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At least 19 recordsLinked to original sources
PubMed2005

Live imaging of regenerating lamprey spinal axons.

BACKGROUND: The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Although the growing tips of developing axons in lamprey have not been described, in all species studied, growth cones are complex in shape, consisting of a lamellipodium and filopodia, rich in F-actin and lacking neurofilaments (NF). By contrast, static immunohistochemical and electron microscopic observations of fixed tissue suggested that the tips of regenerating lamprey spinal axons are simple in shape, densely packed with NF, but contain very little F-actin. Thus, it has been proposed that regeneration of axons in the CNS of mature animals is not based on the canonical pulling mechanism of growth cones but involves an internal protrusive force, perhaps generated by the transport and assembly of NF. To eliminate the possibility that these histological features are due to fixation artifact, fluorescently labeled regenerating axon tips were imaged live. METHODS: Spinal cords were transected, and after 0 to 10 weeks, the CNS was isolated in lamprey Ringer at 5 degrees C to 12 degrees C and the large reticulospinal axons were microinjected with fluorescent tracers. The proximal axon tips were imaged with a fluorescence dissecting microscope repeatedly over 2 to 5 days and photographed with confocal microscopy. Experiments were also performed through a dorsal incision in the living animal. Axon tips were microinjected as above or retrogradely labeled with tracer applied to the transection site and photographed through the fluorescence dissecting scope or with two-photon microscopy. The spinal cords were then fixed and processed for wholemount NF immunohistochemistry. RESULTS: The living axon tips were simple in shape, not significantly different from those in fixed spinal cords, and filled with NF. In isolated CNS preparations, very little axon retraction and no regeneration was observed. In the living animal, rapid retraction, up to 3 mm/day, was seen during the 1st few days posttransection. At more than 2 weeks posttransection, some fibers showed regeneration of up to 35 microm/day. CONCLUSIONS: 1) The tips of regenerating lamprey axons are simple in shape and filled with NF. 2) Both axon retraction and axon extension are active processes, requiring factors present in the living animal that are missing in the isolated CNS.

Actins↗
PubMed2005

Imaging living central neurones using viral gene transfer.

Studies of central neurones and other cellular components of the brain, such as glial and vascular cells, can be greatly advanced by the use of the modern optical techniques such as confocal live cell imaging. Fluorescent proteins have allowed imaging of particular cell types or intracellular elements to be visualised and distinguished from irrelevant background structures. To introduce the genetic information encoding for fluorescent proteins into relevant cellular targets, molecular tools are required. Viral vectors are one of the best ways of gene delivery into differentiated postnatal brain neurones and glia. Current progress in this field allows targeting of various cell types and therefore makes it possible to express a variety of fluorescent constructs in selected subpopulations of neurones, for example. In this review, we will discuss and compare the properties of the most popular viral gene delivery systems and the advantages of different brain cell preparations to illustrate how they can be used for high-resolution live cell confocal imaging in order to study new aspects of central nervous system (CNS) structure and function.

Adenoviridae↗
PubMed1997

A simple water-immersion condenser for imaging living brain slices on an inverted microscope.

Due to some physical limitations of conventional condensers, inverted compound microscopes are not optimally suited for imaging living brain slices with transmitted light. Herein is described a simple device that converts an inverted microscope into an effective tool for this application by utilizing an objective as a condenser. The device is mounted on a microscope in place of the condenser, is threaded to accept a water immersion objective, and has a slot for a differential interference contrast (DIC) slider. When combined with infrared video techniques, this device allows an inverted microscope to effectively image living cells within thick brain slices in an open perfusion chamber.

Animals↗
PubMed2006

Imaging living mice using a 1-T compact MRI system.

