Fluorescence Video Microscopy Research Articles

An intravital and confocal microscopic study of the distribution of intracameral antigen in the aqueous outflow pathways and limbus of the rat eye

In a previous investigation into the fate of fluorescently labelled antigen (Ag) injected into the anterior chamber (AC) of the rat eye, a large number of Ag + cells were noted in the conventional and non-conventional aqueous humour outflow pathways together with the external limbus. The aim of this study was to investigate the precise distribution and phenotype of these cells and compare their ability to capture fluorescent-labelled protein (bovine serum albumin, BSA, and ovalbumin, OVA) and polysaccharides (dextran, Dx) injected into the AC. The density of Ag + cells in the iris and limbus was investigated using in vivo video fluorescence microscopy 24 hr post-injection. The distribution and phenotype of Ag + cells in ocular tissues was analysed by confocal microscopy of frozen sections and in iris and corneoscleral/limbal wholemounts from animals sacrificed 24 hr post injection. The general distribution of labelled Ag was equivalent in OVA, BSA and Dx injected animals. Antigen-bearing cells were observed within the iris, iridocorneal angle, pre-equatorial choroid and around limbal/episcleral vessels. Localization of Ag + cells and free Ag in the anterior segment suggests that substances of these molecular weights (40–70 kDa) leave the eye through the conventional and non-conventional aqueous outflow pathways. The cells that internalized BSA, OVA or Dx in ocular tissues were of a similar phenotype, namely, ED1 +, ED2 +, occasionally ED3 + and predominantly MHC class II −, thus suggesting that they are of the macrophage phenotype. However, a few Ag + MHC class II + dendriform cells (putative DC) were also observed in the iris, trabecular meshwork, choroid and episclera. In conclusion our data reveal that the majority of intracamerally injected soluble Ag retained in the eye is taken up by resident macrophages not only in the iris but in all tissues lining the AC of the eye.

Ultrafine particles exert prothrombotic but not inflammatory effects on the hepatic microcirculation in healthy mice in vivo.

Air pollution episodes are strongly associated with increased cardiovascular morbidity and mortality. The effect of ultrafine particles (UFPs), when translocated after inhalation, on the microcirculation of extrapulmonary organs remains unclear. In C57BL/6 mice, either carbon black UFPs (1x10(7) and 5x10(7)) or vehicle was infused intra-arterially. Two hours after infusion, platelet- and leukocyte-endothelial cell interactions, sinusoidal perfusion, endothelial fibrin(ogen) deposition, and phagocytic activity of Kupffer cells were analyzed by intravital video fluorescence microscopy in the liver microvasculature. Expression of fibrin(ogen), von Willebrand factor (vWF), and P-selectin on hepatic endothelium was determined by immunostaining. Apoptotic cells were quantified in TUNEL-stained tissue sections. Application of UFPs caused significantly enhanced platelet accumulation on endothelium of postsinusoidal venules and sinusoids in healthy mice. UFP-induced platelet adhesion was not preceded by platelet rolling but was strongly associated with fibrin deposition and an increase in vWF expression on the endothelial surface. In contrast, inflammatory parameters such as the number of rolling/adherent leukocytes, P-selectin expression/translocation, and the number of apoptotic cells were not elevated 2 hours after UFP exposure. In addition, UFPs did not affect sinusoidal perfusion and Kupffer cell function. UFPs induce platelet accumulation in the hepatic microvasculature of healthy mice that is associated with prothrombotic changes on the endothelial surface of hepatic microvessels. Accumulation of particles in the liver exerts a strong procoagulatory impact but does not trigger an inflammatory reaction and does not induce microvascular/hepatocellular tissue injury.

Rapid, endoplasmic reticulum-independent diffusion of the mitotic Golgi haze.

Early in mitosis, the mammalian Golgi apparatus disassembles, and fluorescence microscopy reveals Golgi clusters and an extensive, nonresolvable haze that either represents scattered vesicles or a merged endoplasmic reticulum (ER)-Golgi compartment. To help decide between these alternatives, we have carried out a combined microscopic and pharmacological analysis, by using a BS-C-1 cell line stably coexpressing ER and Golgi markers. Video fluorescence microscopy showed that these two organelles were morphologically distinguishable at all stages of mitosis, and photobleaching experiments showed that diffusion of the Golgi marker was unaffected by the presence of the ER. Fragmentation of the ER by using filipin III completely blocked diffusion of the ER marker but had no effect on the Golgi marker, unless it was first relocated to the ER by using brefeldin A. The Golgi haze was also studied using BODIPY ceramide. Its diffusion was slower in mitotic Golgi than in mitotic ER, but similar to that of a Golgi enzyme marker in the mitotic Golgi haze or in Golgi vesicles generated by ilimaquinone. Together, these results support the idea that the Golgi and the ER remain separate during mitosis and strongly suggest that Golgi markers move by vesicle diffusion, as opposed to lateral diffusion in continuous membranes.

