Nanoscale imaging magnetometry with diamond spins under ambient conditions.

Magnetic resonance imaging and optical microscopy are key technologies in the life sciences. For microbiological studies, especially of the inner workings of single cells, optical microscopy is normally used because it easily achieves resolution close to the optical wavelength. But in conventional microscopy, diffraction limits the resolution to about half the wavelength. Recently, it was shown that this limit can be partly overcome by nonlinear imaging techniques, but there is still a barrier to reaching the molecular scale. In contrast, in magnetic resonance imaging the spatial resolution is not determined by diffraction; rather, it is limited by magnetic field sensitivity, and so can in principle go well below the optical wavelength. The sensitivity of magnetic resonance imaging has recently been improved enough to image single cells, and magnetic resonance force microscopy has succeeded in detecting single electrons and small nuclear spin ensembles. However, this technique currently requires cryogenic temperatures, which limit most potential biological applications. Alternatively, single-electron spin states can be detected optically, even at room temperature in some systems. Here we show how magneto-optical spin detection can be used to determine the location of a spin associated with a single nitrogen-vacancy centre in diamond with nanometre resolution under ambient conditions. By placing these nitrogen-vacancy spins in functionalized diamond nanocrystals, biologically specific magnetofluorescent spin markers can be produced. Significantly, we show that this nanometre-scale resolution can be achieved without any probes located closer than typical cell dimensions. Furthermore, we demonstrate the use of a single diamond spin as a scanning probe magnetometer to map nanoscale magnetic field variations. The potential impact of single-spin imaging at room temperature is far-reaching. It could lead to the capability to probe biologically relevant spins in living cells.

Tailoring spatiotemporal light confinement in single plasmonic nanoantennas.

Plasmonic nanoantennas are efficient devices to concentrate light in spatial regions much smaller than the wavelength. Only recently, their ability to manipulate photons also on a femtosecond time scale has been harnessed. Nevertheless, designing the dynamical properties of optical antennas has been difficult since the relevant microscopic processes governing their ultrafast response have remained unclear. Here, we exploit frequency-resolved optical gating to directly investigate plasmon response times of different antenna geometries resonant in the near-infrared. Third-harmonic imaging is used in parallel to spatially monitor the plasmonic mode patterns. We find that the few-femtosecond dynamics of these nanodevices is dominated by radiative damping. A high efficiency for nonlinear frequency conversion is directly linked to long plasmon damping times. This single parameter explains the counterintuitive result that rod-type nanoantennas with minimum volume generate by far the strongest third-harmonic emission as compared to the more bulky geometries of bow-tie-, elliptical-, or disk-shaped specimens.

Single defect centers in diamond nanocrystals as quantum probes for plasmonic nanostructures.

We present two applications of a single nitrogen vacancy center in a nanodiamond as quantum probe for plasmonic nanostructures. Coupling to the nanostructures is achieved in a highly controlled manner by picking up a pre-characterized nanocrystal with an atomic force microscope and placing it at the desired position. Local launching of single excitations into a nanowire with a spatial control of few nanometers is demonstrated. Further, a two dimensional map of the electromagnetic environment of a plasmonic bowtie antenna was derived, resembling an ultimate limit of fluorescence lifetime nanoscopy.

Ultrabroadband background-free coherent anti-Stokes Raman scattering microscopy based on a compact Er:fiber laser system.

We demonstrate a scheme for efficient coherent anti-Stokes Raman scattering (CARS) microscopy free of nonresonant background. Our method is based on a compact Er:fiber laser source. Impulsive excitation of molecular resonances is achieved by an 11 fs pulse at 1210 nm. Broadband excitation gives access to molecular resonances from 0 cm(-1) up to 4000 cm(-1). Time-delayed narrowband probing at 775 nm enables sensitive and high-speed spectral detection of the CARS signal free of nonresonant background with a resolution of 10 cm(-1).

