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The influence of surface modifications, including carboxyl-, amine-, and mercaptoundecanoicacid-functionalized chemistries, on the dynamic fluorescence properties of single quantum dots (QDs) are probed using a combined confocal fluorescence microscopy and spectroscopy system. Water-soluble QDs with carboxyl- or amine-functionalized surfaces prepared through a linker arm show significant fluorescence intermittency and appear statistically identical. In contrast, water-soluble QDs functionalized via mercaptoundecanoicacid, an electron-donating thiol group, demonstrate suppressed fluorescence intermittency suggesting the photostability of water-soluble QDs is strongly dependant upon the method of surface functionalization. In addition, the long-term photostability of the mercaptoundecanoicacid-functionalized QD demonstrates an excitation-time-dependent blue-shift in the fluorescence emission spectra. Our measurement and analysis techniques promise a wealth of insight in elucidating the photophysics of bio-conjugated QDs and assessing and designing bio-conjugated QDs for biomedical applications requiring long-term photostabililty.

Some of the most important trafficking processes in cells involve transport across the nuclear envelope. Whether it is the import of transcription factors or the export of RNA, the only known portal across the double lipid bilayer that forms the nuclear envelope are the macromolecular pores known as nuclear pore complexes (NPCs). Understanding how signals influence the conformation of the NPC is important for testing models of, and perhaps modifying, transport across the nuclear envelope. Here we summarize high-resolution atomic force microscopy studies of NPC structure following manipulation of nuclear envelope calcium stores of nuclei from Xenopus laevis oocytes. The results show that the release of calcium from these stores through the specific activation of inositol 1,4,5-trisphosphate receptors leads to changes in NPC structure observable from both sides of the nuclear envelope. The diameter of the NPC is also sensitive to these calcium stores and increases upon calcium release. Western blot analysis reveals the presence of ryanodine receptors in the nuclear envelope of X. laevis oocytes, although in low abundance. Activation of these calcium channels also leads to the displacement of the central mass and changes in NPC diameter. This change in structure may involve a displacement of the cytoplasmic and nuclear rings of the NPC towards each other, leading to the apparent emergence of the central mass from both sides of the NPC. The changes in conformation and diameter of the NPC may alter cargo access and binding to phenylalanine-glycine repeats lining the pore, thus altering transport.

Novel high-resolution microscopic techniques have been utilized to characterize the spatial distribution of orange emitting fluorophores, e.g., A2E, in lipofuscin granules isolated from human retinal pigment epithelium cells. Granules have been imaged using atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM). Near-field scanning optical microscopy images of lipofuscin show that the orange fluorophores, including A2E, are not major components of the granule. These results suggest that the orange fluorophores may not be the dominant photoactive species in lipofuscin.

An interferometric optical feedback mechanism is explored for tip-sample distance control in near-field scanning optical microscopy. An optical signal, based on the interference between the light exiting the tip aperture and that reflecting off the sample surface, is used to implement a feedback scheme to regulate tip-sample distance. The noncontact nature of this feedback mechanism may provide greater flexibility in imaging soft or fragile samples. To characterize the performance of the optical feedback mechanism, images are analyzed of a calibration standard, fluorescently doped lipid monolayers, latex spheres, and fixed cells. Images taken of these samples using optical feedback and the standard tapping-mode feedback are comparable in quality. These samples also demonstrate the ability of optical feedback to follow both large and small height changes and accurately reflect the sample topography. In a nonscanning mode, the interferometric signal can be used to noninvasively probe small dynamic height changes of sample with nanometric spatial resolution. Using a piezo ceramic bimorph to simulate sample movement, we show that nanometric height changes can be detected with millisecond time resolution. This may provide a unique way to probe protein conformational changes free of tip-sample interactions.

We report a near-field scanning optical microscopy (NSOM) probe that enables high-resolution imaging of living cells under physiological buffered conditions. The hybrid design combines a conventional fiber optic near-field probe with a standard atomic force microscopy cantilever. Imaging of fluorescent latex spheres suspended in an acetate matrix demonstrates the subdiffraction limited fluorescence and topography capabilities of the tips. The reduced spring constant of the hybrid tip is also shown to be amenable to measurements on living cells. NSOM fluorescence and topography measurements on living human arterial smooth muscle cells under buffered conditions are demonstrated.

