Imaging membrane potential with voltage-sensitive dyes

Membrane potential can be measured optically using a variety of molecular probes. These measurements can be useful in studying function at the level of an individual cell, for determining how groups of neurons generate a behavior, and for studying the correlated behavior of populations of neurons. Examples of the three kinds of measurements are presented. The signals obtained from these measurements are generally small. Methodological considerations necessary to optimize the resulting signal-to-noise ratio are discussed.     

Solid phase gene extraction isolates mRNA at high spatial and temporal resolution

Rapid, localized changes in gene expression require mRNA extraction at high temporal and spatial resolution. Current small-scale mRNA extractions depend on the removal of the cells/tissue from an organism or preserved specimens. What these methods have in common is that they are destructive and do not distinguish between genomic DNA and RNA. Therefore, extracted (m)RNA is typically contaminated by extracted cytoplasm, nuclear DNA, or other compounds, and the required purification leads to loss of especially low-abundant mRNA. The need to repeatedly remove mRNA from living material has led to the development of solid phase gene extraction (SPGE). SPGE sampling can be achieved using gene-specific or generic sequences and is not species-specific. Here we demonstrate the versatility and validity of this novel RNA extraction by simultaneously profiling nanos and bicoid mRNA in individual Drosophila eggs. The SPGE technique detects previously described distribution profiles of nanos and bicoid. Its low impact is underscored by the normal development of repeatedly sampled eggs. In our study, quantification of actin mRNA in germinating flax seeds linked gene expression to distinct developmental processes. These data demonstrate the universality of SPGE as a simple generic, analytical, and diagnostic procedure.     

Imaging activity of neuronal populations with new long-wavelength voltage-sensitive dyes

We have assessed the utility of five new long-wavelength fluorescent voltage-sensitive dyes (VSD) for imaging the activity of populations of neurons in mouse brain slices. Although all the five were capable of detecting activity resulting from activation of the Schaffer collateral-CA1 pyramidal cell synapse, they differed significantly in their properties, most notably in the signal-to-noise ratio of the changes in dye fluorescence associated with neuronal activity. Two of these dyes, Di-2-ANBDQPQ and Di-1-APEFEQPQ, should prove particularly useful for imaging activity in brain tissue and for combining VSD imaging with the control of neuronal activity via light-activated proteins such as channelrhodopsin-2 and halorhodopsin.     

Imaging in vivo redox status in high spatial resolution with OMRI

Abstract

Redox-reactions are playing a significant role in regulation of homeostasis of organism. Disorder of the redox-status is related with the onset and/or propagation of oxidative diseases such as lifestyle-related diseases, including cancers and cardiac diseases, etc. In vivo imaging of redox-status is thereby important in the analysis of mechanisms of oxidative diseases and developments of new medicines for the diseases. Aminoxyl radicals are redox-sensitive reporter molecules, which lose their paramagnetic moiety by reactions of free radicals or reducing compounds. Electron spin resonance (ESR) technique has been used to measure the molecules in vivo. In vivo spatial resolution in ESR imaging is in the range of a few millimeters and is not sufficient for the detailed diagnosis of disease models. Overhauser enhanced MRI (OMRI) is an emerging free radical imaging technique, which utilised electron–proton coupling to image the distribution of free radicals. In vivo imaging of redox-status is applicable with OMRI/aminoxyl radical technique. The detailed imaging analysis was demonstrated in oxidative diseases, such as tumour-bearing, neurodegeneration or gastric ulcer models. The OMRI/aminoxyl radical technique has a large potential as a diagnostic system for biomedical applications in the future.     

Imaging in vivo redox status in high spatial resolution with OMRI

Redox-reactions are playing a significant role in regulation of homeostasis of organism. Disorder of the redox-status is related with the onset and/or propagation of oxidative diseases such as lifestyle-related diseases, including cancers and cardiac diseases, etc. In vivo imaging of redox-status is thereby important in the analysis of mechanisms of oxidative diseases and developments of new medicines for the diseases. Aminoxyl radicals are redox-sensitive reporter molecules, which lose their paramagnetic moiety by reactions of free radicals or reducing compounds. Electron spin resonance (ESR) technique has been used to measure the molecules in vivo. In vivo spatial resolution in ESR imaging is in the range of a few millimeters and is not sufficient for the detailed diagnosis of disease models. Overhauser enhanced MRI (OMRI) is an emerging free radical imaging technique, which utilised electron-proton coupling to image the distribution of free radicals. In vivo imaging of redox-status is applicable with OMRI/aminoxyl radical technique. The detailed imaging analysis was demonstrated in oxidative diseases, such as tumour-bearing, neurodegeneration or gastric ulcer models. The OMRI/aminoxyl radical technique has a large potential as a diagnostic system for biomedical applications in the future.     

