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PubMed2006

Nanoscale controlled self-assembled monolayers and quantum dots.

Self-assembled monolayer (SAM) and fluorescent quantum dots (QDs) share common ground as emerging tools for nanoscale observation of biological interactions. SAMs provide excellent means of controlling the surface characteristics through individually tailored and engineered building blocks. SAMs on various surfaces have demonstrated clear advantages over uncontrolled multilayer films in fabricating electrochemical sensor, optical sensor, chemical biosensor, and atomic force microscopy. Similarly, QDs have advantages over organic fluorophores in long-term and real-time optical imaging of biological specimens. QDs conjugated with various biomolecules have been successfully applied to bioimaging, biosensing and cell encoding.

Animals↗
PubMed2004

Prediction of three-dimensional fractal dimensions using the two-dimensional properties of fractal aggregates.

Fractal dimension analysis using an optical imaging analysis technique is a powerful tool in obtaining morphological information of particulate aggregates formed in coagulation processes. However, as image analysis uses two-dimensional projected images of the aggregates, it is only applicable to one and two-dimensional fractal analyses. In this study, three-dimensional fractal dimensions are estimated from image analysis by characterizing relationships between three-dimensional fractal dimensions (D(3)) and one (D(1)) and two-dimensional fractal dimensions (D(2) and D(pf)). The characterization of these fractal dimensions were achieved by creating populations of aggregates based on the pre-defined radius of gyration while varying the number of primary particles in an aggregate and three-dimensional fractal dimensions. Approximately 2000 simulated aggregates were grouped into 33 populations based on the radius of gyration of each aggregate class. Each population included from 15 to 115 aggregates and the number of primary particles in an aggregate varied from 10 to 1000. Characterization of the fractal dimensions demonstrated that the one-dimensional fractal dimensions could not be used to estimate two- and three-dimensional fractal dimensions. However, two-dimensional fractal dimensions obtained statistically, well-characterized relationships with aggregates of a three-dimensional fractal characterization. Three-dimensional fractal dimensions obtained in this study were compared with previously published experimental values where both two-dimensional fractal and three-dimensional fractal data were given. In the case of inorganic aggregates, when experimentally obtained three-dimensional fractal dimensions were 1.75, 1.86, 1.83+/-0.07, 2.24+/-0.22, and 1.72+/-0.13, computed three-dimensional fractal dimensions using two-dimensional fractal dimensions were 1.75, 1.76, 1.77+/-0.04, 2.11+/-0.09, and 1.76+/-0.03, respectively. However, when primary particles were biological colloids, experimentally obtained three-dimensional fractal dimensions were 1.99+/-0.08 and 2.14+/-0.04, and computed values were both 1.79+/-0.08. Analysis of the three-dimensional fractal dimensions with the imaging analysis technique was comparable to the conventional methods of both light scattering and electrical sensing when primary particles are inorganic colloids.

Journal Article↗
PubMed2006

Area and layer patterning in the developing cerebral cortex.

Two anatomical patterns characterize the neocortex, and both are essential for normal cortical function. First, neocortex is divided into anatomically distinct and functionally specialized areas that form a species-specific map. Second, neocortex is composed of layers that organize cortical connectivity. Recent studies of layer and area development have used time-lapse microscopy to follow cortical cell division and migration, gene arrays to find layer- or area- specific regulatory genes, time- and region- specific manipulations of candidate genes, and optical imaging to compare area maps in wild type with genetically altered mice. New observations clarify the molecular and cellular mechanisms that generate each pattern, and stress the links between layer and area formation.

Animals↗
PubMed2006

From crawling to cognition: analyzing the dynamical interactions among populations of neurons.

