Fluorescent Labeling Technique Research Articles

Detection of submillisecond spike timing differences based on delay-line anticoincidence detection

Detection of submillisecond interaural timing differences is the basis for sound localization in reptiles, birds, and mammals. Although comparative studies reveal that different neural circuits underlie this ability, they also highlight common solutions to an inherent challenge: processing information on timescales shorter than an action potential. Discrimination of small timing differences is also important for species recognition during communication among mormyrid electric fishes. These fishes generate a species-specific electric organ discharge (EOD) that is encoded into submillisecond-to-millisecond timing differences between receptors. Small, adendritic neurons (small cells) in the midbrain are thought to analyze EOD waveform by comparing these differences in spike timing, but direct recordings from small cells have been technically challenging. In the present study we use a fluorescent labeling technique to obtain visually guided extracellular recordings from individual small cell axons. We demonstrate that small cells receive 1-2 excitatory inputs from 1 or more receptive fields with latencies that vary by over 10 ms. This wide range of excitatory latencies is likely due to axonal delay lines, as suggested by a previous anatomic study. We also show that inhibition of small cells from a calyx synapse shapes stimulus responses in two ways: through tonic inhibition that reduces spontaneous activity and through precisely timed, stimulus-driven, feed-forward inhibition. Our results reveal a novel delay-line anticoincidence detection mechanism for processing submillisecond timing differences, in which excitatory delay lines and precisely timed inhibition convert a temporal code into a population code.

In Vivo Ectopic Implantation Model to Assess Human Mesenchymal Progenitor Cell Potential

Clinical interest on human mesenchymal progenitor cells (hMPC) relies on their potential applicability in cell-based therapies. An in vitro characterization is usually performed in order to define MPC potency. However, in vitro predictions not always correlate with in vivo results and thus there is no consensus in how to really assess cell potency. Our goal was to provide an in vivo testing method to define cell behavior before therapeutic usage, especially for bone tissue engineering applications. In this context, we wondered whether bone marrow stromal cells (hBMSC) would proceed in an osteogenic microenvironment. Based on previous approaches, we developed a fibrin/ceramic/BMP-2/hBMSCs compound. We implanted the compound during only 2 weeks in NOD-SCID mice, either orthotopically to assess its osteoinductive property or subcutaneously to analyze its adequacy as a cell potency testing method. Using fluorescent cell labeling and immunohistochemistry techniques, we could ascertain cell differentiation to bone, bone marrow, cartilage, adipocyte and fibrous tissue. We observed differences in cell potential among different batches of hBMSCs, which did not strictly correlate with in vitro analyses. Our data indicate that the method we have developed is reliable, rapid and reproducible to define cell potency, and may be useful for testing cells destined to bone tissue engineering purposes. Additionally, results obtained with hMPCs from other sources indicate that our method is suitable for testing any potentially implantable mesenchymal cell. Finally, we propose that this model could successfully be employed for bone marrow niche and bone tumor studies.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-013-9464-1) contains supplementary material, which is available to authorized users.

A transgenic Xenopus laevis reporter model to study lymphangiogenesis

SummaryThe importance of the blood- and lymph vessels in the transport of essential fluids, gases, macromolecules and cells in vertebrates warrants optimal insight into the regulatory mechanisms underlying their development. Mouse and zebrafish models of lymphatic development are instrumental for gene discovery and gene characterization but are challenging for certain aspects, e.g. no direct accessibility of embryonic stages, or non-straightforward visualization of early lymphatic sprouting, respectively. We previously demonstrated that the Xenopus tadpole is a valuable model to study the processes of lymphatic development. However, a fluorescent Xenopus reporter directly visualizing the lymph vessels was lacking. Here, we created transgenic Tg(Flk1:eGFP) Xenopus laevis reporter lines expressing green fluorescent protein (GFP) in blood- and lymph vessels driven by the Flk1 (VEGFR-2) promoter. We also established a high-resolution fluorescent dye labeling technique selectively and persistently visualizing lymphatic endothelial cells, even in conditions of impaired lymph vessel formation or drainage function upon silencing of lymphangiogenic factors. Next, we applied the model to dynamically document blood and lymphatic sprouting and patterning of the initially avascular tadpole fin. Furthermore, quantifiable models of spontaneous or induced lymphatic sprouting into the tadpole fin were developed for dynamic analysis of loss-of-function and gain-of-function phenotypes using pharmacologic or genetic manipulation. Together with angiography and lymphangiography to assess functionality, Tg(Flk1:eGFP) reporter tadpoles readily allowed detailed lymphatic phenotyping of live tadpoles by fluorescence microscopy. The Tg(Flk1:eGFP) tadpoles represent a versatile model for functional lymph/angiogenomics and drug screening.