Differentiation and developmental pathways of uropathogenic Escherichia coli in urinary tract pathogenesis.

Uropathogenic Escherichia coli (UPEC) are capable of forming complex intracellular bacterial communities (IBC) within the superficial umbrella cells of the bladders of C3H and BALB/c mice. By using time-lapse fluorescence videomicroscopy to observe infected mouse bladder explants, we discovered that IBCs formed by uropathogenic E. coli progressed through four distinct developmental stages that differed with respect to growth rate, bacterial length, colony organization, motility, and its eventual dispersal. In the first phase, bacteria in the IBC were nonmotile, rod shaped, and grew rapidly in loosely organized colonies free in the cytoplasm of the bladder superficial umbrella cells. In the second phase, the loose collection of bacteria in the IBC matured into a slower growing, highly organized biofilm-like community consisting of coccoid bacteria that ultimately filled most of the cytoplasm. In the third phase, bacteria in the biofilm-like state in the IBC switched to a motile rod-shaped phenotype allowing detachment from the community and eventual fluxing out of the host cell. During the fourth phase, the bacteria filamented. Filamentation appeared to be in response to a Toll-like receptor 4-mediated innate defense mechanism. Bacteria that fluxed out of the superficial umbrella cells were able to reenter the IBC developmental cascade but with slower kinetics and ultimately a quiescent reservoir was established. Intracellular growth and filamentation provided an advantage to the bacteria in evading infiltrating polymorphonuclear leukocytes. This work has developed a technique to observe live infected organs and revealed a complex differentiation pathway that facilitates bacterial persistence in the urinary tract.

Cadmium-induced mitochondrial membrane-potential dissipation does not necessarily require cytosolic oxidative stress: studies using rhodamine-123 fluorescence unquenching.

The impact of cadmium on the cellular redox state and mitochondrial membrane potential (psi(m)) has been studied by monitoring dichlorofluorescein (DCF), CMXRos (dichlorodihydrofluorescein diacetate, chloromethyl-X-rosamine), and Rh-123 fluorescence in 5-day-old TC7 cells, a highly differentiated clone of the human intestinal Caco-2 cell line. Flow cytometry analyses, using DCFH oxidation to DCF, clearly revealed that a 30-min incubation to 50 microM cadmium (Cd) is sufficient to induce reactive oxygen species (ROS) formation; this effect was completely eliminated by the presence of 50 mM mannitol for the 30-min incubation period, but mannitol only partially scavenged ROS for the longer period of time studied. Imaging studies using fluorescence video microscopy revealed a parallel increase in (DCF) fluorescence in the nuclear and cytoplasmic regions as soon as Cd was added to the exposure medium. Flow cytometry analyses monitoring CMXRos fluorescence clearly showed that Cd also leads to psi(m) disruption, but, contrary to what was observed for ROS formation, mannitol was completely inefficient in preventing this effect. Further investigation using fluorescence microscopy and Rh-123 fluorescence unquenching revealed that although mannitol did not protect against Cd-induced dissipation of psi(m), it considerably delayed the process. We found that Rh-123 unquenching, occurring during probe redistribution, is a suitable tool to monitor the decrease of psi(m). We conclude that Cd rapidly induces ROS formation, mainly hydroxyl radical species OH(*), as well as the loss of psi(m). However, psi(m) dissipation does not necessarily require cellular OH(*) and may occur in the absence of apparent oxidative injury.

Deformation of DNA molecules by hydrodynamic focusing

The motion of a DNA molecule in a solvent flow reflects the deformation of a nano/microscale flexible mass–spring structure by the forces exerted by the fluid molecules. The dynamics of individual molecules can reveal both fundamental properties of the DNA and basic understanding of the complex rheological properties of long-chain molecules. In this study, we report the dynamics of isolated DNA molecules under homogeneous extensional flow. Hydrodynamic focusing generates homogeneous extensional flow with uniform velocity in the transverse direction. The deformation of individual DNA molecules in the flow was visualized with video fluorescence microscopy. A coil–stretch transition was observed when the Deborah number (De) is larger than 0.8. With a sudden stopping of the flow, the DNA molecule relaxes and recoils. The longest relaxation time of T2 DNA was determined to be 0.63 s when scaling viscosity to 0.9 cP.

