Superresolution Optical Fluctuation Imaging (SOFI) as initially demonstrated allows for a resolution enhancement in imaging by a factor of square-root of two. Here, we demonstrate how to increase the resolution of SOFI images by re-weighting the Optical Transfer Function (OTF). Furthermore, we demonstrate how cross-cumulants can be exploited to obtain a fair approximation of the underlying Point-Spread Function. We show a twofold increase of resolution (over the diffraction limit) of near-infrared quantum dot labeled tubulin-network of 3T3 fibroblasts
Maxwell's equations successfully describe the statistical properties of fluorescence from an ensemble of atoms or semiconductors in one or more dimensions. But quantization of the radiation field is required to explain the correlations of light generated by a single two-level quantum emitter, such as an atom, ion or single molecule. The observation of photon antibunching in resonance fluorescence from a single atom unequivocally demonstrated the non-classical nature of radiation. Here we report the experimental observation of photon antibunching from an artificial system--a single cadmium selenide quantum dot at room temperature. Apart from providing direct evidence for a solid-state non-classical light source, this result proves that a single quantum dot acts like an artificial atom, with a discrete anharmonic spectrum. In contrast, we find the photon-emission events from a cluster of several dots to be uncorrelated.
In this study, we describe optical detection of antibody-conjugated nanoparticles bound to surgically resected human pancreatic cancer tissue. Gold nanoparticles stabilized by heterobifunctional polyethylene glycol (PEG) were prepared using approximately 15 nm spherical gold cores and covalently coupled to F19 monoclonal antibodies. The heterobifunctional PEG ligands contain a dithiol group for stable anchoring onto the gold surface and a terminal carboxy group for coupling of antibodies to the outside of the PEG shell. The nanoparticle-antibody bioconjugates form highly stable dispersions and exhibit long-term resistance to agglomeration. This has been demonstrated by dynamic light scattering, size exclusion chromatography, and transmission electron microscopy. The nanoparticle bioconjugates were used to label tumor stroma in approximately 5 mum thick sections of resected human pancreatic adenocarcinoma. After rinsing away nonbound nanoparticles and fixation, the tissue samples were imaged by darkfield microscopy near the nanoparticle resonance scattering maximum (approximately 560 nm). The images display pronounced tissue features and suggest that this novel labeling method could provide for facile identification of cancer tissue. Tumor samples treated with gold nanoparticles conjugated to nonspecific control antibodies and noncancerous pancreatic tissue treated with mAb-F19-conjugated gold nanoparticles both exhibited correctly negative results and showed no tissue staining.
We spatially isolate and detect the luminescence from individual porous Si nanoparticles at room temperature. Our experiments show a variety of phenomena not previously observed in the emission from porous Si including a distribution of emission wavelengths, resolved vibronic structure, luminescence intermittency, and irreversible photobleaching. Our results indicate that the emission from porous Si nanoparticles originates from excitons in quantum confined Si, and is strongly mediated by the surface of the quantum dot. [S0031-9007(98)
We use a combination of single nanoparticle luminescence and scanning force microscopy to determine the quantum efficiency (QE) of single porous Si nanoparticles and to determine the ratio of luminescent nanoparticles deposited on a silica surface to the total nanoparticles. An estimate of the QE of bulk porous Si based on these data compares favorably to the QE measured experimentally. From this we conclude that the 1% QE of bulk porous Si measured experimentally results primarily from a statistical distribution of high QE quantum-confined Si chromophores.
Intentionally grown GaN inversion domain boundaries (IDBs) of lateral polarity heterostructures have been spectroscopically imaged at low temperature using high spatial resolution photoluminescence. It is shown that the IDBs are not only optically active, but are more than an order of magnitude brighter than the GaN bulk material. Our findings are in agreement with calculations predicting that IDBs should not adversely affect near-band-gap photoluminescence due to the absence of midgap electronic states. Typical linewidths are on the order of 10–20 meV, however, features less than 0.6 meV are observed. The boundary emission is found to be neither spectrally nor spatially uniform. Also, a strong polarization dependence of the IDB photoluminescence is measured and determined to be oriented parallel to the boundary between GaN of N- or Ga-face polarity.
Diffraction limits the biological structures that can be imaged by normal light microscopy. However, recently developed techniques are breaking the limits that diffraction poses and allowing imaging of biological samples at the molecular length scale. Fluorescence photoactivation localization microscopy (FPALM) and related methods can now image molecular distributions in fixed and living cells with measured resolution better than 30 nm. Based on localization of single photoactivatable molecules, FPALM uses repeated cycles of activation, localization, and photobleaching, combined with high sensitivity fluorescence imaging, to identify and localize large numbers of molecules within a sample. Procedures and pitfalls for construction and use of such a microscope are discussed in detail. Final images of cytosolic proteins, membrane proteins, and other structures, as well as examples of results during acquisition are shown. It is hoped that these details can be used to perform FPALM on a variety of biological samples, in order to significantly advance the understanding of biological systems.
The relevant photophysical properties of single fluorescent molecules and single SERS active surface-coated gold nanostars tagged with the Raman reporter molecule 4-mercaptopyridine are compared for imaging purposes. Mean count rate distributions are built from the single molecule/single probe level. The individually observed variance and count rates of both systems are compared as well as the behavior over multiple image acquisitions.
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