A simple method for high temporal resolution calcium imaging with dual excitation dyes.

Calcium-sensitive dual excitation dyes, such as fura-2, are now widely used to measure the free calcium concentration ([Ca2+]) in living cells. Preferentially, [Ca2+] is calculated in a ratiometric manner, but if calcium images need to be acquired at high temporal resolution, a potential drawback of ratiometry is that it requires equally fast switching of the excitation light between two wavelengths. To circumvent continuous excitation switching, some investigators have devised methods for calculating [Ca2+] from single-wavelength measurements combined with the acquisition of a single ratiometric pair of fluorescence images at the start of the recording. These methods, however, are based on the assumption that the concentration of the dye does not change during the experiment, a condition that is often not fulfilled. We describe here a method of single-wavelength calcium imaging, in which the dye concentration is estimated from ratiometric fluorescence image pairs acquired at regular intervals during the recording period, that furthermore includes a correction for the changing dye concentration in the calculation of [Ca2+].     

Two-dimensional spectrophotometry with high spatial and temporal resolution by digital video techniques and powerful computers

A two-dimensional spectrophotometer having a spatial raster resolution of 512 X 512 picture elements with 256 grey levels and a time resolution of 30 images per min is assembled by combination of digital video techniques and a powerful computer system. The instrument is applied to the analysis of pattern formation processes in cytoplasmic media.     

Kinetics of cardiac RyR channel gating studied at high temporal resolution

Measurements of ryanodine receptor (RyR) activity during dynamic changes of calcium concentration have suggested that RyR has at least four calcium binding sites, and that activation transpires as an increase in the activity within the high open probability H-mode. Binding of several Ca2+ ions within the H-mode should manifest itself in the steady-state RyR activity by the presence of multiple closed times. However, previously only two closed times were detected in the H-mode of RyR activity. Here we recorded steady-state activity of single cardiac RyRs with high temporal resolution and compared it to data simulated under the same conditions using our previously published model of RyR gating. At a 10 kHz resolution, the closed time histograms of both experimental and simulated data had three exponential components. The closed times of simulated data were not significantly different from those obtained experimentally. After filtering at 2 kHz, only two exponential closed time components with time constants not significantly different from those previously published could be detected in both experimental and simulated records. The conformity of the steady-state experimental data to the model derived from the dynamic data provides further support for the idea that RyRs need binding of multiple Ca2+ ions to open.     

Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes

The usefulness of a new voltage-sensitive fluorescent dye, the membrane permeant negatively charged oxonol dye diBA-C4-(3)-, was evaluated by measuring the membrane potentials of BICR/M1R-k and L cells with glass microelectrodes and simultaneously recording the fluorescence of the stained cells. The membrane potential of BICR/M1R-k cells was varied between -25 mV and -90 mV by changing the bicarbonate concentration in the medium or by voltage clamping. To avoid any interference by the inserted electrodes with the fluorescence measurement of the cytoplasm, the cells were fused by polyethyleneglycol to form giant cells (homokaryons). These homokaryons also allowed penetration by two glass microelectrodes without causing a serious leakage of the plasma membrane. The slow responding dye diBA-C4-(3)- had a fluorescence response of about 1% per mV. Mathematical analysis of the fluorescence changes after voltage clamping revealed a first-order reaction with a rate constant between 0.1 min-1 and 0.8 min-1, depending on the cell size which was determined by the number of nuclei per homokaryon. A model for the mechanism of the fluorescence changes is proposed.     