Quantification of ATRP initiator density on polymer latex particles by fluorescence labeling technique using copper-catalyzed azide-alkyne cycloaddition

We developed a novel fluorescence labeling technique for quantification of surface densities of atom transfer radical polymerization (ATRP) initiators on polymer particles. The cationic P(St-CPEM-C4DMAEMA) and anionic P(St-CPEM) polymer latex particles carrying ATRP-initiating chlorine groups were prepared by emulsifier-free emulsion polymerization of styrene (St), 2-(2-chloropropionyloxy)ethyl methacrylate (CPEM), and N-n-butyl-N,N-dimethyl-N-(2-methacryloyloxy)ethylammonium bromide (C4DMAEMA). ATRP initiators on the surface of polymer particles were converted into azide groups by sodium azide, followed by fluorescent labeling with 5-(N,N-dimethylamino)-N′-(prop-2-yn-1-yl)naphthalene-1-sulfonamide (Dansyl-alkyne) by copper-catalyzed azide-alkyne cycloaddition (CuAAC). The reaction time required for both azidation of ATRP-initiating groups and successive fluorescence labeling of azide groups with Dansyl-alkyne by CuAAC were investigated in detail by FTIR and fluorescence spectral measurement, respectively. The ATRP initiator densities on the cationic P(St-CPEM-C4DMAEMA) and anionic P(St-CPEM) particle surfaces were estimated to be 0.21 and 0.15 molecules nm−2, respectively, which gave close agreement with values previously determined by a conductometric titration method. The fluorescence labeling through click chemistry proposed herein is a versatile technique to quantify the surface ATRP initiator density both on anionic and cationic polymer particles. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 4042–4051

Deducing Underlying Mechanisms from Protein Recruitment Data

By using fluorescent labelling techniques, the distribution and dynamics of proteins can be measured within living cells, allowing to study in vivo the response of cells to a triggering event, such as DNA damage. In order to evaluate the reaction rate constants and to identify the proteins and reactions that are essential for the investigated process, mechanistic models are used, which often contain many proteins and associated parameters and are therefore underdetermined by the data. In order to establish criteria for assessing the significance of a model, we present here a systematic investigation of the information that can be reliably deduced from protein recruitment data, assuming that the complete set of reactions that affect the data of the considered protein species is not known. To this purpose, we study in detail models where one or two proteins that influence each other are recruited to a substrate. We show that in many cases the kind of interaction between the proteins can be deduced by analyzing the shape of the recruitment curves of one protein. Furthermore, we discuss in general in which cases it is possible to discriminate between different models and in which cases it is impossible based on the data. Finally, we argue that if different models fit experimental data equally well, conducting experiments with different protein concentrations would allow discrimination between the alternative models in many cases.