We present a quantitative scheme for full-field polarization rotating fluorescence microscopy. A quarter-wave plate, in combination with a liquid crystal variable retarder, provides a tunable method to rotate polarization states of light prior to its being coupled into a fluorescence microscope. A calibration of the polarization properties of the incident light is performed in order to correct for elliptical polarization states. This calibration allows the response of the sample to linear polarization states of light to be recovered. Three known polarization states of light can be used to determine the average fluorescent dipole orientations in the presence of a spatially varying dc offset or background polarization-invariant fluorescence signal. To demonstrate the capabilities of this device, we measured a series of full-field fluorescence polarization images from fluorescent analogs incorporated in the lipid membrane of Burkitts lymphoma CA46 cells. The fluorescent lipid-like analogs used in this study are molecules that are labeled by either a DiI (1,1(')-Dioctadecyl 3,3,3('),3(')-Tetramethylindocarbocyanine) fluorophore in its head group or a Bodipy (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) molecule in its acyl chain. A spatially varying contrast in the normalized amplitude was observed on the cell surface, where the orientation of the DiI molecules is tangential to the cell membrane. The internally labeled cellular structures showed zero response to changes in linear polarization, and the net linear polarization amplitude for these regions was zero. This instrument provides a low cost calibrated method that may be coupled to existing fluorescence microscopes to perform investigations of cellular processes that involve a change in molecular orientations.

We present real-time, full-field, fluorescence polarization microscopy and its calibration and validation methods to monitor the absorption dipole orientation of fluorescent molecules. A quarter-wave plate, in combination with a liquid crystal variable retarder (LCVR), provides a tunable method to rotate a linear polarized light prior to being coupled into a fluorescence microscope. A series of full-field fluorescence polarization images are obtained of fluorescent molecules interleaved into the lipid bilyaer of liposomes. With this system, the dynamic dipole orientation of the fluorescent lipid analog tetramethylindocarbocyanine (DiI)-labeled lipids inserted in liposomes are probed and found to be aligned with the liposome in a tangential manner. The dipole orientation of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)- labeled lipids are expected to be aligned perpendicularly in the liposome membrane. Spectral separation of fluorescent lipid analogs into separate images provide an internal control and the ability to quantitatively correlate the membrane structure and fluctuations, within an optical section, in real-time. Application of this technique to the identification of characteristic features of cellular processes such as adhesion, endocytosis, and apoptosis are being investigated.

Rapid, specific, and sensitive detection of airborne bacteria, viruses, and toxins is critical for biodefense, yet the diverse nature of the threats poses a challenge for integrated surveillance, as each class of pathogens typically requires different detection strategies. Here, we present a laboratory-on-a-chip microfluidic device (LOC-DLA) that integrates two unique assays for the detection of airborne pathogens: direct linear analysis (DLA) with unsurpassed specificity for bacterial threats and Digital DNA for toxins and viruses. The LOC-DLA device also prepares samples for analysis, incorporating upstream functions for concentrating and fractionating DNA. Both DLA and Digital DNA assays are single molecule detection technologies, therefore the assay sensitivities depend on the throughput of individual molecules. The microfluidic device and its accompanying operation protocols have been heavily optimized to maximize throughput and minimize the loss of analyzable DNA. We present here the design and operation of the LOC-DLA device, demonstrate multiplex detection of rare bacterial targets in the presence of 100-fold excess complex bacterial mixture, and demonstrate detection of picogram quantities of botulinum toxoid.

A probe for near-field scanning optical microscopy is demonstrated based on a high index glass sphere attached to the end of a conventional atomic force microscopy tip. The sphere is machined into a pyramid geometry using a focused ion beam (FIB) instrument, coated with aluminum to confine the excitation light, and milled further with the FIB to open an aperture at the end of the tip. Near-field fluorescence images of 50 nm fluorescent latex spheres reveal subdiffraction limit spatial resolution, illustrating the utility of these probes for near-field scanning optical microscopy.

