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Results for “Fluorescence imaging”

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At least 253 records · Page 14Linked to original sources

PubMed1996

Identification of F-reticulocytes by two-stage fluorescence image cytometry.

Precise determination of reticulocytes (young red blood cells) containing fetal hemoglobin (Hb F), F-reticulocytes, is important for assessment of the efficacy of drugs such as hydroxyurea and butyrate in elevating the levels of Hb F in patients with sickle cell disease (SCD) and beta-thalassemia. We developed a reliable and easily applicable method for determining F-reticulocytes using fluorescence image cytometry. Reticulocytes were first identified by preparing a monolayer smear of blood stained by acridine orange. Images of reticulocytes and of all cells were obtained for a selected area on the smear. After removing the acridine orange, cells containing Hb F (F-cells) in the same area were then identified by immunofluorescence. Using images of F-cells, reticulocytes, and all cells for the same fields it was possible to identify F-reticulocytes. To assess the validity of our two-stage staining method, we compared our results with those obtained by traditional methods. There was significant correlation of our method with the conventional immunofluorescence staining method for F-cells (r2 = 0.99; slope = 0.99) and with the accepted brilliant cresyl blue method for reticulocytes (r2 = 0.97; slope = 0.96). Heretofore, the ability to determine F-reticulocyte levels has been limited to a small number of laboratories possessing special equipment and techniques. The method presented here should be of great interest to many basic science and clinical investigators involved in studies evaluating synthesis of Hb F.

Acridine Orange↗

PubMed2006

Visualization of dynamics of plant-pathogen interaction by novel combination of chlorophyll fluorescence imaging and statistical analysis: differential effects of virulent and avirulent strains of P. syringae and of oxylipins on A. thaliana.

Pathogen infection leads to defence induction as well as to changes in carbohydrate metabolism of plants. Salicylic acid and oxylipins are involved in the induction of defence, but it is not known if these signalling molecules also mediate changes in carbohydrate metabolism. In this study, the effect of application of salicylic acid and the oxylipins 12-oxo-phytodienoic acid (OPDA) and jasmonic acid on photosynthesis was investigated by kinetic chlorophyll fluorescence imaging and compared with the effects of infection by virulent and avirulent strains of Pseudomonas syringae. Both pathogen strains and OPDA caused a similar change in fluorescence parameters of leaves of Arabidopsis thaliana. The response to OPDA appeared faster compared with that to the pathogens and persisted only for a short time. Infiltration with jasmonic acid or salicylic acid did not lead to a localized and distinct fluorescence response of the plant. To capture the faint early symptoms of the plant response, a novel algorithm was applied identifying the unique fluorescence signature-the set of images that, when combined, yield the highest contrast between control and infected leaf segments. Unlike conventional fluorescence parameters, this non-biased approach indeed detected the infection as early as 6 h after inoculation with bacteria. It was posssible to identify distinct fluorescence signatures characterizing the early and late phases of the infection. Fluorescence signatures of both infection phases were found in leaves infiltrated with OPDA.

Arabidopsis↗

PubMed2005

Endogenous fluorescence imaging of somatosensory cortical activities after discrimination learning in rats.

Aerobic energy metabolism in the brain is reflected as changes in the green fluorescence of mitochondrial flavoproteins, and the activity-dependent changes in endogenous fluorescence are applicable for functional brain imaging. To understand the roles of cortical plasticity in discrimination learning, we used flavoprotein fluorescence imaging to visualize changes of neural activities in the rat primary somatosensory cortex (SI) after learning. Rats were trained to discriminate floor vibration at rewarded and unrewarded frequencies. After this discrimination learning was accomplished in 3-5 days, the rats were anesthetized with urethane (1.5 g/kg, i.p.), and neural responses were recorded in SI during flutter stimuli applied to the contralateral hindpaw. The fluorescence responses to the stimuli at unrewarded frequencies were selectively depressed in the trained rats, which had behaviorally neglected unrewarded stimuli. The depression of cortical responses was not observed in the rats trained with rewarded stimuli only. Therefore, the stimulus-specific depression in SI might explain a part of neural mechanisms underlying discrimination behavior. To reproduce the stimulus-specific depression of cortical responses in anesthetized rats, tetanic cortical stimulation was paired with flutter stimulation applied to the hindpaw. Selective depression of fluorescence responses or field potentials in SI was induced by the paired stimulation. Our findings suggest that some intracortical circuits in SI are specifically tuned to and modulated by unrewarded stimuli of a particular frequency while SI neurons are responsive to both of rewarded and unrewarded stimuli. The present results indicate the usefulness of flavoprotein fluorescence imaging for investigating somatosensory cortical plasticity after learning.

