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PubMed2006

Principles of two-photon excitation microscopy and its applications to neuroscience.

The brain is complex and dynamic. The spatial scales of interest to the neurobiologist range from individual synapses (approximately 1 microm) to neural circuits (centimeters); the timescales range from the flickering of channels (less than a millisecond) to long-term memory (years). Remarkably, fluorescence microscopy has the potential to revolutionize research on all of these spatial and temporal scales. Two-photon excitation (2PE) laser scanning microscopy allows high-resolution and high-sensitivity fluorescence microscopy in intact neural tissue, which is hostile to traditional forms of microscopy. Over the last 10 years, applications of 2PE, including microscopy and photostimulation, have contributed to our understanding of a broad array of neurobiological phenomena, including the dynamics of single channels in individual synapses and the functional organization of cortical maps. Here we review the principles of 2PE microscopy, highlight recent applications, discuss its limitations, and point to areas for future research and development.

Animals↗
PubMed2005

Expression of GFP-tagged neuronal glutamate transporters in cerebellar Purkinje neurons.

Of the five excitatory amino acid transporters (EAATs) identified, two genes are expressed by neurons (EAAT3 and EAAT4) and give rise to transporters confined to neuronal cell bodies and dendrites. At an ultrastructural level, EAAT3 and EAAT4 proteins are clustered at the edges of postsynaptic densities of excitatory synapses. This pattern of localization suggests that postsynaptic EAATs may help to limit spillover of glutamate from excitatory synapses. In an effort to study transporter localization in living neurons and ultimately to manipulate uptake at intact synapses, we have developed viral reagents encoding neuronal EAATs tagged with GFP. We demonstrate that these fusion proteins are capable of Na(+)-dependent glutamate uptake, that they generate ionic conductances indistinguishable from their wild-type counterparts, and that GFP does not alter their glutamate dose-dependence. Two-photon microscopy was used to examine fusion protein expression in Purkinje neurons in acute cerebellar slices. Both EAAT3-GFP and EAAT4-GFP were observed at high levels in the dendritic spines of transfected Purkinje neurons. These findings indicate that functional EAAT fusion proteins can be synthesized and appropriately trafficked to postsynaptic compartments. Furthermore, they validate a powerful system for looking at EAAT function in situ.

Amino Acid Transport System X-AG↗
PubMed2005

Confocal laser scanning microscopy in orthopaedic research.

Confocal laser scanning microscopy (CLSM) is a type of high-resolution fluorescence microscopy that overcomes the limitations of conventional widefield microscopy and facilitates the generation of high-resolution 3D images from relatively thick sections of tissue. As a comparatively non-destructive imaging technique, CLSM facilitates the in situ characterization of tissue microstructure. Images generated by CLSM have been utilized for the study of articular cartilage, bone, muscle, tendon, ligament and menisci by the foremost research groups in the field of orthopaedics including those teams headed by Bush, Errington, Guilak, Hall, Hunziker, Knight, Mow, Poole, Ratcliffe and White. Recent evolutions in techniques and technologies have facilitated a relatively widespread adoption of this imaging modality, with increased "user friendliness" and flexibility. Applications of CLSM also exist in the rapidly advancing field of orthopaedic implants and in the investigation of joint lubrication.

Animals↗
PubMed2005

Close encounters of the first and second kind: T-DC and T-B interactions in the lymph node.

Cellular interactions in lymphoid organs initiate the immune response and determine its outcome. Using two-photon microscopy in the lymph node, several groups have begun to investigate the motility characteristics and interactions among T lymphocytes, B lymphocytes, and dendritic cells (DC) in lymphoid organs. In the first "close encounter", T cells of a particular antigen specificity interact with antigen-bearing dendritic cells and begin to activate. Activation of both CD4+ and CD8+ T cells evolves through several stages; from transient interactions to stable clusters and later to dissociation and proliferation of T cells (clonal expansion). The second "close encounter" requires that antigen-engaged B cells become accessible to T cells by directed migration to the edge of the follicle. T cells and B cells then pair up and waltz together for an extended period, while helper T cells provide signals for B cells to differentiate into plasma cells. In this topical review, we compare the activation choreography of CD4+ T cells interacting first with dendritic cells, and then with B cells, during initiation of the humoral immune response.

Animals↗
PubMed2005

A dynamic view of the immunological synapse.