Direct Linear Analysis (DLA) technology obtains high content sequence information by optically mapping sequence specific fluorescent tags bound to elongated genomic DNA molecules in shear flows.[1] To facilitate sensitivity and throughput, we have implemented a high molecular weight DNA concentrating system by photopatterning a semi-permeable membrane inside the microfluidic device. This minimizes the fluid volume in which the DNA molecules reside prior to optical mapping leading to decreased read times. The membrane is selectively permeable to buffer ions but not high molecular weight DNA molecules allowing enhanced sample concentration at the membrane surface during electrokinetic transfer. In addition, the semi-permeable membrane allows electrophoretic sample transfer into and throughout the microfluidic device avoiding hydrodynamic induced shear forces that can degrade the integrity of large DNA molecules. The device employs novel microfluidic channel geometries to limit the electric field strength to appropriate levels near the membrane surface to minimize both sample and membrane degradation while maintaining a sufficiently high electric field for rapid sample transfer. Additionally, ion polarization across the membrane due to selective membrane permeability is addressed by active buffer replenishment through devoted channels behind the membrane. This architecture is amenable to integration into electrophoretic systems requiring rapid sample concentration, positioning, and transfer between microfluidic components. This research was supported by the Department of Homeland Security Science and Directorate Technology.[1] Chan et al., Genome Research, 2004, 14:1137.

Direct Linear Analysis (DLA) technology employs continuous microfluidic elongational flow to stretch and optically map individual DNA molecules. Sequence specific tagging of DNA molecules with bisPNAs enables genomic differentiation between species. We report on the operation of a microfluidic lab-on-a-chip platform with the integrated functionality of sample concentration, fractionation, and high throughput optical mapping. Integration of the components on a single chip enables high throughput analysis of sub-nanogram samples. Previously described DLA devices [1] had throughput rates that were directly proportional to the initial sample concentration. Integrated on chip concentration in conjunction with tunable sample delivery flow rates to the DLA component allows for dynamic optimization of throughput to 12,000 kbp/s, independent of the initial sample concentration. In addition, fractionation of the sample enhances information throughput by discarding shorter fragments with lower information content prior to the optical mapping step. This integrated microfluidic device has been demonstrated in conjunction with a macrofluidic upstream sample preparation chamber in an automated system. This research was supported by the Department of Homeland Security Science and Technology Directorate.[1] Phillips et al, Nucleic Acids Research, 2005, 33 (18), 5828.

A fluorescence-based integrated optics microfluidic device is presented, capable of detecting single DNA molecules in a high throughput and reproducible manner. The device integrates microfluidics for DNA stretching with two optical elements for single molecule detection (SMD): a plano-aspheric refractive lens for fluorescence excitation (illuminator) and a solid parabolic reflective mirror for fluorescence collection (collector). Although miniaturized in size, both optical components were produced and assembled onto the microfluidic device by readily manufacturable fabrication techniques. The optical resolution of the device is determined by the small and relatively low numerical aperture (NA) illuminator lens (0.10 effective NA, 4.0 mm diameter) that delivers excitation light to a diffraction limited 2.0 microm diameter spot at full width half maximum within the microfluidic channel. The collector (0.82 annular NA, 15 mm diameter) reflects the fluorescence over a large collection angle, representing 71% of a hemisphere, toward a single photon counting module in an infinity-corrected scheme. As a proof-of-principle experiment for this simple integrated device, individual intercalated lambda-phage DNA molecules (48.5 kb) were stretched in a mixed elongational-shear microflow, detected, and sized with a fluorescence signal to noise ratio of 9.9 +/-1.0. We have demonstrated that SMD does not require traditional high numerical aperture objective lenses and sub-micron positioning systems conventionally used in many applications. Rather, standard manufacturing processes can be combined in a novel way that promises greater accessibility and affordability for microfluidic-based single molecule applications.