Animals↗

PubMedDate not supplied

Fluorescent image tracking velocimetry of the Nimbus AxiPump.

High shear rates and extended residence times causing hemolysis and platelet activation can develop in an assist pump or cannula when inferior flow conditions exist. The high volume output of a miniature axial flow pump presents challenges in avoiding these adverse conditions. To assess the hemodynamics within the continuous flow Nimbus Axi-Pump, vector flow fields inside a translucent inflow cannula and a modified 12 mm AxiPump were mapped. Fluorescent image tracking velocimetry was used to track the motion of neutrally buoyant fluorescent particles (30 microns) using pulsed laser light, high resolution video cameras, and computer image analysis. An acrylic pump housing and cannula were integrated into a mock circulatory loop filled with a Newtonian, optically clear blood analog fluid. The flow parameters were controlled to yield known, physiologic loading conditions, including varying degrees of pulsatility. Cannula flow visualization results exhibited critical recirculation patterns at the bend. These results will be used to further optimize the design of the inflow. Particle impact was seen at the pump inlet in the inducer region of the rotor. Very good attachment of flow from the rotor to stator was observed when the pump operated at normal operating speeds. Intermittent regurgitant flow fields were evident in the presence of increased pulsatility and low pump speed. These results have lead to improvements in impeller design and speed control criteria to avoid potential deleterious flows.

Blood Flow Velocity↗

PubMed2001

Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging.

Viral vectors based on lentiviruses, such as the human immunodeficiency virus, are able to transduce a broad spectrum of nondividing cells in vivo. This ability of lentiviral vectors makes them an attractive vehicle for gene transfer into the liver. In order to determine the requirements for efficient lentiviral gene transfer, we used a fluorescence imaging system, which allows the detection of cells and tissues that express fluorescent reporter genes (e.g., green fluorescence protein) in the living animal. We show that the latest generation of lentiviral vectors efficiently transduces the murine liver. Further analysis demonstrated that neither cell-cycle activation nor division of liver cells is a prerequisite for lentiviral gene transfer in vivo.

Animals↗

PubMed1997

Time-resolved fluorescence imaging in islet cell autoantibody quantitation.

The prodromal period of insulin-dependent diabetes mellitus (IDDM) is characterized by circulating islet cell autoantibodies (ICA) and other beta cell specific autoantibodies. Despite biochemical characterization of the major beta cell autoantigens insulin, glutamic acid decarboxylase and protein tyrosine phosphatase and development of the respective antibody assays, ICA has remained the standard in IDDM prediction. Conventional ICA quantitation using classic fluorochromes is prone to errors since fluorescence intensity is estimated subjectively using the human eye, which is also unable to differentiate specific signals from non-specific signals and autofluorescence. Using Eu(3+)-chelate labelled anti-human polyclonal IgG (decay time 1000 microseconds) as the secondary antibody in time-resolved fluorescence imaging (TRFI), the chelate and autofluorescence signals (typical decay time < 100 ns) are fully separated. The image is recorded using an optically gated cooled digital CCD camera. The specificity of the ICA signal is further improved by interactive analysis of the image. Signal detection is objective, the signal-to-background ratio improves, and ICA quantitation is possible using undiluted serum. Of 57 consecutive new-onset IDDM patients, 55 (96.5%) were ICA positive in the new assay while 51 (89.5%) were positive in the conventional assay suggesting that the sensitivity of TRFI exceeds that of the IAA, GAD65 and IA-2 autoantibody assays combined. For later comparisons, the stained slides may be stored in the light for years without any decrease in specific fluorescence.