Compact coherent anti-Stokes Raman scattering microscope based on a picosecond two-color Er:fiber laser system.

We present a compact coherent anti-Stokes Raman scattering microscope based on a widely tunable picosecond Er:fiber laser. Intense and bandwidth-limited 1 ps pump pulses at a center wavelength of 775 nm are generated via frequency mixing within the broadband fundamental at 1.55 microm. Narrowband Stokes pulses are obtained by frequency shifting of solitons in a highly nonlinear bulk fiber and subsequent second-harmonic generation. The tuning range from 850 nm to 1100 nm gives access to vibrational resonances between 1150 cm(-1) and 3800 cm(-1). A first imaging application in the spectral region of CH stretch vibrations is demonstrated.

Colloidal ZnO quantum dots in ultraviolet pillar microcavities.

Three dimensional light confinement and distinct pillar microcavity modes in the ultraviolet have been observed in pillar resonators with embedded colloidal ZnO quantum dots fabricated by focused ion beam milling. Results from a waveguide model for the mode patterns and their spectral positions are in excellent agreement with the experimental data.

Cloning, functional analysis and post-transcriptional regulation of a type II DNA topoisomerase from Leishmania infantum. A new potential target for anti-parasite drugs.

We identified a type II topoisomerase enzyme from Leishmania infantum, a parasite protozoon causing disease in humans. This protein, named Li topo II, which displays a variable C-terminal end, is located in the kinetoplast. The cloned gene encoding Li-TOP2 compensates for the slow growth of topo II-deficient mutants of Saccharomyces cerevisiae, resulting in a catalytically active DNA topoisomerase in yeast. Analysis of the specific mRNA levels of the Li-TOP2 gene showed variations throughout the parasite cell cycle in synchronized cells as well as between the distinct forms of the parasite. Thus, the enzyme had higher levels of mRNA expression in the highly infective intracellular form of the parasite, the amastigote, than in the extracellular promastigote form, suggesting a relation with the distinct developmental and infectious phases of the protozoon. In addition, western blot analysis showed differences in protein expression between the proliferative and non-proliferative forms of L.infantum promastigotes, which displayed similar levels of mRNA. This indicated possible post-transcriptional regulation mechanisms. The data suggest that Li topo II has a part in DNA decatenation and probably at the initial stages of proliferation in the intracellular form of L.infantum, a parasite that has to proliferate into the host macrophage to survive its hostile environment in its first moments of intracellular infection.

Protection in dogs against visceral leishmaniasis caused by Leishmania infantum is achieved by immunization with a heterologous prime-boost regime using DNA and vaccinia recombinant vectors expressing LACK.

A heterologous prime-boost vaccination regime with DNA and recombinant vaccinia virus (rVV) vectors expressing relevant antigens has been shown to enhance specific cellular immune responses and to elicit protection against a variety of pathogens in animal models. In this paper, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a plasmid carrying the gene for the LACK antigen from Leishmania infantum (DNA-LACK) followed by a booster with a rVV containing the same gene (rVV-LACK). Thereafter, animals were challenged with L. infantum to induce visceral leishmaniasis (VL). In the vaccinated dogs as compared with the controls, the outcome of the infection after challenge with a high inoculum (10(8)) of L. infantum stationary promastigotes was assessed by tissue parasite load, specific anti-Leishmania antibody production, cytokine level and development of clinical signs of leishmaniasis. We observed a 60% protection against infection in dogs immunized by DNA-LACK prime/rVV/-LACK boost while two doses of DNA-LACK did not elicit protection against the disease. The interleukin 4 (IL-4), interferon gamma (IFNgamma) and IL-12 (p40 subunit) cytokine mRNA expression profiles in PBMC as well as lymphocyte proliferative response and the IgG2/IgG1 ratios specific for LACK suggest that in vaccinated animals there is triggering of cellular immune responses. This type of DNA/rVV prime/boost immunization approach may have utility against visceral leishmaniasis in dogs.