By using multi-electrode arrays or optical imaging, investigators can now record from many individual neurons in various parts of nervous systems simultaneously while an animal performs sensory, motor or cognitive tasks. Given the large multidimensional datasets that are now routinely generated, it is often not obvious how to find meaningful results within the data. The analysis of neuronal-population recordings typically involves two steps: the extraction of relevant dynamics from neural data, and then use of the dynamics to classify and discriminate features of a stimulus or behavior. We focus on the application of techniques that emphasize interactions among the recorded neurons rather than using just the correlations between individual neurons and a perception or a behavior. An understanding of modern analysis techniques is crucially important for researchers interested in the co-varying activity among populations of neurons or even brain regions.

Action Potentials↗
PubMed2006

Insect olfactory memory in time and space.

Recent studies using functional optical imaging have revealed that cellular memory traces form in different areas of the insect brain after olfactory classical conditioning. These traces are revealed as increased calcium signals or synaptic release from defined neurons, and include a short-lived trace that forms immediately after conditioning in antennal lobe projection neurons, an early trace in dopaminergic neurons, and a medium-term trace in dorsal paired medial neurons. New molecular genetic tools have revealed that for normal behavioral memory performance, synaptic transmission from the mushroom body neurons is required only during retrieval, whereas synaptic transmission from dopaminergic neurons is required at the time of acquisition and synaptic transmission from dorsal paired medial neurons is required during the consolidation period. Such experimental results are helping to identify the types of neurons that participate in olfactory learning and when their participation is required. Olfactory learning often occurs alongside crossmodal interactions of sensory information from other modalities. Recent studies have revealed complex interactions between the olfactory and the visual senses that can occur during olfactory learning, including the facilitation of learning about subthreshold olfactory stimuli due to training with concurrent visual stimuli.

Animals↗
PubMed2005

Punishment prediction by dopaminergic neurons in Drosophila.

The temporal pairing of a neutral stimulus with a reinforcer (reward or punishment) can lead to classical conditioning, a simple form of learning in which the animal assigns a value (positive or negative) to the formerly neutral stimulus. Olfactory classical conditioning in Drosophila is a prime model for the analysis of the molecular and neuronal substrate of this type of learning and memory. Neuronal correlates of associative plasticity have been identified in several regions of the insect brain. In particular, the mushroom bodies have been shown to be necessary for aversive olfactory memory formation. However, little is known about which neurons mediate the reinforcing stimulus. Using functional optical imaging, we now show that dopaminergic projections to the mushroom-body lobes are weakly activated by odor stimuli but respond strongly to electric shocks. However, after one of two odors is paired several times with an electric shock, odor-evoked activity is significantly prolonged only for the "punished" odor. Whereas dopaminergic neurons mediate rewarding reinforcement in mammals, our data suggest a role for aversive reinforcement in Drosophila. However, the dopaminergic neurons' capability of mediating and predicting a reinforcing stimulus appears to be conserved between Drosophila and mammals.

Animals↗
PubMed2004

In vitro visualization of respiratory neuron activity in the newborn mouse ventral medulla.

To clarify the neuronal organization of the respiratory center of the mouse, we analyzed the spatio-temporal pattern of respiratory neuron activity in the ventral medulla of a newborn mouse preparation, using optical recordings. We also demonstrated optical images of the respiratory activity of two different lines of knock-out mice (Tlx3-/-, Pbx3-/-) that exhibit respiratory failure leading to neonatal death from dysfunction of central respiratory neuron activity. In the wild type mice, the respiratory neuron activity in the para-facial region of the rostral medulla appeared prior to inspiratory activity in the more caudal ventrolateral medulla. This rostral to caudal activity pattern was basically preserved in Tlx3-/- mice though the activity was more dispersed and weaker than in the wild type mice. Such an activity pattern was not clearly detected in Pbx3-/- mouse preparations. The difference in the spatio-temporal pattern between Tlx3-/- and Pbx3-/- suggests different levels of functional disorder of the respiratory center.

Animals↗
PubMed2005

Corneal NF-kappaB activity is necessary for the retention of transparency in the cornea of UV-B-exposed transgenic reporter mice.