PURPOSE: To determine the feasibility of imaging living mice with a 1-T compact MRI system and investigate appropriate imaging techniques for use in routine animal experiments. MATERIALS AND METHODS: An MRI system consisting of a 1-T permanent magnet and compact console was used. Images of the entire trunks of living mice were obtained on the system using a T1-weighted three-dimensional fast low-angle shot (3D FLASH) sequence, and image quality was evaluated in relation to imaging techniques. RESULTS: Restraint of respiratory motion improved the image quality. Decreasing the slice thickness reduced artificial inhomogeneity in signal intensity (SI). Substantial effects of TR and FA on image quality were also demonstrated. With the determined techniques, images covering the entire trunk with a voxel size of 0.26x0.26x0.52 mm were acquired in an acquisition time of five minutes 28 seconds and a total experiment time of <20 minutes, and various organs and subcutaneous tumors were clearly visualized. CONCLUSION: The compact MRI system provides images of living mice with acceptable quality in a reasonable time. Considering its convenience, it appears to be suitable for use in routine mouse experiments.

Animals↗
PubMedDate not supplied

A comparison of the concordance of digital images, live examinations, and skin biopsies for the diagnosis of hospitalized dermatology consultation patients.

OBJECTIVE: The goal of this study was to compare live and digital image examination of hospitalized dermatology consultation patients. DESIGN: A medical student digitally imaged patients with a Nikon Coolpix 950 digital camera (Nikon Corp., Tokyo, Japan). A resident reviewed these images and the student recorded the differential diagnosis. Digital examination was followed by live dermatologic examination by the dermatology resident and then by a supervising board certified dermatologist. The diagnoses in 29 of these cases were compared with definitive diagnostic tests (e.g., skin biopsy). SETTING: Montefiore Hospital, Bronx, NY Patients. In March, 2000, 48 of 58 consecutive hospitalized patients for whom dermatology consultations were requested and from whom consent for photographs could be obtained. MAIN OUTCOME MEASURES: The accuracy and consistency of the examiners top (up to three) digital diagnoses, (up to three) live diagnoses, and histopathologic diagnoses were compared. RESULTS: Eighty-one percent (38/47) of resident digital consult diagnoses agreed with live resident diagnoses. Live resident diagnosis agreement with live supervising board certified dermatologists diagnosis was 90% (18/20). Resident digital image diagnosis correlation with definitive diagnostic test results was 76% (22/29). Live supervising board certified dermatologist diagnosis correlation with definitive diagnostic test results was 73% (14/19). CONCLUSIONS: Digital image review results in reliable and accurate diagnosis compared to live consultations. For inpatient consultations, three-dimensional lesions are more amenable to digital diagnosis than flat, generalized rashes.

Biopsy↗
PubMed1999

Imaging living cells and tissues by two-photon excitation microscopy.

Two-photon excitation microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Since two-photon excitation occurs only at the focal point of the microscope, it inherently provides three-dimensional resolution. This localization of excitation also minimizes photobleaching and photodamage, which are the ultimate limiting factors in imaging living cells. Furthermore, no pinhole is required to attain three-dimensional discrimination, so the efficiency of fluorescence collection is increased. These advantages allow experiments on thick living samples that would not be possible with other imaging techniques. The cost and complexity of the lasers required for two-photon excitation microscopy have limited its use, but appropriate turn-key lasers have now been introduced, and their cost should decrease. Finally, the recent introduction of commercial two-photon excitation laser-scanning microscope systems allows a much larger group of researchers access to this state-of-the-art methodology.

Animals↗
PubMed2004

[Analysis of molecular mechanism regulating spatio-temporal localization and activity of protein kinase C and diacylglycerol kinase using live imaging].

Protein kinase C (PKC) changes its subcellular localization depending on extracellular signals including hormones and neurotransmitters. Such translocation is referred to as "targeting" in this review. The live imaging technique using GFP has allowed the dynamic movement of PKC to be visualized in living cells and revealed a remarkable diversity in PKC targeting. These studies indicate an importance of targeting in regulating the physiological and isotype-specific function of PKC. Like PKC, diacylglycerol kinase (DGK), which phosphorylates diacylglycerol resulting in attenuation of PKC, subtype-specifically translocates to particular subcellular compartments including the plasma membrane and Golgi complex. In addition, it has been shown that the localization and activation of the two functionally-related kinases are well organized by direct interaction and phosphorylation. This review summarizes diversity in targeting of PKC and DGK and the molecular mechanisms regulating their targeting.