Does timing matter? How priority effects influence the outcome of parasite interactions within hosts

In nature, hosts are exposed to an assemblage of parasite species that collectively form a complex community within the host. To date, however, our understanding of how within-host-parasite communities assemble and interact remains limited. Using a larval amphibian host (Pacific chorus frog, Pseudacris regilla) and two common trematode parasites (Ribeiroia ondatrae and Echinostoma trivolvis), we experimentally examined how the sequence of host exposure influenced parasite interactions within hosts. While there was no evidence that the parasites interacted when hosts were exposed to both parasites simultaneously, we detected evidence of both intraspecific and interspecific competition when exposures were temporally staggered. However, the strength and outcome of these priority effects depended on the sequence of addition, even after accounting for the fact that parasites added early in host development were more likely to encyst compared to parasites added later. Ribeiroia infection success was reduced by 14 % when Echinostoma was added prior to Ribeiroia, whereas no such effect was noted for Echinostoma when Ribeiroia was added first. Using a novel fluorescent-labeling technique that allowed us to track Ribeiroia infections from different exposure events, we also discovered that, similar to the interspecific interactions, early encysting parasites reduced the encystment success of later arriving parasites by 41 %, which could be mediated by host immune responses and/or competition for space. These results suggest that parasite identity interacts with host immune responses to mediate parasite interactions within the host, such that priority effects may play an important role in structuring parasite communities within hosts. This knowledge can be used to assess host-parasite interactions within natural communities in which environmental conditions can lead to heterogeneity in the timing and composition of host exposure to parasites.

Two-photon microscopy with double-circle trajectories for in vivo cerebral blood flow measurements

Scanning microscopes normally use trajectories which produce full-frame images of an object at a low frame rate. Time-resolved measurements are possible if scans along a single line are repeated at a high rate. In conjunction with fluorescence labeling techniques, in vivo recording of blood flow in single capillaries is possible. The present work investigates scanning with double-circle trajectories to measure blood flow simultaneously in several vessels of a capillary network. With the trajectory centered near a bifurcation, a double circle crosses each vessel twice, creating a sensing gate for passing dark red blood cells in fluorescently labeled plasma. From the stack of scans repeated at 1,300 Hz, the time-resolved velocity is retrieved using an image correlation approach. Single bifurcation events can be identified from a few fluorescently labeled red blood cells. The applicability of the method for in vivo measurements is illustrated on the basis of two-photon laser scanning microscopy of the cerebral capillary network of mice. Its performance is assessed with synthetic data generated from a two-phase model for the perfusion in a capillary network. The calculation of velocities is found to be sufficiently robust for a wide range of conditions. The achievable limits depend significantly on the experimental conditions and are estimated to be in the 1 μm/s (velocity) and 0.1 s (time resolution) ranges, respectively. Some manual fine-tuning is required for optimal performance in terms of accuracy and time resolution. Further work may lead to improved reliability with which bifurcation events are identified in the algorithm and to include red blood cell flux and hematocrit measurements. With the capability for time-resolved measurements in all vessels of a bifurcation, double-circle scanning trajectories allow a detailed study of the dynamics in vascular networks.

NeuroGPS: automated localization of neurons for brain circuits using L1 minimization model

Drawing the map of neuronal circuits at microscopic resolution is important to explain how brain works. Recent progresses in fluorescence labeling and imaging techniques have enabled measuring the whole brain of a rodent like a mouse at submicron-resolution. Considering the huge volume of such datasets, automatic tracing and reconstruct the neuronal connections from the image stacks is essential to form the large scale circuits. However, the first step among which, automated location the soma across different brain areas remains a challenge. Here, we addressed this problem by introducing L1 minimization model. We developed a fully automated system, NeuronGlobalPositionSystem (NeuroGPS) that is robust to the broad diversity of shape, size and density of the neurons in a mouse brain. This method allows locating the neurons across different brain areas without human intervention. We believe this method would facilitate the analysis of the neuronal circuits for brain function and disease studies.