To determine the dynamics of Nuclear Factor-kappaB (NF-kappaB) in murine corneal pathology and the role of NF-kappaB in maintaining corneal clarity after ultraviolet B radiation insult, transgenic mice containing NF-kappaB-luciferase reporter were exposed to LPS (bacterial lipopolysaccharide), TNF-alpha (Tumor Necrosis Factor-alpha) or 4 kJ m(-2) UV-B radiation. NF-kappaB decoy oligonucleotides were also administered in some of the UV-B experiments. Following various exposure times, the mice were sacrificed and whole eyes or corneal tissues were obtained. Whole eyes were examined for scattering using a point-source optical imaging technique. Tissue homogenates were examined for luciferase activity using a luminometer. TNF-alpha and LPS-injected NF-kappaB-luciferase transgenic mice demonstrated 3-10-fold increases in cornea NF-kappaB with peak activities at 4 and 6 hr post-injection, respectively. Mice exposed to 4 kJ m(-2) UV-B exhibited a 3-fold increase in NF-kappaB activity 4 hr post-exposure. The administration of NF-kappaB-decoy oligonucleotides to mice had the effect of reducing UV-B-induced NF-kappaB activity in the cornea and significantly increasing the amount of light scattering in UV-B exposed corneas 7 days post-UV-B exposure when compared to sham injected mice. These results indicate that NF-kappaB is activated in cornea in pathologies that involves increased plasma levels of LPS and TNF-alpha, as well as direct UV-B exposure, and suggest that NF-kappaB activation play an essential part in the corneal healing process.

Animals↗
PubMed2006

Quantifying retinal nerve fiber layer thickness in whole-mounted retina.

In order to relate optical measurement of the retinal nerve fiber layer (RNFL) to the underlying structure, one must have accurate values for RNFL thickness at the locations measured optically. The purpose of this study was to develop a method for measuring RNFL thickness at any location on retinal tissue previously studied by other optical imaging. The method developed used confocal laser scanning microscopy (cLSM) to provide both en face and cross-sectional images of a whole-mounted retina. Isolated rat retina was fixed with 3% glutaraldehyde. Nerve fiber bundles were identified by using phalloidin to label F-actin and ganglion cell bodies were identified by DAPI fluorescent counterstain of nuclei. The flat-mounted retina was examined by cLSM. 2-D images were collected through the retina to a depth at least covering the ganglion cell layer. The images were stacked to reconstruct cross-sectional images of the measured retina. Thickness of nerve fiber bundles was measured on these synthesized cross sections and compared with the measurement from conventional histologic sections. The en face image displayed individual nerve fiber bundles and ganglion cells between bundles as different colors. Blood vessels, which also bound phalloidin, were easily distinguished from nerve fiber bundles. The en face image displayed the same pattern of nerve fiber bundles as seen in imaging measurements and simplified the identification of corresponding areas in the two modalities. The cross-sectional images provided thickness measurements of the RNFL over the entire field-of-view, not just at the points represented by the conventional histologic section, resulting in a large increase in available data.

Actins↗
PubMed2006

Dynamic behavior of individual cells in developing organotypic brain slices revealed by the photoconvertable protein Kaede.

In recent years, advances in optical imaging methods have facilitated the visualization of events in the developing cortex. In particular, the introduction of DNA encoding fluorescent protein into cells of the embryonic brain allows the visualization of progenitor cells; slice preparations of the cortex then allow the monitoring of the behavior of transfected cells in the context of the living cerebral wall by time-lapse microscopy. Such approaches have provided substantial information about the patterns of neuronal migration. However, as these techniques label large numbers of cells in the ventricular zone (VZ), it is difficult to follow individual cell shape changes or cell behaviors within the VZ, where neuron production and initial migration take place. Here, we report a unique method using the photoconvertable fluorescent protein Kaede, which emits green fluorescence and shifts to emitting red fluorescence upon radiation with UV. Using this method, we were able to follow the behavior of a particular pair of daughter cells among neighboring Kaede-positive cells in the SVZ of mouse brain slices. The spindle shape progenitor divided into two multipolar-shaped daughter cells. The cell-cell borders of daughter cells were clearly visualized, and easily describe the position and distance between two or more cells. The photoconvertable property of Kaede offers a powerful cell marking tool to identify the precise morphology and migratory behaviors of individual cells within living cortical slices.