Adolescent↗

PubMed2002

Fluorescence imaging of mobility shifts: an expression cloning method for identification of cell signaling targets.

There is a need for a simple global approach to identify signaling targets that are posttranslationally modified in response to physiologic or pathologic stimuli within living cells. Reported here is a simple method, fluorescence imaging of mobility shifts (FIMS), which relies on in-gel detection of cell-expressed green fluorescent protein fusion proteins undergoing electrophoretic mobility shifts. This detection method is applied to a small pool cDNA library screening protocol. The readout is essentially a differential display of posttranslational modifications. Unlike biochemical approaches to identifying signaling targets, the screen is performed in living cells using standard methods for transient transfection. This enables detection of intracellular targets modified in response to either molecularly defined stimuli, such as growth factors or drugs, or complex pathologic stimuli, such as oxidative stress or hypoglycemia. FIMS is rapid, sensitive, inexpensive, and nonradioactive and easily adapted to automated high throughput methods, including capillary electrophoresis. The technique is sufficiently sensitive to easily detect fluorescent proteins expressed in a single well in 384-well format. FIMS is applicable to traditional cDNA library screening, but the method will be especially attractive for screening preselected collections of autofluorescent fusion proteins. A bonus of the technique is that examination of transfected cells by fluorescence microscopy provides immediate information about intracellular localization and stimulus-induced translocation of putative targets. We illustrate the utility of the technique with pilot screens for apoptotic and mitogenic targets modified by staurosporine and serum stimulation, respectively.

Animals↗

PubMed2005

Fluorescence imaging for real-time monitoring of high-intensity focused ultrasound cardiac ablation.

Side effects and limitations of radio-frequency ablation of cardiac arrhythmias prompted search for alternative energy sources and means of their application. High-intensity focused ultrasound (HIFU) is becoming an increasingly attractive modality for ablation because of its unique ability for non-invasive or minimally invasive, non-contact focal ablation in 3D volume without affecting intervening and surrounding cells. The purpose of this study is to develop a real-time monitoring technique to elucidate HIFU-induced modifications of electrical conduction in cardiac tissues and to investigate the HIFU cardiac ablation process to help to achieve optimal HIFU ablation outcome. We conducted experimental studies applying HIFU at 4.23 MHz to ablate the atrio-ventricular (AV) node and ventricular tissue of Langendorff-perfused rabbit hearts. We employed fluorescent voltage-sensitive dye imaging and surface electrodes to monitor the electrical conduction activity induced by HIFU application in real time. In ventricular epicardium HIFU ablation, fluorescent imaging revealed gradual reduction of the plateau phase and amplitude of the action potential. Subsequently, conduction block and cell death were observed at the site of ablation. When HIFU was applied to the AV node, fluorescent imaging and electrograms revealed the development of the AV block. The study establishes that real-time fluorescent imaging provides novel monitoring and assessment to study HIFU cardiac ablation, which may be able to provide improved understanding of HIFU cardiac ablation process and mechanism useful for development of successful clinical applications.

Animals↗

PubMed1999

Fluorescence imaging of GABAA receptor-mediated intracellular [Cl-] in P19-N cells reveals unique pharmacological properties.