Harmonic Analysis of the Fluorescence Response of Bimane Adducts of Colicin E1 at Helices 6, 7, and 10

The pre-channel state of helices 6, 7, and 10 (Val(447)-Gly(475) and Ile(508)-Ile(522)) of colicin E1 was investigated by a site-directed fluorescence labeling technique. A total of 44 cysteine variants were purified and covalently labeled with monobromobimane fluorescent probe. A variety of fluorescence properties of the bimane fluorophore were measured for both the soluble and membrane-bound states of the channel peptide, including the fluorescence emission maximum, fluorescence anisotropy, and membrane bilayer penetration depth. Using site-directed fluorescence labeling combined with our novel helical periodicity analysis method, the data revealed that helices 6, 7, and 10 are separate amphipathic α-helices with a calculated periodicity of T = 3.34 ± 0.08 for helix 6, T = 3.56 ± 0.03 for helix 7, and T = 2.99 ± 0.12 for helix 10 in the soluble state. In the membrane-bound state, the helical periodicity was determined to be T = 3.00 ± 0.15 for helix 6, T = 3.68 ± 0.03 for helix 7, and T = 3.47 ± 0.04 for helix 10. Dual fluorescence quencher analysis showed that both helices 6 and 7 adopt a tilted topology that correlates well with the analysis based on the fluorescence anisotropy profile. These data provide further support for the umbrella model of the colicin E1 channel domain.

ChemInform Abstract: Fluorescent Labeling Techniques in Biomolecules: A Flashback

AbstractReview: 112 refs.

High‐throughput flow cytometry compatible biosensor based on fluorogen activating protein technology

Monitoring the trafficking of multiple proteins simultaneously in live cells is of great interest because many receptor proteins are found to function together with others in the same cell. However, existing fluorescent labeling techniques have restricted the mechanistic study of functional receptor pairs. We have expanded a hybrid system combining fluorogen-activating protein (FAP) technology and high-throughput flow cytometry to a new type of biosensor that is robust, sensitive, and versatile. This provides the opportunity to study multiple trafficking proteins in the same cell. Human beta2 adrenergic receptor (β2AR) fused with FAP AM2.2 and murine C-C chemokines receptor type 5 fused with FAP MG13 was chosen for our model system. The function of the receptor and the binding between MG13 and fluorogen MG-2p have been characterized by flow cytometry and confocal microscopy assays. The binding of fluorogen and the FAP pair is highly specific, while both FAP-tagged fusion proteins function similarly to their wild-type counterparts. The system has successfully served as a counter screen assay to eliminate false positive compounds identified in a screen against NIH Molecular Libraries Small Molecule Repository targeting regulators of the human β2AR.

Single Influenza Virus Tracking Reveals Cell Specific Modes of Movement - a Key to Understand Cellular Tropism

Following single viruses on their journey through a host cell provides valuable insights that help to understand the infection process. Fluorescent labeling techniques together with high-resolution microscopic imaging allows tracking of single viruses on living cells with high temporal and spatial accuracy.Influenza A viruses were fluorescently labeled without interfering with their biological activity. Single-virus trajectories were recorded on the surface and inside living cells of different permissive cell lines. By using conventional computational methods we extracted the position of viruses within each image frame. In addition, we have applied a recently developed mathematical tool to further analyze a special type of recurrent trajectory obtained at the surface of living cells. using this approach we can identify and characterize microdomains on the cell surface. We show that the viruses are surprisingly mobile and display a variety of different types of movement, which depend on the studied cell line. The cell surface was characterized by scanning electron microscopy (SEM) and revealed a unique topography that also strongly depends on the cell type and the cells growth state.By comparing trajectories obtained from two permissive cell lines we were able to correlate the distinctive behavior of single viruses with the cell surface ultrastructure. We used cells that are infected to a different degree and combine this with virus uptake and intracellular fusion kinetics. This allows us to draw a comprehensive picture of the first steps of virus infection. Further we can draw conclusions about the infectivity and could explain why some cells are permissive while others are not.