Diacylglycerol Kinase↗
PubMed2005

Live imaging of bidirectional traffic from the ERGIC.

The endoplasmic reticulum-Golgi intermediate compartment (ERGIC) defined by the cycling lectin ERGIC-53 consists of tubulovesicular clusters, but it is unknown if these membranes are transport vehicles or stationary entities. Here, we show by live imaging that GFP-ERGIC-53 mainly localizes to long-lived stationary and some short-lived highly mobile elements. Unlike the anterograde marker VSV-G-GFP, GFP-ERGIC-53 does not vectorially move to the Golgi upon exit from the ERGIC, as assessed by a novel quantitative vector field method. Dual-color imaging of GFP-ERGIC-53 and a secretory protein (signal-sequence-tagged dsRed) reveals that the stationary elements are sites of repeated sorting of retrograde and anterograde cargo, and are interconnected by highly mobile elements. These results suggest that the ERGIC is stationary and not simply a collection of mobile carriers that mediate protein traffic from endoplasmic reticulum to Golgi.

Biological Transport, Active↗
PubMed2026

Impaired stem cell migration and divisions in Duchenne muscular dystrophy revealed by live imaging.

Dysregulation of stem cell properties is a hallmark of many pathologies, but the dynamic behaviour of stem cells in their microenvironment during disease progression remains poorly understood. Using the mdx mouse model of Duchenne Muscular Dystrophy, we developed innovative live imaging of muscle stem cells (MuSCs) in vivo, and ex vivo on isolated myofibres. We show that mdx MuSCs have impaired migration and precocious differentiation through unbalanced symmetric divisions, driven by p38 and PI3K signalling pathways, in contrast to the p38-only dependence of healthy MuSCs. Cross-grafting shows that MuSC fate decisions are governed by fibre-independent cues, whereas their migration behaviour is determined by the myofibre niche. This study provides the first dynamic analysis of dystrophic MuSC properties in vivo, reconciling conflicting reports on their function. Our findings establish DMD as a MuSC disease with niche dysfunctions, offering strategies to restore stem cell functions for improved muscle regeneration.

Stem Cells↗
PubMed2005

Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control.

Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactin-dependent processes is not clearly understood. Here we generate and/or characterize mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function. In addition, we develop an improved time-lapse video microscopy technique that allows live imaging of GFP-Lis1, GFP-Rod checkpoint protein, green fluorescent protein (GFP)-labeled chromosomes, or GFP-labeled mitotic spindle dynamics in neuroblasts within whole larval brain explants. Our mutant analyses show that Lis1/dynactin have at least two independent functions during mitosis: first promoting centrosome separation and bipolar spindle assembly during prophase/prometaphase, and subsequently generating interkinetochore tension and transporting checkpoint proteins off kinetochores during metaphase, thus promoting timely anaphase onset. Furthermore, we show that Lis1/dynactin/dynein physically associate and colocalize on centrosomes, spindle MTs, and kinetochores, and that regulation of Lis1/dynactin kinetochore localization in Drosophila differs from both Caenorhabditis elegans and mammals. We conclude that Lis1/dynactin act together to regulate multiple, independent functions in mitotic cells, including spindle formation and cell cycle checkpoint release.

Animals↗
PubMed2006

Comparative study of the conditions required to image live human epithelial and fibroblast cells using atomic force microscopy.

