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.

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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 ͑FIB͒ 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.

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.

We report a near-field scanning optical microscopy ͑NSOM͒ 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. Near-field scanning optical microscopy ͑NSOM͒ is a scanning probe technique that utilizes specially fabricated probes to deliver light down to the nanometric dimension.1,2 NSOM provides opportunities to simultaneously measure both optical and topographic features of samples with subdiffraction limited spatial resolution. These capabilities have been utilized to probe thin films, solid-state devices, and due to the unique electromagnetic fields emerging from the nearfield aperture, single molecule orientations.2 The potential impact of NSOM is arguably the greatest in the biological sciences, where there is a well-developed history of using fluorescence probes to tag specific proteins or structures. This field seems particularly well suited to take advantage of the single molecule fluorescence sensitivity, high spatial resolution, and simultaneous force information that NSOM offers. However, current applications of NSOM to biological samples are largely limited to isolated protein samples, model membranes, or chemically fixed biological cells. 2-5The extension to viable, unfixed biological tissues has previously proven problematic.The difficulty in conducting NSOM measurements on viable biological tissues arises from the forces generated in maintaining the NSOM tip close to the specimen. Highresolution NSOM measurements require that the tip be held within nanometers of the sample while scanning. This necessitates the implementation of a feedback system for sensing the sample surface and maintaining the tip-sample gap. [6][7][8] Traditionally, a force feedback approach is implemented in which the tip is either dithered laterally or vertically to the sample surface, depending on probe geometry. The dampening in the amplitude of the oscillating NSOM tip as the tip interacts with the sample surface is monitored and used to hold the tip-sample gap constant during scanning. While straightforward and highly successful for most applications, the large forces generated using conventional fiber optic NSOM probes often damage fragile biological samples such as living cells.A number of approaches have been reported in attempts to circumvent the large forces generated under normal NSOM feedback operation and thus open applications in the biological sciences. These have largely involved either changing the feedback mechanism utilized to hold the tip close to the sample or modifying the probes themselves to lower the spring constant and thus the forces generated in force feedback. 2,5,9 ...