ERG9 and COQ1 disruption reveals isoprenoids biosynthesis is closely related to mitochondrial function in Saccharomyces cerevisiae

Understanding in vivo regulation of isoprenoid biosynthesis is important for identifying strategies to improve carbon fluxes to desirable endproducts. To study the relevance of gene functions, substrate availability, metabolic flux, and the energetics of resource utilization for isoprenoids biosynthesis, Saccharomyces cerevisiae was co-engineered with endogenous deregulated erg9 and/or coq1 and exogenous plant genes that will lead to sesqui- and diterpene production. The flow of substrates through the MVA pathway statistically showed isoprenoids precursor levels were tightly regulated by erg9 and coq1 genes in S. cerevisiae. Physiological analysis by fluorescent probes and labeling techniques revealed elimination of the competing and/or degrading pathways by gene knockout can increase cellular stresses and change the energy metabolism. To further evaluate the role of erg9 and coq1 in the regulation of isoprenoids synthesis, heterologous terpene synthases from plants were introduced into S. cerevisiae, including the wild-type and mutant strains along with feeding of various metal cofactors under anoxic/oxic conditions. The product distribution in the strains indicated that the energy barrier could be the reason that different metal ion-assisted electrophilic attacks initiated carbocation formation, carbocation stabilization, and rearrangement, incorporating exquisite regio- and stereospecificity, to generate structurally diverse products. The yeast altered in the profile of terpenoids and their precursor pools aided the mechanisms of terpenoid biosynthesis and its regulation, highlighting the need for mitochondrial function when engineering metabolic flux using assumptions based on a comprehensive network of the mitochondrial system.

A micro-cantilever sensor chip based on contact angle analysis for a label-free troponin I immunoassay

Cantilever sensors have been extensively explored as a promising technique for real-time and label-free analyses in biological systems. A major sensing principle utilized by state-of-the-art cantilever sensors is based on analyte-induced surface stress changes, which result in static bending of a cantilever. The sensor performance, however, suffers from the intrinsically small change in surface stress induced by analytes, especially for molecular recognition such as antigen-antibody binding. Through the contact angle change on a tailored solid surface, it is possible to convert a tiny surface stress into a capillary force-a much larger physical quantity needed for a practical sensor application. In this work, a micro-cantilever sensor based on contact angle analysis (CAMCS) was proposed to effectively enhance the sensitivity of a sensor in proportion to the square of the length to thickness ratio of the cantilever structure. CAMCS chips were fabricated using a standard complementary-metal-oxide-semiconductor (CMOS) process to demonstrate a 1250-fold enhancement in the sensitivity of surface stress to bioanalyte adsorption using a piezoresistive sensing method. A real-time and label-free troponin I (cTnI) immunoassay, which is now widely used in clinics and considered a gold standard for the early diagnosis and prognosis of cardiovascular disease, was performed to demonstrate cTnI detection levels as low as 1 pg mL(-1). The short detection time of this assay was within several minutes, which matches the detection time of commercially available instruments that are based on fluorescence-labeling techniques.

Expansion and differentiation of human primary osteoblasts in two- and three-dimensional culture

Despite the regenerative capability of bone, treatment of large defects often requires bone grafts. The challenge for bone grafting is to establish rapid and sufficient vascularization. Three-dimensional (3D) multicellular spheroids consisting of the relevant cell types can be used as “mini tissues” to study the complexity of angiogenesis. We investigated two-dimensional (2D) expansion, differentiation and characterization of primary osteoblasts as steps toward the establishment of 3D multicellular spheroids. Supplementation of cell culture medium with vitamin D3 induces the osteocalcin expression of osteoblasts. An increased osteocalcin concentration of 10.8 ± 0.58 ng/ml could be measured after 19 days in supplemented medium. Vitamin D3 has no influence on the expression of alkaline phosphatase or the deposition of calcium. Expression of these additional osteogenic markers requires addition of a cocktail of osteogenic factors that, conversely, have no influence on the expression of osteocalcin. Supplementation of the cell culture medium with both vitamin D3 and a cocktail of osteogenic factors is recommended to produce an osteoblast phenotype that secretes osteocalcin, expresses alkaline phosphatase and deposits calcium. In such a supplemented medium, a mean osteocalcin concentration of 11.63 ± 4.85 ng/ml was secreted by the osteoblasts. Distinguishing osteoblasts and fibroblasts remains a challenge. Neither differentiated nor undifferentiated osteoblasts can be distinguished from fibroblasts by the expression of CD90, ED-A-fibronectin or α-smooth muscle actin; however, these cell types exhibit clear differences in their growth characteristics. Osteoblasts can be arranged as 3D spheroids by coating the bottom of the cell culture device with agarose. The cellular composition of 3D multicellular spheroids can be evaluated quantitatively using vital fluorescence labeling techniques. Spheroids are a promising tool for studying angiogenic and osteogenic phenomena in vivo and in vitro.