CRP2 is an autonomous actin-binding protein

Cysteine-rich proteins (CRPs) have been shown to be involved in cell differentiation, transcriptional regulation and the organisation of the actin cytoskeleton. Thus far, the latter function has been inferred solely from the in vitro interaction of CRP1, CRP2, and CRP3 with α-actinin and zyxin. We show here that purified, recombinant CRP2 binds directly to F-actin in vitro in co-sedimentation assays. Using a green fluorescent protein (GFP)-tagged construct of CRP2 we analysed its localisation and dynamics in A7r5 rat smooth muscle cells. CRP2 was associated with the actin cytoskeleton and decorated actin stress fibres in a continuous fashion, unlike the periodic labelling pattern observed for α-actinin and zyxin, which also accumulate in focal adhesions. Using live video fluorescence microscopy we observed the behaviour of GFP-CRP2 during the dynamic rearrangement of the actin cytoskeleton in phorbol 12,13-dibutyrate-treated A7r5 cells. In contrast to the actin-binding proteins SM22α and α-actinin, GFP-CRP2 did not translocate into the podosomes induced by this treatment, but remained preferentially bound to the stress fibres, suggesting an actin filament-stabilising role for CRP2. When fused to the mitochondrial targeting sequence from the Listeria protein ActA, GFP-CRP2 was almost completely localised to mitochondria, but no significant recruitment of either α-actinin or zyxin could be observed. Taken together, our results demonstrate that CRP2 can bind to F-actin directly and that the association with the actin cytoskeleton is independent of α-actinin or zyxin localisation in the cell, thus questioning the role of CRP2 as a regulator of α-actinin function in vivo.

Mechanism of mitochondrial dysfunction in diabetic sensory neuropathy.

Symmetrical sensory polyneuropathy, the most common form of diabetic neuropathy in humans, is associated with a spectrum of structural changes in peripheral nerve that includes axonal degeneration, paranodal demyelination, and loss of myelinated fibers--the latter probably the result of a dying-back of distal axons. Mitochondrial dysfunction has recently been proposed as an etiological factor in this degenerative disease of the peripheral nervous system. Lack of neurotrophic support has been proposed as a contributing factor in the etiology of diabetic neuropathy based on studies in animal models of Type I diabetes. We have recently demonstrated that insulin and neurotrophin-3 (NT-3) modulate mitochondrial membrane potential in cultured adult sensory neurons. We therefore tested the hypothesis that diabetes-induced mitochondrial dysfunction is caused by impairments in neurotrophic support. We have used real-time fluorescence video microscopy to analyze mitochondrial membrane potential in cultured adult sensory neurons isolated from normal and diabetic rats. Diabetes caused a significant loss of mitochondrial membrane potential in all sub-populations of sensory neurons which can be prevented by in vivo treatment with insulin or NT-3. The mechanism of insulin and NT-3-dependent modulation of mitochondrial membrane potential involves the activation of the phosphoinositide 3 kinase (PI 3 kinase) pathway. Downstream targets of PI 3 kinase, such as Akt and the transcription factor cAMP response element-binding protein (CREB), are activated by insulin and NT-3 and regulate sensory neuron gene expression. These alterations in gene expression modulate critical components of metabolite pathways and the electron transport chain associated with the neuronal mitochondrion. Our results show that in adult sensory neurons, treatment with insulin can elevate the input of reducing equivalents into the mitochondrial electron transport chain, which leads to greater mitochondrial membrane polarization and enhanced ATP synthesis.

Local Retention of Soluble Antigen by Potential Antigen-Presenting Cells in the Anterior Segment of the Eye