This study describes the pharmacological properties of GABAA receptors expressed in P19-N cells using fluorescence imaging of intracellular chloride with 6-methoxy-N-ethylquinolinium iodide (MEQ). We show that application of the GABA agonist, muscimol (10-200 microM), produces time- and concentration-dependent increases in intracellular [Cl-] that are blocked by bicuculline. Diazepam (10 microM) and pentobarbital (1 mM) potentiate muscimol-stimulation. These receptors exhibit novel pharmacological properties. The neurosteroid, 3alpha-hydroxy-5alpha-pregnane-20-one (1-10 microM) exhibited weak potency in enhancement of muscimol-stimulation. Ethanol (50 and 100 mM) exhibited high efficacy on muscimol responses, a 4- to 5-fold potentiation, respectively, of muscimol (10 microM) alone. GABA and muscimol allosterically modulated specific binding of [3H]flunitrazepam to differentiated P19 cells. Modulation of GABAA receptor mediated increases in intracellular [Cl-] demonstrated stability in response magnitude from 7 to 15 days following removal of retinoic acid. In concert, GABAA receptor subunit mRNA and protein expression patterns in these neuron-like cells were stable over the same period. Using RT-PCR we determined that differentiated P19 cells lack gamma1, gamma2L, alpha6 and delta subunit mRNAs while expressing alpha1, alpha2, alpha3, alpha4, alpha5, beta1, beta2, beta3, gamma2S and gamma3. Furthermore, subunit specific antibody immunocytochemical labeling of cells with a neuronal morphology indicated the presence of alpha1, alpha2, alpha4, and gamma2 subunits (the only subunits tested). Therefore, P19-N cells should prove useful to researchers in need of a model cell culture system in which to study function and regulation of neuronal GABAA receptors.

Animals↗

PubMed1989

Simultaneous measurements of cytosolic calcium and secretion in single bovine adrenal chromaffin cells by fluorescent imaging of fura-2 in cocultured cells.

The cytosolic free calcium concentration ([Ca2+]i) and exocytosis of chromaffin granules were measured simultaneously from single, intact bovine adrenal chromaffin cells using a novel technique involving fluorescent imaging of cocultured cells. Chromaffin cell [Ca2+]i was monitored with fura-2. To simultaneously follow catecholamine secretion, the cells were cocultured with fura-2-loaded NIH-3T3t cells, a cell line chosen because of their irresponsiveness to chromaffin cell secretagogues but their large Ca2+ response to ATP, which is coreleased with catecholamine from the chromaffin cells. In response to the depolarizing stimulus nicotine (a potent secretagogue), chromaffin cell [Ca2+]i increased rapidly. At the peak of the response, [Ca2+]i was evenly distributed throughout the cell. This elevation in [Ca2+]i was followed by a secretory response which originated from the entire surface of the cell. In response to the inositol 1,4,5-trisphosphate (InsP3)-mobilizing agonist angiotensin II (a weak secretagogue), three different responses were observed. Approximately 30% of chromaffin cells showed no rise in [Ca2+]i and did not secrete. About 45% of the cells responded with a large (greater than 200 nM), transient elevation in [Ca2+]i and no detectable secretory response. The rise in [Ca2+]i was nonuniform, such that peak [Ca2+]i was often recorded only in one pole of the cell. And finally, approximately 25% of cells responded with a similar Ca2+-transient to that described above, but also gave a secretory response. In these cases secretion was polarized, being confined to the pole of the cell in which the rise in [Ca2+]i was greatest.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed2004

Fluorescent imaging of acute mercuric chloride exposure on cultured human kidney tubular epithelial cells.

BACKGROUND: Imaging of intracellular mercuric ion is necessary for mechanism of renal toxicity of exposure to HgCl2. The distribution of Hg2+ inside a living cell, however, is still invisible due to the lack of high selective and sensitive fluorescent molecular probe for Hg2+. METHODS: A new fluorescent probe, EPNP, was applied to the cultured cells of human kidney proximal tubular epithelial cell line (HKC) in the presence of HgCl2 and some other bivalent ions. The relative fluorescence intensity of EPNP was measured and fluorescence images were taken by laser scanning confocal microscope. RESULTS: Results showed it led to an Hg2+ concentration- and time-dependent increase in fluorescence intensity, and responded weakly for some other heavy and transition metal ions. It could be seen during acute exposure on HKC cells, Hg2+ locate perinuclear, and on nuclear membrane, which was beyond what one knew before. CONCLUSION: EPNP is a real-time and on-line probe for imaging Hg2+ in a living cell due to its high selectivity and sensitivity for Hg2+ and slow bleaching/fading. Both the probe and the new results about the distribution of intracellular Hg2+ may be helpful for relevant biologic research.

Cells, Cultured↗

PubMed2004

Fluorescence imaging of two-photon linear dichroism: cholesterol depletion disrupts molecular orientation in cell membranes.