Neuregulin-1β regulates tyrosine kinase receptor expression in cultured dorsal root ganglion neurons with excitotoxicity induced by glutamate

Neuregulin-1 (NRG-1) signaling regulates neuronal development, migration, myelination, and synaptic maintenance. Three members of tyrosine kinase receptor (Trk) family, TrkA, TrkB, and TrkC, have been identified in DRG neurons. Whether NRG-1β and its signaling pathways influence the expression of these Trk receptors in DRG neurons is still unclear. In the present study, primary cultured DRG neurons were used to determine the effects of NRG-1β on TrkA, TrkB, and TrkC expression in DRG neurons with excitotoxicity induced by glutamate (Glu). The involvement of phosphatidylinositol 3-kinase (PI3K)/Akt and the effects of extracellular signal-regulated protein kinase (ERK1/2) signaling pathways on NRG-1β were also determined. DRG neurons were cultured for 48h and then exposed to Glu, Glu plus NRG-1β, LY294002 plus Glu plus NRG-1β, PD98059 plus Glu plus NRG-1β, and PD98059 plus LY294002 plus Glu plus NRG-1β for an additional 24h. The DRG neurons were continuously exposed to culture media as a control. After that, all cultures were processed for detection of mRNA levels of TrkA, TrkB, and TrkC using real time-PCR analysis. Protein levels of TrkA, TrkB, and TrkC were detected using a Western blot assay. The expression of TrkA, TrkB, and TrkC in situ was determined by a fluorescent labeling technique. The levels of phosphorylated Akt (pAkt), phosphorylated ERK1/2 (pERK1/2), total protein levels of Akt and ERK1/2 were detected using a Western blot assay. The results indicated that in primary cultured DRG neurons with excitotoxicity induced by Glu, NRG-1β increased the expression of TrkA and TrkB their mRNAs, but not TrkC and its mRNA. Inhibitors (LY294002, PD98059) either alone or in combination blocked the effects of NRG-1β. NRG-1β may play an important role in regulating the expression of different Trk receptors in DRG neurons through the PI3K/Akt and ERK1/2 signaling pathways.

Alterations in tyrosine kinase receptor (Trk) expression induced by insulin-like growth factor-1 in cultured dorsal root ganglion neurons

Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor expressed in small dorsal root ganglion (DRG) neurons. IGF-1 promotes neuronal survival by activating its receptor (IGF-1R). Whether IGF-1 and its signaling pathways influence the expression of tyrosine kinase receptors TrkA, TrkB and TrkC in DRG neurons remains unknown. In the present study, primary cultured DRG neurons were used to determine the effects of IGF-1 on TrkA, TrkB and TrkC expression. The involvement of extracellular signal-regulated protein kinase (ERK1/2) and the effects of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways on IGF-1 were also evaluated. DRG neurons were cultured for 48h and then exposed to IGF-1, PD98059 plus IGF-1, LY294002 plus IGF-1, and PD98059 plus LY294002 plus IGF-1 for an additional 24h. The DRG neurons were continuously exposed to culture medium as a control. All cultures were then processed for detection of mRNA levels of TrkA, TrkB and TrkC using real-time PCR analysis. Protein levels of TrkA, TrkB and TrkC were detected using a Western blot assay. The expression of TrkA, TrkB and TrkC in situ was determined by a fluorescent labeling technique. The levels of phosphorylated ERK1/2 (pERK1/2) and phosphorylated Akt (pAkt) were detected using a Western blot assay. The results indicated that in primary cultured DRG neurons, IGF-1 increased the expression of TrkA and TrkB and their mRNAs but not TrkC or its mRNA. Neither the ERK1/2 inhibitor PD98059 nor the PI3K inhibitor LY294002 alone blocked the effect of IGF-1, but the use of both inhibitors together was effective. IGF-1 may play an important role in regulating the expression of different Trk receptors in DRG neurons through the ERK1/2 and PI3K/Akt signaling pathways. These results suggest that IGF-1 signaling might be a potential target on modifying distinct Trk receptor-mediated biological effects.

