Fast functional imaging of single neurons using random-access multiphoton (RAMP) microscopy.
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The successful study of dendritic signaling and computation requires the ability to simultaneously monitor neuronal activity at multiple cellular sites. While the difficulties of accessing dendritic submicron structures with conventional micropipette approaches are generally overcome by optical recording techniques, their spatio-temporal resolution has limited such studies to few sites or slow signals. Here we present a novel approach to functional imaging, termed random-access multiphoton (RAMP) microscopy, which combines multiphoton excitation with an inertia-free scanning mechanism. RAMP microscopy employs two-dimensional acousto-optic deflection to rapidly position a focused near-infrared ultrafast laser beam between dwell periods at multiple user-selected sites. Because neuronal structures are generally sparse, activity located throughout various compartments, including thin dendritic branches and spines, can be mapped at high frame rates while maintaining the signal-to-noise ratio of conventional scanning microscopy. Moreover, RAMP microscopy maintains the excellent structural imaging capability of multiphoton excitation, i.e., intrinsic optical sectioning and high lateral resolution from within highly light-scattering brain tissue. RAMP microscopy thus comprises a versatile tool for investigating correlations of dendritic structure and function with significantly enhanced experimental throughput. (+info)
Close encounters of the first and second kind: T-DC and T-B interactions in the lymph node.
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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. (+info)
An algorithm for automatic evaluation of the spot quality in two-color DNA microarray experiments.
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BACKGROUND: Although DNA microarray technologies are very powerful for the simultaneous quantitative characterization of thousands of genes, the quality of the obtained experimental data is often far from ideal. The measured microarrays images represent a regular collection of spots, and the intensity of light at each spot is proportional to the DNA copy number or to the expression level of the gene whose DNA clone is spotted. Spot quality control is an essential part of microarray image analysis, which must be carried out at the level of individual spot identification. The problem is difficult to formalize due to the diversity of instrumental and biological factors that can influence the result. RESULTS: For each spot we estimate the ratio of measured fluorescence intensities revealing differential gene expression or change in DNA copy numbers between the test and control samples. We also define a set of quality characteristics and a model for combining these characteristics into an overall spot quality value. We have developed a training procedure to evaluate the contribution of each individual characteristic in the overall quality. This procedure uses information available from replicated spots, located in the same array or over a set of replicated arrays. It is assumed that unspoiled replicated spots must have very close ratios, whereas poor spots yield greater diversity in the obtained ratio estimates. CONCLUSION: The developed procedure provides an automatic tool to quantify spot quality and to identify different types of spot deficiency occurring in DNA microarray technology. Quality values assigned to each spot can be used either to eliminate spots or to weight contribution of each ratio estimate in follow-up analysis procedures. (+info)
Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure.
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BACKGROUND: We characterize the application of multiphoton microscopy to the observation of the extracellular matrix of fresh unstained vessels. METHOD: Combined two-photon-excited fluorescence (2PEF) and second harmonic generation (SHG) imaging of large arteries reveals the architecture of elastin and collagen fibers in the vessel wall with remarkable specificity. RESULTS: We present elastin/collagen imaging in unstained rat vessels at both micrometer and whole vessel scales, and we characterize the optical properties of rat carotid artery and aorta walls. We apply this method to evidence deleterious effects of residual doses of a pesticide on the vessel wall. CONCLUSION: This study illustrates the potential of 2PEF/SHG microscopy for pharmacological studies in unlabeled arteries. (+info)
In vivo delivery of fluoresceinated dextrans to the murine growth plate: imaging of three vascular routes by multiphoton microscopy.
