Super-resolution optical microscopy is a rapidly evolving area of fluorescence microscopy with a tremendous potential for impacting many fields of science. Several super-resolution methods have been developed over the last decade, all capable of overcoming the fundamental diffraction limit of light. We present here an approach for obtaining subdiffraction limit optical resolution in all three dimensions. This method relies on higher-order statistical analysis of temporal fluctuations (caused by fluorescence blinking/ intermittency) recorded in a sequence of images (movie). We demonstrate a 5-fold improvement in spatial resolution by using a conventional wide-field microscope. This resolution enhancement is achieved in iterative discrete steps, which in turn allows the evaluation of images at different resolution levels. Even at the lowest level of resolution enhancement, our method features significant background reduction and thus contrast enhancement and is demonstrated on quantum dot-labeled microtubules of fibroblast cells.cumulants ͉ fluorescence ͉ quantum dots ͉ superresolution microscopy ͉ intermittency

This study evaluates the influence of particle size, PEGylation, and surface coating on the quantitative biodistribution of near-infrared-emitting quantum dots (QDs) in mice. Polymer- or peptide-coated 64Cu-labeled QDs 2 or 12 nm in diameter, with or without polyethylene glycol (PEG) of molecular weight 2000, are studied by serial micropositron emission tomography imaging and region-of-interest analysis, as well as transmission electron microscopy and inductively coupled plasma mass spectrometry. PEGylation and peptide coating slow QD uptake into the organs of the reticuloendothelial system (RES), liver and spleen, by a factor of 6–9 and 2–3, respectively. Small particles are in part renally excreted. Peptide-coated particles are cleared from liver faster than physical decay alone would suggest. Renal excretion of small QDs and slowing of RES clearance by PEGylation or peptide surface coating are encouraging steps toward the use of modified QDs for imaging living subjects.

After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed, the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo.

We have developed a new functionalization approach for semiconductor nanocrystals based on a single-step exchange of surface ligands with custom-designed peptides. This peptide-coating technique yield small, monodisperse and very stable water-soluble NCs that remain bright and photostable. We have used this approach on several types of core and core-shell NCs in the visible and near-infrared spectrum range and used fluorescence correlation spectroscopy for rapid assessment of the colloidal and photophysical properties of the resulting particles. This peptide coating strategy has several advantages: it yields probes that are immediately biocompatible; it is amenable to improvements of the different properties (solubilization, functionalization, etc) via rational design, parallel synthesis, or molecular evolution; it permits the combination of several functions on individual NCs. These functionalized NCs have been used for diverse biomedical applications. Two are discussed here: single-particle tracking of membrane receptor in live cells and combined fluorescence and PET imaging of targeted delivery in live animals.

This study evaluates the quantitative biodistribution of commercially available CdSe quantum dots (QD) in mice. Methods: 64 Cu-Labeled 800-or 525-nm emission wavelength QD (21-or 12-nm diameter), with or without 2,000 MW (molecular weight) polyethylene glycol (PEG), were injected intravenously into mice (5.55 MBq/25 pmol QD) and studied using well counting or by serial microPET and region-of-interest analysis. Results: Both methods show rapid uptake by the liver (27.4-38.9 %ID/ g) (%ID/g is percentage injected dose per gram tissue) and spleen (8.0-12.4 %ID/g). Size has no influence on biodistribution within the range tested here. Pegylated QD have slightly slower uptake into liver and spleen (6 vs. 2 min) and show additional low-level bone uptake (6.5-6.9 %ID/g). No evidence of clearance from these organs was observed. Conclusion: Rapid reticuloendothelial system clearance of QD will require modification of QD for optimal utility in imaging living subjects. Formal quantitative biodistribution/imaging studies will be helpful in studying many types of nanoparticles, including quantum dots. Quant um dots (QD) are fluorescent semiconductor nanocrystals with high quantum yield, resistance to photobleaching, narrow emission peak, tunable emission wavelength, and constant excitation profile regardless of emission wavelength, making them interesting for in vivo smallanimal imaging (1,2). Naturally hydrophobic, QD are made water-soluble by surface conjugation (1). Targeting molecules, such as antibodies (3), aptamers (4), peptides (5,6), folate (7), or high-molecular-weight dextran (8) can then be added. Polyethylene glycol (PEG) is commonly attached to the surface of QD for in vivo applications. Pegylation increases in vivo circulation times of nanoparticles and liposomes, likely by sterically hindering the absorption of opsonizing proteins and, thus, delaying recognition and clearance by the reticuloendothelial system (RES) (9,10).QD have been used for in vivo fluorescence imaging for fluorescence sentinel lymph node mapping (11-13), or diffusion analysis of the brain extracellular space (14), in which particle size confines QD to a specific compartment or clearance route. Other studies have used surface modifications to target QD. Akerman et al. used ex vivo fluorescence microscopy to show lung and tumor vasculature targeting of peptide-and 5,000 MW (molecular weight) PEG-coated CdSe/ZnS QD (5). Significant liver and spleen uptake of QD was noted, and it remains unclear whether tumor uptake in this study would have been sufficient for in vivo noninvasive fluorescence imaging. In vivo fluorescence imaging of targets expressed in tumor vasculature and tumor tissues has been reported by our group (a v b 3 integrin targeting using 15-to 20-nm-wide CdSe/ZnS QD coated with RGD peptide and 2,000 MW PEG (6), and others (prostate-specific membrane antigen [PSMA] targeting with 10-to 15-nm-diameter CdSe/ZnS QD coated with anti-PSMA monoclonal antibodies and 5,000 MW PEG (3)). Both reports show significant QD uptake in liver and...