Deterministic and stochastic naive T cell population dynamics: symmetric and asymmetric cell division

There is growing evidence that asymmetric cell division has a key role in fate decision during T cell responses, however it is unknown if this process also has a role in the normal homeostasis of the naive T cell population. In order to explore asymmetric cell division, we develop a mathematical model in which naive T cells exist in one of two states: a resting and a cycling state. We consider three variants of the model, differing in the state of daughter cells produced by cell division, and study the steady states and time evolution of the populations in each case. We begin with a deterministic model: two coupled ordinary differential equations for the two cell populations (resting and cycling). We then introduce a stochastic generalization of the model and show, by means of the appropriate moment generating function, that the equations of the deterministic model are those of the mean sizes of the populations. The stochastic model allows us to explore questions the deterministic model fails to address, such as extinction of the population and expected lifetimes of a naive T cell clone, when the number of cells is small. Finally, we consider a multi-variate Markov process, in which each cell is classified according to its generation, or number of divisions, up to a maximum number of generations, consistent with the detection limit of fluorescent labelling techniques. Immunologically, this is a challenging question to address, and our results indicate that the mathematical models developed allow us to discern between symmetric and asymmetric division scenarios. These results can be, in principle, experimentally tested and we provide a brief description of the experimental procedure that will allow to determine the relative role of symmetric versus asymmetric cell division in naive T cell homeostasis, without directly observing this process in vivo.

A novel leaflet-selective fluorescence labeling technique reveals differences between inner and outer leaflets at high bilayer curvature

Understanding the differences in the physical properties of the inner and outer leaflet of membranes and how the leaflets are coupled to each other requires methods that can selectively label both the outer and inner leaflets. In this report we introduce a combined chromatography/cyclodextrin method for selective labeling of the inner leaflet. Combining this method with selective labeling of the outer leaflet, we are able to show that there is a distinct difference in polar headgroup physical properties of the inner and outer leaflet headgroups in small unilamellar vesicles composed of a wide variety of phosphatidylcholines and a phosphaticylcholine/sphingomyelin mixture. It appears that the inner leaflet headgroups are more tightly packed than those of the outer leaflet. This differential packing disappears when vesicle size increases, showing that it is a consequence of membrane curvature. Differential packing is also reduced as acyl chain length is decreased. In the future, selective leaflet labeling is likely to be a powerful tool for investigating the properties of asymmetric lipid vesicles.

Live-Cell Super-Resolution Imaging with Synthetic Fluorophores

Super-resolution imaging methods now can provide spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. They can be applied to biological samples and provide new and exciting views on the structural organization of cells and the dynamics of biomolecular assemblies on wide timescales. These revolutionary developments come with novel requirements for fluorescent probes, labeling techniques, and data interpretation strategies. Synthetic fluorophores have a small size, are available in many colors spanning the whole spectrum, and can easily be chemically modified and used for stoichiometric labeling of proteins in live cells. Because of their brightness, their photostability, and their ability to be operated as photoswitchable fluorophores even in living cells under physiological conditions, synthetic fluorophores have the potential to substantially accelerate the broad application of live-cell super-resolution imaging methods.