T cell activation requires interactions of T cell antigen receptors (TCR) and peptides presented by major histocompatibility complex molecules (MHCp) in an adhesive junction between the T cell and antigen-presenting cell. Stable junctions with bull's eye supramolecular activation clusters (SMACs) have been defined as immunological synapses (IS). These structures maintain T cell-APC interaction and allow directed secretion. T cells can also be activated by asymmetric hemi-synapses (HS) that allow migration during signal integration. IS and HS operate in different stages of T cell priming. Optimal effector functions may also depend upon cyclical use of IS and HS.

Animals↗
PubMed2005

Real-time measurement of signaling and motility during T cell development in the thymus.

Intracellular signals arising from interactions of immature thymocytes with distinct populations of stromal cells in the thymus are central to T cell development. The characteristics of these signals and the mechanisms underlying thymocyte migration between stromal cell compartments have been difficult to identify from static measurements of fixed tissue. Recent advances in two-photon microscopy and the development of three-dimensional models for real-time studies of T cell development have shed light on how single cells navigate the thymus. These studies reveal crosstalk between thymocyte signaling and motility that may integrate the search for potentially rare self-antigens with the requirement for sustained signaling in T cell maturation.

Animals↗
PubMed2005

Tracking thymocyte migration in situ.

The dynamic process of thymocyte migration can now be visualized in real-time and in the context of the native thymic environment. With improved computational resources, key information can be extracted from real-time imaging data and the migratory behaviors of developing thymocytes can be quantitated. The extraction and exploitation of three dimensional data through time is providing new insight into the nature and regulation of intrathymic migration. In this review we discuss this interdisciplinary approach and the promise it holds for the study of thymocyte migration in situ.

Animals↗
PubMed2005

Mechanisms of T cell motility and arrest: deciphering the relationship between intra- and extracellular determinants.

T lymphocytes are capable of rapid motility in vitro and in vivo. Upon antigen recognition, they may stop crawling and form a stable cell-cell contact called the 'immunological synapse' (IS). However, it is becoming clear that this outcome may not occur with the reliability that was once presumed. T cells, particularly naïve cells, are apparently triggered partly 'on the fly' during short contacts with peptide-MHC (pMHC) bearing antigen-presenting cells (APCs) and are also influenced in both activity and synapse duration by a multitude of external cues. Underlying the emerging issues is a paucity of data concerning the cell biology of T lymphocytes. Here, we review the molecular mechanisms of crawling and adhesion versus the various potential modes of 'stopping' in T lymphocytes. Both motility and arrest involve similar processes: adhesion, actin elongation and internal tension control, but with different coordination. We will attempt to integrate this with the known and potential external cues that signal for T cell motility versus stopping to form a synapse in vivo. Finally, we discuss how this interplay may give rise to unexpectedly complex motile and morphological behavior.

Animals↗
PubMed2005

Comparative sensitivity of rat cerebellar neurons to dysregulation of divalent cation homeostasis and cytotoxicity caused by methylmercury.

The objective of the present study was to determine the relative effectiveness of methylmercury (MeHg) to alter divalent cation homeostasis and cause cell death in MeHg-resistant cerebellar Purkinje and MeHg-sensitive granule neurons. Application of 0.5-5 microM MeHg to Purkinje and granule cells grown in culture caused a concentration- and time-dependent biphasic increase in fura-2 fluorescence. At 0.5 and 1 microM MeHg, the elevations of fura-2 fluorescence induced by MeHg were biphasic in both cell types, but significantly delayed in Purkinje as compared to granule cells. Application of the heavy-metal chelator, TPEN, to Purkinje cells caused a precipitous decline in a proportion of the fura-2 fluorescence signal, indicating that MeHg causes release of Ca(2+) and non-Ca(2+) divalent cations. Purkinje cells were also more resistant than granule cells to the neurotoxic effects of MeHg. At 24.5 h after-application of 5 microM MeHg, 97.7% of Purkinje cells were viable. At 3 microM MeHg there was no detectable loss of Purkinje cell viability. In contrast, only 40.6% of cerebellar granule cells were alive 24.5 h after application of 3 microM MeHg. In conclusion, Purkinje neurons in primary cultures appear to be more resistant to MeHg-induced dysregulation of divalent cation homeostasis and subsequent cell death when compared to cerebellar granule cells. There is a significant component of non-Ca(2+) divalent cation released by MeHg in Purkinje neurons.

Air Pollutants↗
PubMed2005

A multi-photon window onto neuronal-glial-vascular communication.

The cellular components of the brain are neurons, glia and vascular cells. These three entities form a metabolic network to sustain brain activity. Interactions among these cell types have been studied extensively in vitro, where the cells are easily accessible to physiological and pharmacological manipulations. With the advent of optical tools, it has become possible to investigate the cerebral metabolic network in vitro at the cellular and subcellular levels. However, the metabolic and homeostatic nature of neuronal-glial-vascular interactions must eventually be examined in vivo, and multi-photon imaging now provides a means to monitor neurovascular units in living experimental animals.