Successful imaging of living human cells using atomic force microscopy (AFM) is influenced by many variables including cell culture conditions, cell morphology, surface topography, scan parameters, and cantilever choice. In this study, these variables were investigated while imaging two morphologically distinct human cell lines, namely LL24 (fibroblasts) and NCI H727 (epithelial) cells. The cell types used in this study were found to require different parameter settings to produce images showing the greatest detail. In contact mode, optimal loading forces ranged between 2-2.8 x 10(-9) and 0.1-0.7 x 10(-9) (N) for LL24 and NCI H727 cells respectively. In tapping (AC) mode, images of LL24 cells were obtained using cantilevers with a spring constant of at least 0.32 N/m, while NCI H727 cells required a greater spring constant of at least 0.58 N/m. To obtain tapping mode images, cantilevers needed to be tuned to resonate at higher frequencies than their resonance frequencies to obtain images. For NCI H727 cells, contact mode imaging produced the clearest images. For LL24 cells, contact and tapping mode AFM produced images of comparable quality. Overall, this study shows that cells with different morphologies and surface topography require different scanning approaches and optimal conditions must be determined empirically to achieve images of high quality.

Cell Line↗
PubMed2006

Live imaging of neural structure and function by fibred fluorescence microscopy.

Only a few methods permit researchers to study selected regions of the central and peripheral nervous systems with a spatial and time resolution sufficient to image the function of neural structures. Usually, these methods cannot analyse deep-brain regions and a high-resolution method, which could repeatedly probe dynamic processes in any region of the central and peripheral nervous systems, is much needed. Here, we show that fibred fluorescence microscopy-which uses a small-diameter fibre-optic probe to provide real-time images-has the spatial resolution to image various neural structures in the living animal, the consistency needed for a sequential, quantitative evaluation of axonal degeneration/regeneration of a peripheral nerve, and the sensitivity to detect calcium transients on a sub-second timescale. These unique features should prove useful in many physiological studies requiring the in situ functional imaging of tissues in a living animal.

Animals↗
PubMed2002

Illumination of the malaria parasite Plasmodium falciparum alters intracellular pH. Implications for live cell imaging.

Live cell fluorescence microscopy has been widely used to study physiological processes in the human malarial parasite Plasmodium falciparum, including pH homeostasis, Ca(2+) signaling and protein targeting. However, the reproducibility of the data is often poor. Controversial statements exist regarding cytosolic and vacuolar baseline pH, as well as regarding the subcellular localization of some of the fluorochromes used. When trying to reproduce published baseline values, we observed an unexpected light sensitivity of P. falciparum, which manifests itself in the form of a strong cytoplasmic acidification. Even short exposure times with moderate to low light intensities caused the parasite cytosol to acidify. We show that this effect arises from the selective disruption of the parasite's acidic food vacuole, brought about by lipid peroxidation initiated by light-induced generation of hydroxyl radicals. Our data suggest that heme serves as a photosensitizer in this process. Our findings have major implications for the use of live cell microscopy in P. falciparum and add a cautionary note to previous studies where live cell fluorometry has been used to determine physiological parameters in P. falciparum.

Animals↗
PubMed2002

Live imaging of telomeres: yKu and Sir proteins define redundant telomere-anchoring pathways in yeast.

BACKGROUND: The positioning of chromosomal domains within interphase nuclei is thought to facilitate transcriptional repression in yeast. Although this is particularly well characterized for telomeres, the molecular basis of their specific subnuclear organization is poorly understood. The use of live fluorescence imaging overcomes limitations of in situ staining on fixed cells and permits the analysis of chromatin dynamics in relation to stages of the cell cycle. RESULTS: We have characterized the dynamics of yeast telomeres and their associated domains of silent chromatin by using rapid time-lapse microscopy. In interphase, native telomeres are highly dynamic but remain within a restricted volume adjacent to the nuclear envelope. This constraint is lost during mitosis. A quantitative analysis of selected mutants shows that the yKu complex is necessary for anchoring some telomeres at the nuclear envelope (NE), whereas the myosin-like proteins Mlp1 and Mlp2 are not. We are able to correlate increased telomeric repression with increased anchoring and show that silent chromatin is tethered to the NE in a Sir-dependent manner in the absence of the yKu complex. Sir-mediated anchoring is S phase specific, while the yKu-mediated pathway functions throughout interphase. Subtelomeric elements of yeast telomere structure influence the relative importance of the yKu- and Sir-dependent mechanisms. CONCLUSIONS: Interphase positioning of telomeres can be achieved through two partially redundant mechanisms. One requires the heterodimeric yKu complex, but not Mlp1 and Mlp2. The second requires Silent information regulators, correlates with transcriptional repression, and is specific to S phase.