To determine the capacity of bone marrow-derived cells in the anterior segment of the eye to capture a fluorescence-labeled antigen (Ag) injected into the anterior chamber (AC). Uveal tract and corneoscleral tissues from Lewis rats were cultured in vitro, with or without FITC-dextran (4 microg/mL final concentration), for 48 hours and examined by confocal microscopy. To investigate antigen uptake in vivo 2 microL (20 microg) of Cascade Blue-labeled dextran (CB-Dx) was injected into the right AC of Lewis rats. The density of Ag-positive cells in the iris at 1, 3, 5, or 12 days after injection was examined by in vivo video fluorescence microscopy. The distribution and phenotype of Ag-positive cells in frozen and paraffin-embedded sections of ocular tissues and in iris wholemounts from animals killed at 24 hours and day 7 were analyzed by fluorescence and confocal microscopy. In organ culture conditions numerous cells in the iris, ciliary body, choroid, and corneal limbus were capable of capturing fluorescence-labeled Ag. In vivo observations and microscopic examination of experimental eyes at days 1 and 7 after AC injection revealed Ag-positive cells within the iris, iridocorneal angle, the suprachoroidal space and around limbal-episcleral vessels. Ag-bearing cells in the iris express combinations of macrophage markers but rarely expressed major histocompatibility complex (MHC) class II molecules. A reduced number of Ag-bearing cells were still present in the iris at day 12. Potential antigen-presenting cells (APCs) in the iris and ciliary body are capable of internalizing intracameral Ag. The characteristics of these cells in the iris are consistent with a predominantly macrophage phenotype. These observations also suggest that the Ag leaving the eye through both the conventional and nonconventional aqueous outflow pathways may be captured by potential APCs in the episcleral tissues.

Intravital microscopy identifies selectins that regulate T cell traffic into allografts.

T cell homing to sites of injury and inflammation is a critical step for adaptive immune responses. While much has been learned regarding T cell homing to lymphoid tissues, few studies have directly observed trafficking events during an effector response. In this study, we developed a model that uses intravital fluorescence videomicroscopy to determine the molecules critical to T cell rolling within skin allograft microvasculature during the effector phase of the rejection response. Additional studies were performed to quantify T cell infiltrates as rejection progressed. We found that P-selectin and E-selectin expressed on postcapillary venules play overlapping roles in the recruitment of activated T cells in a SCID reconstitution model of skin graft rejection and are important in T cell accumulation at the graft site. Surprisingly, we also found that naive T cells are recruited and accumulate via constitutive T cell L-selectin and upregulated L-selectin ligands on rejecting allograft vasculature. These data indicated that a specific retinue of molecules is upregulated during the rejection response, and they suggest potential future therapeutic targets.

Multistep nature of microvascular recruitment of ex vivo-expanded embryonic endothelial progenitor cells during tumor angiogenesis.

Tissue neovascularization involves recruitment of circulating endothelial progenitor cells that originate in the bone marrow. Here, we show that a class of embryonic endothelial progenitor cells (Tie-2+, c-Kit+, Sca-1+, and Flk-1−/low), which were isolated at E7.5 of mouse development at the onset of vasculogenesis, retain their ability to contribute to tumor angiogenesis in the adult. Using intravital fluorescence videomicroscopy, we further defined the multistep process of embryonic endothelial progenitor cell (eEPC) homing and incorporation. Circulating eEPCs are specifically arrested in “hot spots” within the tumor microvasculature, extravasate into the interstitium, form multicellular clusters, and incorporate into functional vascular networks. Expression analysis and in vivo blocking experiments provide evidence that the initial cell arrest of eEPC homing is mediated by E- and P-selectin and P-selectin glycoprotein ligand 1. This paper provides the first in vivo insights into the mechanisms of endothelial progenitor cell recruitment and, thus, indicates novel ways to interfere with pathological neovascularization.

Stepwise cytoskeletal polarization as a series of checkpoints in innate but not adaptive cytolytic killing.

Cytolytic killing is a major effector mechanism in the elimination of virally infected and tumor cells. The innate cytolytic effectors, natural killer (NK) cells, and the adaptive effectors, cytotoxic T cells (CTL), despite differential immune recognition, both use the same lytic mechanism, cytolytic granule release. Using live cell video fluorescence microscopy in various primary cell models of NK cell and CTL killing, we show here that on tight target cell contact, a majority of the NK cells established cytoskeletal polarity required for effective lytic function slowly or incompletely. In contrast, CTLs established cytoskeletal polarity rapidly. In addition, NK cell killing was uniquely sensitive to minor interference with cytoskeletal dynamics. We propose that the stepwise NK cell cytoskeletal polarization constitutes a series of checkpoints in NK cell killing. In addition, the use of more deliberate progression to effector function to compensate for inferior immune recognition specificity provides a mechanistic explanation for how the same effector function can be used in the different functional contexts of the innate and adaptive immune response.