Novel rhizobox design to assess rhizosphere characteristics at high spatial resolution

Available tools to study rhizosphere characteristics at a sub-mm spatial resolution suffer from a number of shortfalls, including geometrically and physiologically ill-defined root layers containing soil or other growth medium. Such designs may result in over- or underestimation of root-induced changes in the rhizosphere. We present a novel rhizobox design that overcomes these shortfalls. Plants are pre-grown in a soil–root compartment with an opening slit at the bottom. As plants reach the targeted physiological stage, this compartment is transferred on top of a rhizosphere soil compartment attached to a vertical root-only compartment. The latter is made up of a membrane (pore size 7 m to restrict root hair growth into the rhizosphere compartment or 30 m to restrict only root growth) and a transparent acrylic window which is gently pressed against the membrane and rhizosphere soil compartment using an adjustable screw. This design allows roots to penetrate from the upper soil–root compartment through the slit into the root-only compartment. Root growth and distribution can be monitored through the acrylic window using digital camera equipment. Upon termination of the experiment, the rhizosphere compartment is removed and frozen prior to separation of sub-mm soil layers using microtome techniques. In a test experiment, canola (Brassica napus L. cv. Sprinter) developed a fairly dense root monolayer within 8 days. Using measurement of soil characteristics at 0.5–1-mm increments across the rhizosphere we demonstrate that the proposed rhizobox design is yielding reproducible data. Due to exudation of LMWOC, we found a statistically significant increase of DOC towards the root plane, whereas more stable soil characteristics were not affected by root activity. Limitations and further extensions of this rhizobox design, including the use of micro suction cups and microsensors for pH and redox potential to measure spatial and temporal changes in a non-destructive manner are discussed along with potential applications such as validation of rhizosphere models.     

Electro-deformation and poration of giant vesicles viewed with high temporal resolution

Fast digital imaging was used to study the deformation and poration of giant unilamellar vesicles subjected to electric pulses. For the first time the dynamics of response and relaxation of the membrane at micron-scale level is revealed at a time resolution of 30 micros. Above a critical transmembrane potential the lipid bilayer ruptures. Formation of macropores (diameter approximately 2 microm) with pore lifetime of approximately 10 ms has been detected. The pore lifetime has been interpreted as interplay between the pore edge tension and the membrane viscosity. The reported data, covering six decades of time, show the following regimes in the relaxation dynamics of the membrane. Tensed vesicles first relax to release the acquired stress due to stretching, approximately 100 micros. In the case of poration, membrane resealing occurs with a characteristic time of approximately 10 ms. Finally, for vesicles with excess area an additional slow regime was observed, approximately 1 s, which we associate with relaxation of membrane curvature. Dimensional analysis can reasonably well explain the corresponding characteristic timescales. Being performed on cell-sized giant unilamellar vesicles, this study brings insight to cell electroporation. The latter is widely used for gene transfection and drug transport across the membrane where processes occurring at different timescales may influence the efficiency.     

The spatial and temporal dynamics of rabies in China

Background and Objectives

Recent years have seen a rapid increase in the number of rabies cases in China and an expansion in the geographic distribution of the virus. In spite of the seriousness of the outbreak and increasing number of fatalities, little is known about the phylogeography of the disease in China. In this study, we report an analysis of a set of Nucleocapsid sequences consisting of samples collected through the trial Chinese National Surveillance System as well as publicly available sequences. This sequence set represents the most comprehensive dataset from China to date, comprising 210 sequences (including 57 new samples) from 15 provinces and covering all epidemic regions. Using this dataset we investigated genetic diversity, patterns of distribution, and evolutionary history.

Results

Our analysis indicates that the rabies virus in China is primarily defined by two clades that exhibit distinct population subdivision and translocation patterns and that contributed to the epidemic in different ways. The younger clade originated around 1992 and has properties that closely match the observed spread of the recent epidemic. The older clade originated around 1960 and has a dispersion pattern that suggests it represents a strain associated with a previous outbreak that remained at low levels throughout the country and reemerged in the current epidemic.

Conclusions

Our findings provide new insight into factors associated with the recent epidemic and are relevant to determining an effective policy for controlling the virus.     