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Bone elongation by endochondral ossification occurs through the differentiation cascade of chondrocytes of cartilaginous growth plates. Molecules from the systemic vasculature reach the growth plate from three different directions: epiphyseal, metaphyseal, and a ring vessel and plexus associated with the perichondrium. This study is an analysis of the real-time dynamics of entrance of fluoresceinated tracers of different molecular weights into the growth plate from the systemic vasculature and tests the hypothesis that molecular weight is a key variable in the determination of both the directionality and the extent of tracer movement into the growth plate. Multiphoton microscopy was used for direct in vivo imaging of the murine proximal tibial growth plate in anesthetized 4- to 5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter. Mice were given an intracardiac injection of either fluorescein (332.3 Da) or fluoresceinated dextrans of 3, 10, 40, 70 kDa, singly or sequentially. For each tracer, directionality and rate of arrival, together with extent of movement within the growth plate, were imaged in real time. For small molecules (up to 10 kDa), vascular access from all three directions was observed and entrance was equally permissive from the metaphyseal and the epiphyseal sides. Within our detection limit (a few percent of vascular concentration), 40 kDa and larger dextrans did not enter. These results have implications both for understanding systemic and paracrine regulation of growth plate chondrocytic differentiation, as well as variables associated with effective drug delivery to growth plate chondrocytes. (+info)
Setup and characterization of a multiphoton FLIM instrument for protein-protein interaction measurements in living cells.
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BACKGROUND: Fluorescence lifetime microscopy (FLIM) is currently one of the best techniques to perform accurate measurements of interactions in living cells. It is independent of the fluorophore concentration, thus avoiding several common artifacts found in Forster Resonance Energy Transfer (FRET) imaging. However, for FLIM to achieve high performance, a rigorous instrumental setup and characterization is needed. METHODS: We use known fluorophores to perform characterization experiments in our instrumental setup. This allows us to verify the accuracy of the fluorescence lifetime determination, and test the linearity of the instrument by fluorescence quenching. RESULTS: We develop and validate here a protocol for rigorous characterization of time-domain FLIM instruments. Following this protocol, we show that our system provides accurate and reproducible measurements. We also used HeLa cells expressing proteins fused to Green Fluorescent Proteins variants (CFP and YFP) to confirm its ability to detect interactions in living cells by FRET. CONCLUSIONS: We report a well-designed protocol in which precise and reproducible lifetime measurements can be performed. It is usable for all confocal-based FLIM instruments and is a useful tool for anyone who wants to perform quantitative lifetime measurements, especially when studying interactions in living cells using FRET. (+info)
Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye.
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PURPOSE: The purpose of this work was to demonstrate the use of the combined imaging modality of multiphoton autofluorescence and second-harmonic generation (SHG) microscopy in obtaining spectrally resolved morphologic features of the cornea, limbus, conjunctiva, and sclera in whole, ex vivo porcine eyes. METHODS: The 780-nm output of a femtosecond, titanium-sapphire laser was used to induce broadband autofluorescence (435-700 nm) and SHG (390 nm) from various regions of the surface of ex vivo porcine eyes. A water-immersion objective was used for convenient imaging of the curved surface of the eye. RESULTS: Multiphoton autofluorescence was useful in identifying cellular structures of the different domains of the ocular surface, and the SHG signal can be used to resolve collagen organization within the cornea stroma and sclera of ex vivo porcine eyes. CONCLUSIONS: Multiphoton autofluorescence and SHG microscopy have been demonstrated to be an effective technique for resolving, respectively, the cellular and collagen structures within the ocular surface of ex vivo porcine eyes. SHG imaging resolved the difference in structural orientations between corneal and sclera collagen fibers. Specifically, the corneal collagen is organized in a depth-dependent fashion, whereas the scleral collagen is randomly packed. Because this technique does not require histologic preparation procedures, it has the potential to be applied for in vivo studies with minimal disturbance to the eye. (+info)
Natural killer cell behavior in lymph nodes revealed by static and real-time imaging.
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Natural killer (NK) cells promote dendritic cell (DC) maturation and influence T cell differentiation in vitro. To better understand the nature of the putative interactions among these cells in vivo during the early phases of an adaptive immune response, we have used immunohistochemical analysis and dynamic intravital imaging to study NK cell localization and behavior in lymph nodes (LNs) in the steady state and shortly after infection with Leishmania major. In the LNs of naive mice, NK cells reside in the medulla and the paracortex, where they closely associate with DCs. In contrast to T cells, intravital microscopy revealed that NK cells in the superficial regions of LNs were slowly motile and maintained their interactions with DCs over extended times in the presence or absence of immune-activating signals. L. major induced NK cells to secrete interferon-gamma and to be recruited to the paracortex, where concomitant CD4 T cell activation occurred. Therefore, NK cells form a reactive but low mobile network in a strategic area of the LN where they can receive inflammatory signals, interact with DCs, and regulate colocalized T cell responses. (+info)