Control of Growth Cone Motility and Morphology by LIM Kinase and Slingshot via Phosphorylation and Dephosphorylation of Cofilin

Growth cone motility and morphology are based on actin-filament dynamics. Cofilin plays an essential role for the rapid turnover of actin filaments by severing and depolymerizing them. The activity of cofilin is repressed by phosphorylation at Ser3 by LIM kinase (LIMK, in which LIM is an acronym of the three gene products Lin-11, Isl-1, and Mec-3) and is reactivated by dephosphorylation by phosphatases, termed Slingshot (SSH). We investigated the roles of cofilin, LIMK, and SSH in the growth cone motility and morphology and neurite extension by expressing fluorescence protein-labeled cofilin, LIMK1, SSH1, or their mutants in chick dorsal root ganglion (DRG) neurons and then monitoring live images of growth cones by time-lapse video fluorescence microscopy. The expression of LIMK1 remarkably repressed growth cone motility and neurite extension, whereas the expression of SSH1 or a nonphosphorylatable S3A mutant of cofilin enhanced these events. The fan-like shape of growth cones was disorganized by the expression of any of these proteins. The repressive effects on growth cone behavior by LIMK1 expression were significantly rescued by the coexpression of S3A-cofilin or SSH1. These findings suggest that LIMK1 and SSH1 play critical roles in controlling growth cone motility and morphology and neurite extension by regulating the activity of cofilin and may be involved in signaling pathways that regulate stimulus-induced growth cone guidance. Using various mutants of cofilin, we also obtained evidence that the actin-filament-severing activity of cofilin is critical for growth cone motility and neurite extension.

Transient pores in vesicles

AbstractWe present our observations of transient pores in giant unilamellar vesicles, placed under tension, by optical illumination. When the membrane tension reached a certain level, transient pores appeared. Pore opening is driven by the membrane tension, σ, and its closure by the pore's line tension, 𝒯. By use of viscous mixtures of glycerol and water, we slowed down the leak out of the inner liquid in the presence of a pore. This allowed pores to reach large sizes (a few micrometres) and last at least a few seconds so that they could be visualized by fluorescence videomicroscopy. Line tension was inferred from the measurements of the closure velocity of the pores. By addition of cholesterol, which increased 𝒯 (reducing pore lifetimes), or of surfactants, which decreased 𝒯 (increasing pore lifetimes), we demonstrate how 𝒯, and consequently pore lifetimes, can be controlled over nearly two orders of magnitude. Addition of surfactants also has a dramatic effect on vesicle fusion. We discuss how our results can be extended to less viscous aqueous solutions which are more relevant for liposomal drug delivery formulations.© 2003 Society of Chemical Industry

Size-dependent free solution DNA electrophoresis in structured microfluidic systems

A new concept based on microstructuring techniques is presented to achieve size-dependent electrophoretic migration of DNA in free solution. Topographically structured microfluidic channels with periodical cavities in the range of the radius of gyration of the tested DNA molecules (<3 μm) are produced by moulding of polydimethylsiloxane on a master wafer fabricated by contact lithography of SU-8 photoresist. The electrophoretic migration of single DNA molecules stained with the intercalator YOYO was investigated by real-time fluorescence video microscopy. It could be demonstrated for large DNA molecules that λ-DNA (48 kbp) always migrated significantly faster than T2-DNA (164 kbp) in the two tested microchannel layouts. Integration into a microdevice for DNA separation in free solution is now in progress.

Arrays of mobile tethered vesicles on supported lipid bilayers.

We report a new system of laterally mobile, arrayed vesicles that are encoded with DNA to control tethering to fluid-supported phospholipid bilayers. The motion of individual fluorescently labeled vesicles, specifically bound, are easily visualized by fluorescence video microscopy and observed to collide reversibly on the surface. This system is an ideal model for studying interactions involving membranes, in particular integral membrane proteins.

Visualizing Superoxide Production in Normal and Diabetic Rat Islets of Langerhans

Oxygen free radicals have been implicated in beta-cell dysfunction and apoptosis associated with type 1 and type 2 diabetes mellitus. The roles of free radicals in diabetes have thus far been defined indirectly by monitoring oxidative tissue damage and the effects of antioxidants, free radical scavengers, and overexpression of superoxide dismutase. We employed the superoxide-mediated oxidation of hydroethidine to ethidium to dynamically and directly assess the relative rates of mitochondrial superoxide anion generation in isolated islets in response to glucose stimulation. Superoxide content of isolated islets increased in response to glucose stimulation. We next compared the oxyradical levels in Zucker lean control and Zucker diabetic fatty rat islets by digital imaging microfluorometry. The superoxide content of Zucker diabetic fatty islets was significantly higher than Zucker lean control islets under resting conditions, relatively insensitive to elevated glucose concentrations, and correlated temporally with a decrease in glucose-induced hyperpolarization of the mitochondrial membrane. Importantly, superoxide levels were elevated in islets from young, pre-diabetic Zucker diabetic fatty animals. Overproduction of superoxide was associated with perturbed mitochondrial morphology and may contribute to abnormal glucose signaling found in the Zucker diabetic fatty model of type 2 diabetes mellitus.

