diffusion across the nuclear envelope for intermediate sized molecules (10-40 kDa). Using atomic force microscopy to measure the surface topography of chemically fixed Xenopus laevis oocyte nuclear envelopes, we present measurements of the relative position of the central granule within the NPC lumen under a variety of conditions known to modify nuclear Ca(2+) stores. These measurements reveal a large, approximately 9-nm displacement of the central granule toward the cytoplasmic face of the nuclear envelope under calcium depleting conditions. Additionally, activation of nuclear inositol triphosphate (IP(3)) receptors by the specific agonist, adenophostin A, results in a concentration-dependent displacement of central granule position with an EC(50) of ~1.2 nM. The displacement of the central granule within the NPC is observed on both the cytoplasmic and nucleoplasmic faces of the nuclear envelope. The displacement is blocked upon treatment with xestospongin C, a specific inhibitor of IP(3) receptor activation. These results extend previous models of NPC conformational dynamics linking central granule position to depletion of IP(3) sensitive nuclear envelope calcium stores.

Probing single molecule orientations in model lipid membranes with near-field scanning optical microscopy

Single molecule near-field fluorescence measurements are utilized to characterize the molecular level structure in Langmuir–Blodgett monolayers of L-α-dipalmitoylphosphatidylcholine (DPPC). Monolayers incorporating 3×10−4 mol % of the fluorescent lipid analog N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine, triethylammonium salt (TRITC–DHPE) are transferred onto a freshly cleaved mica surface at low (π=8 mN/m) and high (π=30 mN/m) surface pressures. The near-field fluorescence images exhibit shapes in the single molecule images that are indicative of the lipid analog probe orientation within the films. Modeling the fluorescence patterns yields the single molecule tilt angle distribution in the monolayers which indicates that the majority of the molecules are aligned with their absorption dipole moment pointed approximately normal to the membrane plane. Histograms of the data indicate that the average orientation of the absorption dipole moment is 2.2° (σ=4.8°) ...

Label-free detection of ovarian cancer biomarkers using whispering gallery mode imaging.

Small optical microresonators that support whispering gallery mode (WGM) resonances are emerging as powerful new platforms for biosensing. These resonators respond to changes in refractive index and potentially offer many advantages for label-free sensing. Recently we reported an approach for detecting WGM resonances based on fluorescence imaging and demonstrated its utility by quantifying the ovarian cancer marker CA-125 in buffer. Here we extend those measurements by reporting a simplified approach for launching WGM resonances using excitation light coupled into a Dove prism. The enhanced phase matching enables significant improvements in signal-to-noise, revealing the mode structure present in each resonator. As with all label-free biosensing techniques, non-specific interactions can be limiting. Here we show that standard blocking protocols reduce non-specific interactions sufficiently to enable CA-125 quantification in serum samples. Finally, fluorescence imaging of WGM resonances offers the potential for large scale multiplexed detection which is demonstrated here by simultaneously exciting and imaging over 120 microsphere resonators. For multiplexed applications, analyte identity can be encoded in the resonator size and/or location. By encoding analyte identity into microresonator size, we simultaneously quantify the putative ovarian cancer markers osteopontin (38 μm diameter sphere), CA-125 (53 μm diameter sphere), and prolactin (63 μm diameter sphere) in a single PBS assay. Together, these results show that fluorescence imaging of WGM resonances offers a promising new approach for the highly multiplexed detection of biomarkers in complex biological fluids.

Orientation of fluorescent lipid analogue BODIPY-PC to probe lipid membrane properties: insights from molecular dynamics simulations.

Single-molecule fluorescence measurements have been used to characterize membrane properties, and recently showed a linear evolution of the fluorescent lipid analogue BODIPY-PC toward small tilt angles in Langmuir-Blodgett monolayers as the lateral surface pressure is increased. In this work, we have performed comparative molecular dynamics (MD) simulations of BODIPY-PC in DPPC (dipalmitoylphosphatidylcholine) monolayers and bilayers at three surface pressures (3, 10, and 40 mN/m) to explore (1) the microscopic correspondence between monolayer and bilayer structures, (2) the fluorophores position within the membrane, and (3) the microscopic driving forces governing the fluorophores tilting. The MD simulations reveal very close agreement between the monolayer and bilayer systems in terms of the fluorophores orientation and lipid chain order, suggesting that monolayer experiments can be used to approximate bilayer systems. The simulations capture the trend of reduced tilt angle of the fluorophore with increasing surface pressure, as seen in the experimental results, and provide detailed insights into fluorophore location and orientation, not obtainable in the experiments. The simulations also reveal that the enthalpic contribution is dominant at 40 mN/m, resulting in smaller tilt angles of the fluorophore, and the entropy contribution is dominant at lower pressures, resulting in larger tilt angles.