Cell Nucleus↗
PubMed2006

Live imaging of yeast Golgi cisternal maturation.

There is a debate over how protein trafficking is performed through the Golgi apparatus. In the secretory pathway, secretory proteins that are synthesized in the endoplasmic reticulum enter the early compartment of the Golgi apparatus called cis cisternae, undergo various modifications and processing, and then leave for the plasma membrane from the late (trans) cisternae. The cargo proteins must traverse the Golgi apparatus in the cis-to-trans direction. Two typical models propose either vesicular transport or cisternal progression and maturation for this process. The vesicular transport model predicts that Golgi cisternae are distinct stable compartments connected by vesicular traffic, whereas the cisternal maturation model predicts that cisternae are transient structures that form de novo, mature from cis to trans, and then dissipate. Technical progress in live-cell imaging has long been awaited to address this problem. Here we show, by the use of high-speed three-dimensional confocal microscopy, that yeast Golgi cisternae do change the distribution of resident membrane proteins from the cis nature to the trans over time, as proposed by the maturation model, in a very dynamic way.

COP-Coated Vesicles↗
PubMed2005

Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem.

BACKGROUND: Plants produce leaf and flower primordia from a specialized tissue called the shoot apical meristem (SAM). Genetic studies have identified a large number of genes that affect various aspects of primordium development including positioning, growth, and differentiation. So far, however, a detailed understanding of the spatio-temporal sequence of events leading to primordium development has not been established. RESULTS: We use confocal imaging of green fluorescent protein (GFP) reporter genes in living plants to monitor the expression patterns of multiple proteins and genes involved in flower primordial developmental processes. By monitoring the expression and polarity of PINFORMED1 (PIN1), the auxin efflux facilitator, and the expression of the auxin-responsive reporter DR5, we reveal stereotypical PIN1 polarity changes which, together with auxin induction experiments, suggest that cycles of auxin build-up and depletion accompany, and may direct, different stages of primordium development. Imaging of multiple GFP-protein fusions shows that these dynamics also correlate with the specification of primordial boundary domains, organ polarity axes, and the sites of floral meristem initiation. CONCLUSIONS: These results provide new insight into auxin transport dynamics during primordial positioning and suggest a role for auxin transport in influencing primordial cell type.

Arabidopsis↗
PubMed2005

Live imaging and morphometric analysis of embryonic development in the ascidian Ciona intestinalis.

The ascidian Ciona intestinalis is one of the model organisms of choice for comparative investigations of chordate development and for unraveling the molecular mechanisms underlying morphogenesis and cell fate specification. Taking advantage of the availability of various genetically encoded fluorescent proteins and of defined cis-regulatory elements, we combined transient transgenesis with laser scanning confocal imaging to acquire and quantitate 3D time-lapse data from living Ciona embryos. We used Ciona tissue-specific enhancers to drive expression of spectrally distinct fluorescent protein reporters to label and simultaneously visualize axially and paraxially positioned mesodermal derivatives, as well as neural precursors in individual embryos. We observed morphogenetic movements, without perturbing development, from the early gastrula throughout the larval stage, including gastrulation, neurulation, convergent extension of the presumptive notochord, and tail elongation. These multidimensional data allowed us to establish a reference system of metrics to quantify key developmental events including blastopore closure and muscle extension. The approach we describe can be used to document morphogenetic cell and tissue rearrangements in living embryos and paves the way for a live digitized anatomical atlas of Ciona.

Animals↗