The plasma membrane of cells is an ordered environment, giving rise to anisotropic orientation and restricted motion of molecules and proteins residing in the membrane. At the same time as being an organized matrix of defined structure, the cell membrane is heterogeneous and dynamic. Here we present a method where we use fluorescence imaging of linear dichroism to measure the orientation of molecules relative to the cell membrane. By detecting linear dichroism as well as fluorescence anisotropy, the orientation parameters are separated from dynamic properties such as rotational diffusion and homo energy transfer (energy migration). The sensitivity of the technique is enhanced by using two-photon excitation for higher photo-selection compared to single photon excitation. We show here that we can accurately image lipid organization in whole cell membranes and in delicate structures such as membrane nanotubes connecting two cells. The speed of our wide-field imaging system makes it possible to image changes in orientation and anisotropy occurring on a subsecond timescale. This is demonstrated by time-lapse studies showing that cholesterol depletion rapidly disrupts the orientation of a fluorophore located within the hydrophobic region of the cell membrane but not of a surface bound probe. This is consistent with cholesterol having an important role in stabilizing and ordering the lipid tails within the plasma membrane.

Algorithms↗

PubMed1998

Fluorescence imaging of light absorption for axial-beam geometry in capillary electrophoresis.

A new method for investigation of axial-beam absorption detection for improved detection limits in microcolumn separations is reported. The method is based on fluorescence imaging of light absorption along a separation capillary. The probing UV light is introduced at one end of the capillary and shows an exponential fall-off along the capillary. As the UV light propagates through the sample peaks, an additional loss in intensity will be observed. In order to view the absorption profile along the capillary, a background fluorophore is added to the buffer. A charge-coupled device (CCD) detector and imaging optics are placed beside the capillary to view the capillary in a direction perpendicular to the capillary. Signal integration is employed for consecutive exposures as well as for neighboring detector pixels in order to increase the signal-to-noise ratio. Measurements for stilbene 3 with sulforhodamine B as a background fluorophore are presented. The characteristics of the detection method and potential improvements are discussed.

Electrophoresis, Capillary↗

PubMed2005

Simultaneous measurement of water volume and pH in single cells using BCECF and fluorescence imaging microscopy.

Regulation and maintenance of cell water volume and intracellular pH (pHi) are vital functions that are interdependent; cell volume regulation affects, and is in turn affected by, changes in pHi. Disruption of either function underlies various pathologies. To study the interaction and kinetics of these two mechanisms, we developed and validated a quantitative fluorescence imaging microscopy method to measure simultaneous changes in pHi and volume in single cells loaded with the fluorescent probe BCECF. CWV is measured at the excitation isosbestic wavelength, whereas pHi is determined ratiometrically. The method has a time resolution of <1 s and sensitivity to osmotic changes of approximately 1%. It can be applied in real time to virtually any cell type attached to a coverslip, independently of cellular shape and geometry. Calibration procedures and algorithms developed to transform fluorescence signals into changes in cell water volume (CWV) and examples of applications are presented.

Algorithms↗

PubMed2003

Fluorescence imaging analysis of upstream regulators and downstream targets of STAT3 in melanoma precursor lesions obtained from patients before and after systemic low-dose interferon-alpha treatment.

Atypical nevi are the precursors and risk markers of melanoma. Apart from persistently monitoring these nevocytic lesions and resecting them at the earliest signs of clinical changes, there is as yet no systemic clinical treatment available to interfere with their progression to melanoma. To explore clinical treatments that might interfere with and possibly prevent atypical nevus progression, a previous study documented that 3 months systemic low-dose interferon-alpha (IFN-alpha) treatment of patients with a clinical history of melanoma and numerous atypical nevi, led to inactivation of the STAT1 and STAT3 transcription factors in atypical nevi. Based upon this finding, we initiated a second study to determine whether systemic low-dose IFN-alpha treatment also impairs the expression of upstream regulators and downstream targets of STAT1 and STAT3 in atypical nevi. Using cyanine dye-conjugated antibodies, fluorescence imaging analysis revealed expression of JAK2, JNK1, AKT1, NF-kappa B, and IFN-alpha/beta receptor in benign and atypical nevi, and early- and advanced-stage melanomas. To determine possible changes in the level of expression of these molecules in atypical nevi, excised before and after 3 months of systemic low-dose IFN-alpha treatment, newly designed optical imaging software was used to quantitate the captured fluorescent hybridization signals on a cell-by-cell basis and across an entire nevus section. The results of this analysis did not provide evidence that systemic low-dose IFN-alpha treatment alters the level of expression of upstream regulators or downstream targets of STAT1 and STAT3.