Animals↗
PubMed2003

Multi-photon microscopic evaluation of saphenous vein endothelium and its preservation with a new solution, GALA.

BACKGROUND: Injury to endothelium can compromise the patency of bypass grafts harvested during coronary artery bypass graft (CABG) surgery. Maintaining structural and functional viability of endothelium in grafts may lead to improved long-term patency. The information gained from the application of multi-photon microscopy in transmission and epifluorescence mode was used to assess the structural and functional integrity of human saphenous vein segments stored in multiple preservation solutions, and to design a superior storage solution. METHODS: Multi-photon microscopy was used to image deep within saphenous vein tissue harvested from patients undergoing CABG for analysis of endothelial structure and function. Endothelial cell structural viability, calcium mobilization, and nitric oxide generation were determined using specific fluorescence markers. RESULTS: Within 60 minutes of harvest and storage in standard preservation solutions, calcium mobilization and nitric oxide generation were markedly diminished with more than 90% of endothelial cells no longer viable in the vein. In contrast, veins could be stored for 24 hours without substantial loss in cell viability in a newly formulated heparinized physiologic buffered salt solution containing glutathione, ascorbic acid, and L-arginine (GALA). CONCLUSIONS: Standard solutions in clinical use today led to a profound decline in saphenous vein endothelial cell viability, whereas the newly designed physiologic salt solution (GALA) maintained endothelial function and structural viability for up to 24 hours. The improvements seen from using GALA as a vessel storage medium may lead to greater long-term vein graft patency following CABG surgery.

Aged↗
PubMed2002

Cellular characterization of adenylate kinase and its isoform: two-photon excitation fluorescence imaging and fluorescence correlation spectroscopy.

Adenylate kinase (AK) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. AK1beta (long form) from murine cells shares the same protein sequence as AK1 (short form) except for the addition of 18 amino acid residues at its N-terminus. It is hypothesized that these residues serve as a signal for protein lipid modification and targeting of the protein to the plasma membrane. To better understand the cellular function of these AK isoforms, we have used several modern fluorescence techniques to characterize these two isoforms of AK enzyme. We fused cytosolic adenylate kinase (AK1) and its isoform (AK1beta) with enhanced green fluorescence protein (EGFP) and expressed the chimera proteins in HeLa cells. Using two-photon excitation scanning fluorescence imaging, we were able to directly visualize the localization of AK1-EGFP and AK1beta-EGFP in live cells. AK1beta-EGFP mainly localized on the plasma membrane, whereas AK1-EGFP distributed throughout the cell except for trace amounts in the nuclear membrane and some vesicles. We performed fluorescence correlation spectroscopy measurements and photon-counting histogram analysis in specific domains of live cells. For AK1-EGFP, we observed only one diffusion component in the cytoplasm. For AK1beta-EGFP, we observed two distinct diffusion components on the plasma membrane. One corresponded to the free diffusing protein, whereas the other represented the membrane-bound AK1beta-EGFP. The diffusion rate of AK1-EGFP was slowed by a factor of 1.8 with respect to that of EGFP, which was 50% more than what we would expect for a free diffusing AK1-EGFP. To rule out the possibility of oligomer formation, we performed photon-counting histogram analysis to direct analyze the brightness difference between AK1-EGFP and EGFP. From our analysis, we concluded that cytoplasmic AK1-EGFP is monomeric. fluorescence correlation spectroscopy proved to be a powerful technique for quantitatively studying the mobility of the target protein in live cells. This technology offers advantages in studying protein interactions and function in the cell.

Adenylate Kinase↗
PubMed2002

Early steps of supported bilayer formation probed by single vesicle fluorescence assays.