Animals↗
PubMed2004

Nuclear organization and the control of HIV-1 transcription.

The regulation of transcription of the human immunodeficiency virus (HIV) is a complex event of significant pathological relevance, which recapitulates general concepts of cellular transcription with some peculiarities. The viral promoter is embedded in a chromatin structure that exerts powerful repression on transcription; activation of gene expression relies on the combined activity of a series of cellular factors that respond to different external stimuli, and on the function of a single viral regulatory protein, the Tat transactivator. Transcriptional activation is consequent to both chromatin remodeling and to the recruitment of elongation-competent RNA polymerase II complexes onto the integrated promoter, two events that require the coordinate, but transient, assembly of different protein complexes. Application of optical imaging techniques now allows us to appreciate the spatial and temporal evolvement of these reactions in vivo. The picture that is emerging is not only descriptive, but also relevant to the understanding of the regulation of the process. In particular, it appears that the confinement of biomolecules within specific subcellular compartments represents a way to control and coordinate the assembly of functional complexes that regulate viral gene expression.

Cell Nucleus↗
PubMed2005

Hibernator Citellus undulatus maintains safe cardiac conduction and is protected against tachyarrhythmias during extreme hypothermia: possible role of Cx43 and Cx45 up-regulation.

BACKGROUND: Most mammals experience cardiac arrest during hypothermia. In contrast, hibernators remain in sinus rhythm even at body temperatures of 0 degrees C. OBJECTIVES: The purpose of this study was to quantify electrical activity and connexin expression in the heart of hibernating Siberian ground squirrel Citellus undulatus. METHODS: Optical imaging and microelectrode recordings were conducted in Langendorff-perfused hearts and isolated papillary muscles of summer active (SA, n = 19), winter hibernating (WH, n = 21), interbout arousal (IBA, n = 12), and winter active (WA, n = 3) ground squirrels and rabbits (n = 14) at temperatures from +37 degrees C to +3 degrees C. RESULTS: All studied SA and WH hearts maintained spontaneous sinus rhythm, safe propagation through the entire conduction system, and normal pattern of ventricular excitation at all temperatures. However, three of the seven IBA and all rabbit hearts lost excitability at 10 degrees C +/- 1 degrees C and 12 degrees C +/- 1 degrees C, respectively. In WH, SA, and IBA ground squirrels, temperature reduction from 37 degrees C to 3 degrees C resulted in a 10-fold slowing of ventricular conduction velocity and increased excitation threshold. At any temperature, WH ventricles had faster conduction velocity and lower excitation threshold compared with SA and IBA. Immunolabeling demonstrated that connexin43 (Cx43) was significantly up-regulated in WH and WA compared with SA myocardium: Cx43 area density was 12.4 +/- 1.3, 15.0 +/- 3.0 and 8.6 +/- 1.1 microm(2)/1,000 microm(2), respectively. Moreover, Cx45 was expressed in the WH but not in the SA or WA ventricles. CONCLUSION: Hibernator Citellus undulatus has evolved to maintain safe conduction at extreme hypothermia via up-regulation of Cx43 and Cx45 in order to protect the heart against arrhythmia associated with hypothermia.

Animals↗
PubMed2006

Characterization of the relationship between preshock state and virtual electrode polarization-induced propagated graded responses resulting in arrhythmia induction.