DNA-Binding Proteins↗

PubMed2004

Increased brain tumor resection using fluorescence image guidance in a preclinical model.

BACKGROUND AND OBJECTIVES: Fluorescence image-guided brain tumor resection is thought to assist neurosurgeons by visualizing those tumor margins that merge imperceptibly into normal brain tissue and, hence, are difficult to identify. We compared resection completeness and residual tumor, determined by histopathology, after white light resection (WLR) using an operating microscope versus additional fluorescence guided resection (FGR). STUDY DESIGN/MATERIALS AND METHODS: We employed an intracranial VX2 tumor in a preclinical rabbit model and a fluorescence imaging/spectroscopy system, exciting and detecting the fluorescence of protoporphyrin IX (PpIX) induced endogenously by administering 5-aminolevulinic acid (ALA) at 4 hours before surgery. RESULTS: Using FGR in addition to WLR significantly increased resection completeness by a factor 1.4 from 68+/-38 to 98+/-3.5%, and decreased the amount of residual tumor post-resection by a factor 16 from 32+/-38 to 2.0+/-3.5% of the initial tumor volume. CONCLUSIONS: Additional FGR increased completeness of resection and enabled more consistent resections between cases.

Aminolevulinic Acid↗

PubMed1996

Fluorescent imaging of GUS activity and RT-PCR analysis of gene expression in the shoot apical meristem.

The use of promoter-reporter gene constructs in transgenic plants is a powerful tool in the analysis of gene expression which can, however, be limited in the resolution of small structures, such as the apical meristem. This paper reports on a fluorescent imaging technique for the analysis of GUS reporter gene expression to cellular resolution in the apical meristem of tomato. Using this technique in combination with an RT-PCR analysis of RBS gene-specific transcript levels, it is shown that: 5' upstream sequences of RBCS genes are sufficient to mimic the pattern of transcripts revealed by in situ hybridisation (no expression in the apical meristem, high expression in the initiated leaf primordia); the genes RBCS2, RBCS3A and RBCS3B are transcriptionally activated upon primordium initiation with transcripts for RBCS1 and RBCS3C accumulating later in leaf development; and that RBCS promoter activity cannot be induced in the apical meristem by light, an environmental signal which elevates RBCS transcript level in other aerial parts of the plant. These data provide a detailed picture of the complex transcriptional events occurring on leaf initiation and the establishment of the photosynthetic machinery; they describe two complementary techniques which allow the analysis of such complex events at the tissue and cellular level; and they characterize an in vivo assay system which can be used to analyse the factors involved in the initiation and maintenance of gene expression patterns in the apical meristem.

Gene Expression Regulation, Plant↗

PubMed1996

Paternity testing by the detection of D1S80 VNTR using fluorescence image analyzer (Dualcolour system).

An improved method for DNA polymorphism typing of D1S80 VNTR locus and its application to paternity testing are described. For accurate estimation of the length of polymorphic DNA fragments, the size marker was labeled with fluorescence different from that of PCR primers, and co-electrophoresed as an internal standard. The dualcolour system of fluorescence image analyzer was used to detect the fragments and determine their size. This internal marker method could successfully overcome the problems of band pattern distortion and tailing, besides it allows easy and accurate interpretation of the DNA types. Our results indicate that the internal marker method is much more accurate than the method of using size marker in gel, even with the presence of distortion or tailing of the band patterns. Family studies applying this method showed complete agreement between the observed and predicted types.

Electrophoresis, Polyacrylamide Gel↗