Cascades of Transient Pores in Giant Vesicles: Line Tension and Transport

Under ordinary circumstances, the membrane tension of a giant unilamellar vesicle is essentially nil. Using visible light, we stretch the vesicles, increasing the membrane tension until the membrane responds by the sudden opening of a large pore (several micrometers in size). Only a single pore is observed at a time in a given vesicle. However, a cascade of transient pores appear, up to 30–40 in succession, in the same vesicle. These pores are transient: they reseal within a few seconds as the inner liquid leaks out. The membrane tension, which is the driving force for pore opening, is relaxed with the opening of a pore and the leakage of the inner liquid; the line tension of the pore’s edge is then able to drive the closure of a pore. We use fluorescent membrane probes and real-time videomicroscopy to study the dynamics of the pores. These can be visualized only if the vesicles are prepared in a viscous solution to slow down the leakout of the internal liquid. From measurements of the closure velocity of the pores, we are able to infer the line tension, T . We have studied the effect of the shape of inclusion molecules on T . Cholesterol, which can be modeled as an inverted cone-shaped molecule, increases the line tension when incorporated into the bilayers. Conversely, addition of cone-shaped detergents reduces T . The effect of some detergents can be dramatic, reducing T by two orders of magnitude, and increasing pore lifetimes up to several minutes. We give some examples of transport through transient pores and present a rough measurement of the leakout velocity of the inner liquid through a pore. We discuss how our results can be extended to less viscous aqueous solutions which are more relevant for biological systems and biotechnological applications.

A WASp Homolog Powers Actin Polymerization-Dependent Motility of Endosomes In Vivo

Background: WASp/SCAR proteins activate the Arp2/3 complex to nucleate actin filament assembly and are thought to have important roles in endocytosis. WASp is required for efficient endocytosis of antigen receptors, N-WASp promotes actin polymerization-dependent movement of endomembrane vesicles, and Las17 (a yeast WASp homolog) is required for endocytic internalization. However, it is unknown whether movement of endosomes or other organelles requires activation of the Arp2/3 complex by members of the WASp/SCAR family.Results: Fluorescence video microscopy of yeast cells expressing a GFP-tagged G protein-coupled receptor (Ste2-GFP) as an endocytic marker revealed that endosomes and the lysosome-like vacuole are highly motile. Endosome/vacuole motility required actin polymerization, as indicated by sensitivity to latrunculin A, whereas microtubules were uninvolved. Endosome/vacuole motility did not require actin cables or myosin V (a MYO2 gene product), which moves secretory vesicles and the Golgi apparatus and mediates vacuole segregation. However, endosome motility required Las17, a WASp homolog. In contrast to other processes involving Las17, endosome/vacuole motility required the WCA domain of Las17, which is necessary and sufficient to activate the Arp2/3 complex.Conclusions: Endosome/vacuole motility in vivo requires actin polymerization stimulated by the WASp homolog Las17. WASp/SCAR family members in mammalian cells may have similar functions. Defects in endosome/lysosome motility may contribute to deficits in lymphocyte or macrophage function observed in human patients lacking WASp or developmental defects in N-WASp-deficient mice.

Multi-wavelength intermittent photoluminescence of single CdSe quantum dots

A single chromophore detection using video-microscopy is one of the latest methodologies to reveal unique characteristics, which could not be obtained from ensemble measurements. Among many kinds of subjects, dynamic optical properties observed in colloidal semiconductor nanoparticles are attractive and important not only for the basis of photo-physics but also for application studies, e.g. biological labeling, electronic devices. In this study, fluorescence video-microscopy was performed on cadmium selenide (CdSe) quantum dots (QDs) spin-coated on a glass substrate. From single CdSe QDs detection, emissions at wavelengths separated over 60 nm were observed for the first time. This spectral feature was attributed to the existence of double-emissive relaxation processes in CdSe QDs. Photoluminescence intermittency was also observed both from relaxation processes. Fluorescence video-microscopy, which was advanced in biology, can be applicable for the real-time monitoring of dynamic properties in semiconductor photo-physics.