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...

Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft-associated glycosyl-phosphatidyl-inositolanchored avidin test probe (Av-GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed highresolution and long-term tracking of individual Av-GPI and the classification of their various diffusive behaviors. Using dual-color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av-GPI and the location of glycosphingolipid GM1-rich microdomains and caveolae. We show that Av-GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1-rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av-GPI dynamically partition in and out of these microdomains in a cholesterol-dependent manner. Our results provide direct evidence that cholesterol-/sphingolipid-rich microdomains can compartmentalize the diffusion of GPI-anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft-associated proteins across the plasma membrane.

Nonself recognition during somatic growth is an essential and ubiquitous phenomenon in both prokaryotic and eukaryotic species. In filamentous fungi, nonself recognition is also important during vegetative growth. Hyphal fusion between genetically dissimilar individuals results in rejection of heterokaryon formation and in programmed cell death of the fusion compartment. In filamentous fungi, such as Neurospora crassa, nonself recognition and heterokaryon incompatibility (HI) are regulated by genetic differences at het loci. In N. crassa, mutations at the vib-1 locus suppress nonself recognition and HI mediated by genetic differences at het-c/pin-c, mat, and un-24/het-6. vib-1 is a homolog of Saccharomyces cerevisiae NDT80, which is a transcriptional activator of genes during meiosis. For this study, we determined that vib-1 encodes a nuclear protein and showed that VIB-1 localization varies during asexual reproduction and during HI. vib-1 is required for the expression of genes involved in nonself recognition and HI, including pin-c, tol, and het-6; all of these genes encode proteins containing a HET domain. vib-1 is also required for the production of downstream effectors associated with HI, including the production of extracellular proteases upon carbon and nitrogen starvation. Our data support a model in which mechanisms associated with starvation and nonself recognition/HI are interconnected. VIB-1 is a major regulator of responses to nitrogen and carbon starvation and is essential for the expression of genes involved in nonself recognition and death in N. crassa.Nonself recognition during somatic growth is an essential and ubiquitous phenomenon in both prokaryotic and eukaryotic species. In vertebrate species, self/nonself recognition relies on the major histocompatibility complex (MHC), which is an array of polymorphic genetic loci that generate proteins important in pathogen recognition and the activation of defense mechanisms (37). In filamentous fungi, nonself recognition is important during vegetative growth. Hyphal fusion between genetically dissimilar individuals results in rejection of heterokaryon formation and in programmed cell death of the fusion compartment (Fig. 1), an event analogous to nonself recognition following fusion in colonial marine invertebrates, such as Hydractinia and Botryllus (32, 73), and nonself recognition and death following fusion of plasmodia in the slime mold Physarum polycephalum (9). Nonself recognition during heterokaryon formation in filamentous fungi is regulated genetically by het loci (for heterokaryon incompatibility [HI]) (29,70,86). In the filamentous fungus Neurospora crassa, 11 het loci mediate nonself recognition and HI (28, 63); each het locus has two or three alternative allelic specificities. Since these het loci are effectively unlinked, the number of possible het genotypes within a segregating population is at least 2 11 genotypes, thus forming an effective barrier to heterokaryon formation between genetically different isolates. HI in filamentous fungi...

Resistance to drug therapy is a major concern in cancer treatment. To probe clones resistant to chemotherapy, the current approach is to conduct pooled cell analysis. However, this can yield false negative outcomes, especially when we are analyzing a rare number of circulating tumor cells (CTCs) among an abundance of other cell types. Here, we develop a microfluidic device that is able to perform high throughput, selective picking and isolation of single CTC to 100% purity from a larger population of other cells. This microfluidic device can effectively separate the very rare CTCs from blood samples from as few as 1 in 20,000 white blood cells. We first demonstrate isolation of pure tumor cells from a mixed population and track variations of acquired T790M mutations before and after drug treatment using a model PC9 cell line. With clinical CTC samples, we then show that the isolated single CTCs are representative of dominant EGFR mutations such as T790M and L858R found in the primary tumor. With this single cell recovery device, we can potentially implement personalized treatment not only through detecting genetic aberrations at the single cell level, but also through tracking such changes during an anticancer therapy.