Principal component analysis of ensemble recordings reveals cell assemblies at high temporal resolution

Simultaneous recordings of many single neurons reveals unique insights into network processing spanning the timescale from single spikes to global oscillations. Neurons dynamically self-organize in subgroups of coactivated elements referred to as cell assemblies. Furthermore, these cell assemblies are reactivated, or replayed, preferentially during subsequent rest or sleep episodes, a proposed mechanism for memory trace consolidation. Here we employ Principal Component Analysis to isolate such patterns of neural activity. In addition, a measure is developed to quantify the similarity of instantaneous activity with a template pattern, and we derive theoretical distributions for the null hypothesis of no correlation between spike trains, allowing one to evaluate the statistical significance of instantaneous coactivations. Hence, when applied in an epoch different from the one where the patterns were identified, (e.g. subsequent sleep) this measure allows to identify times and intensities of reactivation. The distribution of this measure provides information on the dynamics of reactivation events: in sleep these occur as transients rather than as a continuous process.     

Imaging of plant microtubules with high resolution scanning electron microscopy

Summary High resolution scanning electron microscopy was used to obtain images of cortical microtubules and associated structures in onion root tips. Specimens were prepared using a modified quick-freeze deep-etch technique utilising cytosolic extraction with saponin and conductive staining with osmium.Abbreviations DMSO dimethylsulfoxide - HRSEM high resolution scanning electron microscope/microscopy - MTSB microtubule stabilising buffer - TEM transmission electron microscope/microscopy     

Imaging Matrix Assisted Laser Desorption Ionization Mass Spectrometry: a technique to map plant metabolites within tissues at high spatial resolution

Imaging Matrix Assisted Laser Desorption Ionization Mass Spectrometry provides a new and powerful tool to analyse the distribution of metabolites within plant tissues. The two matrices alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA) and 9-aminoacridine provide a useful combination that allows the measurement of amino acids, sugars, and phosphorylated metabolites. Results are presented showing that representatives of these metabolites are unevenly distributed in wheat seeds at different stages of development and under temperature stress.     

Quantitative analysis of the local rates of growth of dicot leaves at a high temporal and spatial resolution,using image sequence analysis

A new technique is presented for quantitative mapping of dicot leaf growth at high spatial and temporal resolution, at a speed making online-mapping feasible. Time lapse video sequences of growing leaves are captured by a personal computer (PC) with a frame-grabber board and a standard CCD camera, and evaluated using algorithms that have been recently developed to analyse motion in dynamic image sequences. Growth can be detected at under 1% per hour, with a time resolution of minutes and a spatial resolution of a few millimeters. The new technique has been verified by comparing it with classical approaches to map integrated growth. Diurnal courses of leaf growth of Ricinus communis and tobacco are presented to demonstrate the localised character of growth in leaves. Expansion growth is restricted to the base of the leaf and is restricted to a few hours at the end of the night and the start of the day. The high resolution of the method is illustrated by mapping the responses to step-changes in leaf turgor. A 3 bar turgor jump led to a rapid transient expansion over the entire length of the leaf that was partially reversed when the turgor was relaxed.     

Systematic neighborhood observations at high spatial resolution: methodology and assessment of potential benefits

There is a growing body of public health research documenting how characteristics of neighborhoods are associated with differences in the health status of residents. However, little is known about how the spatial resolution of neighborhood observational data or community audits affects the identification of neighborhood differences in health. We developed a systematic neighborhood observation instrument for collecting data at very high spatial resolution (we observe each parcel independently) and used it to collect data in a low-income minority neighborhood in Dallas, TX. In addition, we collected data on the health status of individuals residing in this neighborhood. We then assessed the inter-rater reliability of the instrument and compared the costs and benefits of using data at this high spatial resolution. Our instrument provides a reliable and cost-effect method for collecting neighborhood observational data at high spatial resolution, which then allows researchers to explore the impact of varying geographic aggregations. Furthermore, these data facilitate a demonstration of the predictive accuracy of self-reported health status. We find that ordered logit models of health status using observational data at different spatial resolution produce different results. This implies a need to analyze the variation in correlative relationships at different geographic resolutions when there is no solid theoretical rational for choosing a particular resolution. We argue that neighborhood data at high spatial resolution greatly facilitates the evaluation of alternative geographic specifications in studies of neighborhood and health.