We have developed a single vesicle assay to study the mechanisms of supported bilayer formation. Fluorescently labeled, unilamellar vesicles (30-100 nm diameter) were first adsorbed to a quartz surface at low enough surface concentrations to visualize single vesicles. Fusion and rupture events during the bilayer formation, induced by the subsequent addition of unlabeled vesicles, were detected by measuring two-color fluorescence signals simultaneously. Lipid-conjugated dyes monitored the membrane fusion while encapsulated dyes reported on the vesicle rupture. Four dominant pathways were observed, each exhibiting characteristic two-color fluorescence signatures: 1) primary fusion, in which an unlabeled vesicle fuses with a labeled vesicle on the surface, is signified by the dequenching of the lipid-conjugated dyes followed by rupture and final merging into the bilayer; 2) simultaneous fusion and rupture, in which a labeled vesicle on the surface ruptures simultaneously upon fusion with an unlabeled vesicle; 3) no dequenching, in which loss of fluorescence signal from both dyes occur simultaneously with the final merger into the bilayer; and 4) isolated rupture (pre-ruptured vesicles), in which a labeled vesicle on the surface spontaneously undergoes content loss, a process that occurs with high efficiency in the presence of a high concentration of Texas Red-labeled lipids. Vesicles that have undergone content loss appear to be more fusogenic than intact vesicles.

Adsorption↗
PubMed2003

Near-wall micro-PIV reveals a hydrodynamically relevant endothelial surface layer in venules in vivo.

High-resolution near-wall fluorescent microparticle image velocimetry (micro-PIV) was used in mouse cremaster muscle venules in vivo to measure velocity profiles in the red cell-depleted plasma layer near the endothelial lining. micro-PIV data of the instantaneous translational speeds and radial positions of fluorescently labeled microspheres (0.47 microm) in an optical section through the midsagittal plane of each vessel were used to determine fluid particle translational speeds. Regression of a linear velocity distribution based on near-wall fluid-particle speeds consistently revealed a negative intercept when extrapolated to the vessel wall. Based on a detailed three-dimensional analysis of the local fluid dynamics, we estimate a mean effective thickness of approximately 0.33 micro m for an impermeable endothelial surface layer or approximately 0.44 micro m assuming the lowest hydraulic resistivity of the layer that is consistent with the observed particle motions. The extent of plasma flow retardation through the layer required to be consistent with our micro-PIV data results in near complete attenuation of fluid shear stress on the endothelial-cell surface. These findings confirm the presence of a hydrodynamically effective endothelial surface layer, and emphasize the need to revise previous concepts of leukocyte adhesion, stress transmission to vascular endothelium, permeability, and mechanotransduction mechanisms.

Animals↗
PubMed2003

Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells.

The main potential of intrinsically fluorescent proteins (IFPs), as noninvasive and site-specific markers, lies in biological applications such as intracellular visualization and molecular genetics. However, photophysical studies of IFPs have been carried out mainly in aqueous solution. Here, we provide a comprehensive analysis of the intracellular environmental effects on the steady-state spectroscopy and excited-state dynamics of green (EGFP) and red (DsRed) fluorescent proteins, using both one- and two-photon excitation. EGFP and DsRed are expressed either in the cytoplasm of rat basophilic leukemia (RBL-2H3) mucosal mast cells or anchored (via LynB protein) to the inner leaflet of the plasma membrane. The fluorescence lifetimes (within approximately 10%) and spectra in live cells are basically the same as in aqueous solution, which indicate the absence of both IFP aggregation and cellular environmental effects on the protein folding under our experimental conditions. However, comparative time-resolved anisotropy measurements of EGFP reveal a cytoplasmic viscosity 2.5 +/- 0.3 times larger than that of aqueous solution at room temperature, and also provide some insights into the LynB-EGFP structure and the heterogeneity of the cytoplasmic viscosity. Further, the oligomer configuration and internal depolarization of DsRed, previously observed in solution, persists upon expression in these cells. DsRed also undergoes an instantaneous three-photon induced color change under 740-nm excitation, with efficiently nonradiative green species. These results confirm the implicit assumption that in vitro fluorescence properties of IFPs are essentially valid for in vivo applications, presumably due to the beta-barrel protection of the embodied chromophore. We also discuss the relevance of LynB-EGFP anisotropy for specialized domains studies in plasma membranes.

Animals↗
PubMed2003

Two-photon thermal bleaching of single fluorescent molecules.

We have studied the fluorescence emission by two-photon excitation of four dyes widely used for bioimaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level. The single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output until a sudden transition to a dark state very close to the background. The bleaching time that is found to vary in the series pyrene, indo-1, fluorescein and rhodamine 6G from the fastest to the slowest one respectively, has a Gaussian distribution indicating that the observed behavior is not due to photobleaching. Moreover, the bleaching time decreases with the glass substrate temperature reaching a vanishing nonmeasurable value for a limiting temperature whose value is found in the same series as for the bleaching time, from the lowest to the highest temperature respectively. The observed bleaching shows a clear correlation to the amount of absorbed power not reirradiated as fluorescence and to the complexity of the molecule. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon absorption of the single dyes as confirmed also by numerical simulations.

Fluorescein↗