BACKGROUND: Studies have demonstrated that failed defibrillation shocks often are followed by an electrically quiescent period (isoelectric window); however, the underlying mechanisms remain incompletely understood. We recently suggested a new mechanism termed "virtual electrode polarization-induced propagated graded responses" (VEPiPGRs) that might play a role in the origin of the global postshock activation following the isoelectric window. OBJECTIVES: The purpose of this study to elucidate the circumstances under which VEPiPGR activations originate for shocks given to paced right ventricular preparations. Specifically, we examined the dependence of VEPiPGRs on coupling interval (CI) and shock polarity and whether VEPiPGRs emerge preferentially on the epicardium or the endocardium. METHODS: Simultaneous endocardial and epicardial activity in isolated right ventricular preparations (n = 4) was imaged optically following shocks of strength +/-5A. All VEPiPGRs were analyzed, and the time T from shock end to activation onset was recorded (isoelectric window is the smallest T among activations that propagated globally). RESULTS: VEPiPGR activations occurred for CIs in the range from 80 to 150 ms. Average duration of T was 64.5 +/- 18.15 ms, with T decreasing as CI increased (Tmax = 82 ms, Tmin = 46 ms, linear-fit slope = -0.675). The average earliest CI at which cathodal (+5A) shocks resulted in VEPiPGRs was 87 ms compared with 116 ms for anodal (-5A) shocks. All VEPiPGR activations emerged first on the epicardium in a focal pattern, and all induced ventricular fibrillation. CONCLUSION: The global activation that terminates the isoelectric window could result from VEPiPGRs that find an exit pathway. VEPiPGRs originate at the sites of maximum action potential abbreviation by the shock, always on the epicardium for the preparation used here.

Action Potentials↗
PubMed2006

Characterization of proteinase activation dynamics by capillary electrophoresis conjugating with fluorescent protein-based probe.

In this paper, a novel strategy was reported to characterize dynamics of proteinase activation based on capillary electrophoresis (CE), using caspase-2 as the model system. A fusion protein conjugating an amino acid sequence VDVAD with two fluorescent proteins enhanced cyan fluorescence protein (ECFP) and red fluorescence protein (DsRed), ECFP-VDVAD-DsRed, was specially designed and expressed in HeLa cells as the substrate of proteinase caspase-2. In this substrate, the sequence VDVAD could be specifically recognized and cleaved by caspase-2 as soon as its activation was initiated with treatment of a certain dose of cisplatin to HeLa cells, which led to a break of the substrate into two fragments. Analyses of the cell lysates using CE in a time course of the apoptosis illustrated the dynamics of caspase-2 activation. It showed that the employment of fusion fluorescent protein greatly facilitated both CE separation and identification of the analytes. This result from cell colony by CE was compared with that from single cell achieved by optical imaging.

Electrophoresis, Capillary↗
PubMed2006

Electrical conductance study of theta-liquid bridges.

This work investigates the behavior of small liquid bridges that are formed between two horizontal supporting surfaces, aligned at the vertical direction. The contact lines of the liquid bridges are not edge-pinned but free to move across the supporting surfaces with the contact angle as a parameter (theta-bridges). An a.c. electrical conductance technique coupled with high resolution optical images is used to characterize the geometrical details of constant volume liquid bridges when their length is increased gradually until rupture. A mathematical framework is developed for the identification of the geometrical characteristics of theta-liquid bridges explicitly from conductance data. Theoretical predictions show good agreement with measurements for most of the bridge lengths (separation distance between supports) except close to the rupture point where the bridge is highly stretched. It is further shown that for short and moderate separation distances the present model can be used with confidence to determine the bridge volume and neck radius from the electrical signal.

Journal Article↗
PubMed2006

Population balance modeling of aggregation and breakage in turbulent Taylor-Couette flow.

An experimental and computational study of aggregation and breakage processes for fully destabilized polystyrene latex particles under turbulent-flow conditions in a Taylor-Couette apparatus is presented. To monitor the aggregation and breakage processes, an in situ optical imaging technique was used. Consequently, a computational study using a population balance model was carried out to test the various parameters in the aggregation and breakage models. Very good agreement was found between the time evolution of the cluster size distribution (CSD) calculated with the model and that obtained from experiment. In order to correctly model the left-hand side of the CSD (small clusters), it was necessary to use a highly unsymmetric fragment-distribution function for breakage. As another test of the model, measurements with different solid volume fractions were performed. Within the range of the solid volume fractions considered here, the steady-state CSD was not significantly affected. In order to correctly capture the right-hand side of the CSD (large aggregates) at the higher solid volume fraction, a modified aggregation rate prefactor was used in the population balance model.