We have fabricated a combined measurement system capable of confocal microscopy and fluorescence spectroscopy to simultaneously evaluate multiple optical characteristics of single fluorescent nanocrystals. The single particle detection sensitivity is demonstrated by simultaneously measuring the dynamic excitation-time-dependent fluorescence intermittency and the emission spectrum of single cadmium selenide/zinc sulfide (CdSe/ZnS) nanocrystals (quantum dots, QDs). Using this system, we are currently investigating the optical characteristics of single QDs, the surface of which are conjugated with different ligands, such as trioctylphosphine oxide (TOPO), mercaptoundecanoicacid (MDA), and amine modified DNA (AMDNA). In this paper, we present the progress of our measurements of the time-dependent optical characteristics (fluorescence intermittency, photostability, and spectral diffusion) of single MDA-QDs and AMDNA-MDA-QDs in air in an effort to understand the effects of surface-conjugated biomolecules on the optical characteristics at single QD sensitivities.

The fluorescence intermittency of single, bare, CdSe/ZnS quantum dots was probed using single molecule confocal microscopy and found to demonstrate power law kinetics. Various threshold values and line fitting parameters are employed in the data analysis and their effects on the extracted power law exponents, m off and m on , are presented. The threshold is found to be critical in determining m off while having no significant effect on m on . The mean plus 2σ threshold, calculated from the background noise in the measurement, results in a more negative m off slope in comparison to the mean plus 3σ or mean plus 4σ thresholds. This is likely due to the mean plus 2σ threshold lying within the background noise outliers which mimic short on events. In contrast, the mean plus 4σ threshold is above 99.99% of the background noise while adequately below the fluorescence signal. Additionally, it is found that fitting only the ten most probable data points rather than all the data points in the log-log probability density graphs results in no significant change in m off and mon.

Several high resolution imaging techniques are utilized to probe the structure of human ocular lipofuscin granules. Atomic force microscopy reveals typical granule sizes to be about one micrometre in diameter and hundreds of nanometres in height, in agreement with previous electron microscopy results. For issues concerning the role of lipofuscin in age-related macular degeneration, recent attention has focused on the orange-emitting fluorophore, A2E. Confocal microscopy measurements are presented which reveal the presence of a highly emissive component in the granules, consistent with the presence of A2E. It is shown, however, that the interpretation of these results is complicated by the lack of structural details about the particles. To address these issues, near-field scanning optical microscopy (NSOM) measurements are presented which measure both the lipofuscin fluorescence and topography, simultaneously. These measurements reveal distinct structure in the fluorescence image which do not necessarily correlate with the topography of the granules. Moreover, direct comparison between the NSOM fluorescence and topography measurements suggests that A2E is not the major component in lipofuscin. These measurements illustrate the unique capabilities of NSOM for probing into the microstructure of lipofuscin and uncovering new insights into its phototoxicity.

The emission spectra of single lipofuscin granules are examined using spectrally resolved confocal microscopy and near-field scanning optical microscopy (NSOM). The emission spectrum varies among the granules examined revealing that individual granules are characterized by different distributions of fluorophores. The range of spectra observed is consistent with in vivo spectra of human retinal pigment epithelium cells. NSOM measurements reveal that the shape of the spectrum does not vary with position within the emissive regions of single lipofuscin granules. These results suggest that the relative distribution of fluorophores within the emissive regions of an individual granule is homogeneous on the spatial scale ∼150 nm.

Advancements in near-field scanning optical microscopy (NSOM) tip design as well as an interferometric feedback mechanism are presented for the common goal of imaging living biological samples under physiological conditions. The ability of a cantilevered tip to track the subtle topography changes of a fragile lipid film in an aqueous environment is demonstrated. In order to further the imaging capabilities, the probes have been chemically etched to reduce the spring constants of the tips, thereby lowering the forces imparted on the sample. An optical feedback mechanism used as an alternative to the conventional force feedback is also described. Utilizing this optical feedback mechanism, images have been obtained of fixed cells underwater. Finally, progress towards modifying the NSOM tip for chemical sensor applications is discussed in the context of eventually measuring ion fluxes through single protein channels. Together these advancements demonstrate the potential of NSOM for studying live cells.