With current concerns of antibiotic-resistant bacteria and biodefense, it has become important to rapidly identify infectious bacteria. Traditional technologies involving isolation and amplification of the pathogenic bacteria are time-consuming. We report a rapid and simple method that combines in vivo biotinylation of engineered host-specific bacteriophage and conjugation of the phage to streptavidin-coated quantum dots. The method provides specific detection of as few as 10 bacterial cells per milliliter in experimental samples, with an Ϸ100-fold amplification of the signal over background in 1 h. We believe that the method can be applied to any bacteria susceptible to specific phages and would be particularly useful for detection of bacterial strains that are slow growing, e.g., Mycobacterium, or are highly infectious, e.g., Bacillus anthracis. The potential for simultaneous detection of different bacterial species in a single sample and applications in the study of phage biology are discussed.bacteriophage T7 ͉ BirA ͉ Escherichia coli ͉ water sample T he number and diversity of bacteriophages in the environment provide a promising natural pool of specific detection tools for pathogenic bacteria. Currently there are several phagebased methods for detection of pathogenic bacteria (1): a plaque assay for detection of Mycobacterium tuberculosis (2); fluorescence-labeled phage and immunomagnetic separation assay for detection of Escherichia coli O157:H7 (3, 4); phage-based electrochemical assays (5); a luciferase reporter mycobacteriophage and Listeria phage assays (6, 7); and detection of the phagemediated bacterial lysis and release of host enzymes (e.g., adenylate kinase) (8).Two limiting features when detecting pathogenic bacteria are sensitivity and rapidity. Common fluorophores (e.g., GFP and luciferase) used as reporters have two major disadvantages: low signal-to-noise ratio due to autofluorescence of clinical samples and of bacterial cells and low photostability, such as fast photobleaching. To overcome these disadvantages, we used new fluorescent semiconductor nanocrystals, quantum dots (QDs) (9). QDs are colloidal semiconductor (e.g., CdSe) crystals of a few nanometers in diameter. They exhibit broadband absorption spectra, and their emissions are of narrow bandwidth with size-dependent local maxima. The presence of an outer shell of a few atomic layers (e.g., ZnS) increases the quantum yield and further enhances the photostability, resulting in photostable fluorescent probes superior to conventional organic dyes. Recently, development in surface chemistry protocols allows conjugation of biomolecules onto these QDs to target specific biological molecules and probe nanoenvironments (10-12). The power to observe and trace single QDs or a group of bioconjugated QDs, enabling more precise quantitative biology, has been claimed to be one of the most exciting new capabilities offered to biologists today (13,14).Typically, the detection of small numbers of bacteria in environmental or clinical samples require...

Mid-infrared (MIR) microscopy provides rich chemical and structural information about biological samples, without staining. Conventionally, the long MIR wavelength severely limits the lateral resolution owing to optical diffraction; moreover, the strong MIR absorption of water ubiquitous in fresh biological samples results in high background and low contrast. To overcome these limitations, we propose a method that employs photoacoustic detection highly localized with a pulsed ultraviolet (UV) laser on the basis of the Grüneisen relaxation effect. For cultured cells, our method achieves water-background suppressed MIR imaging of lipids and proteins at UV resolution, at least an order of magnitude finer than the MIR diffraction limits. Label-free histology using this method is also demonstrated in thick brain slices. Our approach provides convenient high-resolution and high-contrast MIR imaging, which can benefit diagnosis of fresh biological samples.

Near-field scanning optical microscopy of phospholipid monolayers doped with fluorescent lipid analogs reveals previously undescribed features in various phases, including a concentration gradient at the liquid-expanded/liquid-condensed domain boundary and weblike structures in the solid-condensed phase. Presumably, the web structures are grain boundaries between crystalline solid lipid. These structures are strongly modulated by the addition of low concentrations of cholesterol and ganglioside GM1 in the monolayer.

Near-field scanning optical microscopy (NSOM) uses the near-field interaction of light from a sharp fiber-optic probe with a sample of interest to image surfaces at a resolution beyond the diffraction limit of conventional optics. We used NSOM to image fluorescently labeled plasma membranes of fixed human skin fibroblasts, either dried or in buffer. A patchy distribution of a fluorescent lipid analog suggestive of lipid domains was observed in the fixed, dried cells. The sizes of these patches were consistent with the sizes of domains implied by fluorescence photobleaching recovery measurements. Patches of fluorescent lipid analog were not spatially correlated with patches of transmembrane proteins, HLA class I molecules labeled with fluorescent antibody; the patchiness of the HLA class I molecules was on a smaller scale and was not localized to the same regions of membrane as the lipid analog. Sizes of HLA patches were deduced from a two-dimensional spatial autocorrelation analysis of NSOM images that resolved patches with radii of approximately 70 and approximately 600 nm on fixed, dried cells labeled with IgG and 300-600 nm on cells labeled with Fab and imaged in buffer. The large-size patches were also resolved by far-field microscopy. Both the spatial autocorrelation analysis and estimates from fluorescence intensity indicate that the small patches seen on fixed, dried cells contain approximately 25-125 HLA-I molecules each.

Research involving bacterial pathogens often requires enumeration of bacteria colonies.Here, we present a low-cost, high-throughput colony counting system consisting of colony counting software and a consumer-grade digital camera or document scanner. We demonstrate that this software, called ''NICE'' (NIST's Integrated Colony Enumerator), can count bacterial colonies as part of a high-throughput multiplexed opsonophagocytic killing assay used to characterize pneumococcal vaccine efficacy. The results obtained with NICE correlate well with the results obtained from manual counting, with a mean difference of less than 3%. NICE is also rapid; it can count colonies from multiple reaction wells within minutes and export the results to a spreadsheet for data processing. As this program is freely available from NIST, NICE should be helpful in bacteria colony enumeration required in many microbiological studies, and in standardizing colony counting methods. Published 2010 Wiley-Liss, Inc.