Image Processing, Computer-Assisted↗
PubMed2004

Early diagnosis of osteoarthritis using cathepsin B sensitive near-infrared fluorescent probes.

OBJECTIVE: Osteoarthritis is currently diagnosed utilizing X-ray and MRI-techniques, both of which are based on the morphological changes of tissue. However, once changes are detected, the tissue has an irreversible defect. This study investigates early diagnosis of OA on a molecular basis using a recently developed cathepsin B sensitive near-infrared (NIR) fluorescent probe. METHOD: Twelve male nude mice were induced osteoarthritis by intra-articular injection of collagenase (1.0%, w/v) into the right knee joint. The left knee joint served as the negative control. The cathepsin B NIR probe is activated by arthritis-associated cathepsin B, thus resulting in the emission of an intensive NIR fluorescence signal which can be detected in vivo. NIR fluorescence signals were acquired on an optical imaging system using an excitation wavelength of 610-650 nm and an emission wavelength of 680-720 nm. RESULTS: Mild to moderate degenerative cartilage was observed 1 month after collagenase injection. NIR fluorescence imaging of mice showed approximately a 3-fold difference in signal intensity between osteoarthritic and normal joints 24 h after intravenous injection of the reporter probe. Immunohistochemical evaluation also revealed cathepsin B expression in the arthritic lesion of femorotibial joints, and not in the control contra-lateral knee joints. CONCLUSION: As the cathepsin B activatable NIR fluorescent imaging showed a significant difference between the osteoarthritic and normal joints, the cathepsin B activatable NIR fluorescent probe thus offers a potential new imaging technology for early OA diagnosis.

Animals↗
PubMed2006

Developing a structure-function relationship for anionic porphyrazines exhibiting selective anti-tumor activity.

The porphyrazines (pzs) are a class of porphyrin derivatives being studied for their use as optical imaging agents and photodynamic therapy (PDT) anti-tumor agents. A previous study revealed that the anionic pz, 18--of the form H2[pz(An;B4-n)], where A is [S(CH2)3CO2-], B is a fused beta',beta'-diisopropyloxy benzo group, with n=2 (trans)--selectively killed tumor cells, while analogous neutral and positively charged pzs lacked this property. In this report, we compare the properties of a suite of three H2[pz(An;B4-n)] pzs containing the same A and B groups as 18, but differing in their values of n: pzs 4 (n=4) and 11 (n=3), and 18 (n=2, trans) exhibit a progressive variation in charge due to the carboxylates, balance between hydrophobic/hydrophilic character, as well as a progressive variation in the singlet oxygen quantum yield (PhiDelta): PhiDelta (18)>PhiDelta (11)>PhiDelta (4). The biological activity of the pzs was tested in human lung carcinoma (A549) and SV40 transformed embryonic (WI-38 VA13) cell lines. Pzs 4 and 11 exhibited significant toxicity in both tumor and normal cells, while 18 showed selective anti-tumor cell activity in a dose-dependent manner. As the number of net negative charges decreased, the compounds became less toxic to normal cells, and the killing effect observed with these compounds was light independent. These observations indicate that the toxicity may have little to do with singlet oxygen quantum yields, but rather is more dependent on the net number of negative charges a pz contains. The study reported herein presents an example of how the porphyrazines can be easily modified to vary their biological behavior and specifically suggest that anionic porphyrazines pzs with lower n (fewer carboxylates, larger hydrophobic core) are more specific tumor killers, while those with larger n (increased net negative charge) are more potent tumor killers.

Antineoplastic Agents↗