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OSA Publishing > Biomedical Optics Express > Volume 5 > Issue 8 > Page 2648
Christoph Hitzenberger, Editor-in-Chief
Calcium imaging at kHz frame rates resolves millisecond timing in neuronal circuits and varicosities
Michiel A. Martens, Werend Boesmans, and Pieter Vanden Berghe
Michiel A. Martens,1 Werend Boesmans,1 and Pieter Vanden Berghe1,*
1Laboratory for Enteric Neuroscience (LENS),TARGID, University of Leuven, O&N 1 Herestraat 49-box 701, Leuven 3000, Belgium
*Corresponding author: pieter.vandenberghe@med.kuleuven.be
M Martens
W Boesmans
P Vanden Berghe
Vol. 5,
•https://doi.org/10.1364/BOE.5.002648
Michiel A. Martens, Werend Boesmans, and Pieter Vanden Berghe, "Calcium imaging at kHz frame rates resolves millisecond timing in neuronal circuits and varicosities," Biomed. Opt. Express 5, 2648-2661 (2014)
Wide-field multiphoton imaging of cellular dynamics in thick tissue by temporal focusing and patterned illumination (BOE)
Quasi-simultaneous multiplane calcium imaging of neuronal circuits (BOE)
Efficient multi-site two-photon functional imaging of neuronal circuits (BOE)
Analytical techniques
High speed photography
Digital image processing (100.2000)
Imaging ultrafast phenomena (100.0118)
Time-resolved imaging (170.6920)
Temporal discrimination (330.6790)
Original Manuscript: May 30, 2014
Revised Manuscript: July 10, 2014
2. Experimental setup
3. Millisecond resolved imaging of neuronal circuit activity
Suppl. Mat. (2)
We have configured a widefield fast imaging system that allows imaging at 1000 frames per second (512x512 pixels). The system was extended with custom processing tools including a time correlation method to facilitate the analysis of static subcellular compartments (e.g. neuronal varicosities) with enhanced contrast, as well as a dynamic intensity processing (DIP) algorithm that aids in data size reduction and fast visualization and interpretation of timing and directionality in neuronal circuits. This system, together with our custom developed processing tools enables efficient detection of fast physiological events, such as action potential dependent calcium steps. We show, using a specific blocker of nerve communication, that with this setup it is possible to discriminate between a pre and post synaptic event in an all optical way.
To capture all facets of cellular communication in an optical way, it is of utmost importance to record with high temporal resolution as many biological interactions occur faster than the human eye can see or a classic camera can capture. Action potential firing and the consecutive intracellular calcium signaling events in neurons, which occur at the millisecond timescale, is a typical example of such fast cellular signaling. To fully understand neuronal function it is important to resolve timing at this scale because it allows assessing the chronology of events taking place in multicellular neuronal networks. Electrophysiological techniques such as patch clamping are so far the only approaches that deliver sufficient temporal resolution [1]. However, they are quite limited in terms of spatial resolution as only one or a few [2–4] cells can be monitored simultaneously. Furthermore, these recordings can compromise the physiological environment because of their invasive nature. Optical readouts have a clear advantage as they are minimally-invasive and allow recordings from many different cells in a network at the same time.
To overcome these spatiotemporal limitations, different types of 'fast imaging' have been implemented. Confocal approaches have been optimized for speed based on resonant scanning technology, however they still make a tradeoff between temporal and spatial resolution [5]. A point scanning confocal microscope remains relatively slow because the sample is scanned point by point, which classically takes seconds to minutes to acquire a full field of view. Spinning disk confocal microscopy, on the other hand, is able to acquire whole frames at once, resulting in an increased recording speed up to a few hundreds of Hz [6], again partially trading spatial (mainly axial) resolution for speed [7].
However, when maximum temporal resolution is required widefield fluorescence microscopy is still the optical method of choice [8–11]. Using high speed cameras that are optimally designed for fast acquisition, enough photons can be caught in very short time intervals. Especially EMCCD's and sCMOS now allow full frame recordings at a few hundreds of Hz [12]. Some setups even reach up to 10 kHz, which is at the expense of the number of pixels on the sensor chip (e.g. 80x80) [8, 9], and therefore spatial resolution. Also, signal to noise ratio issues arise at these kHz frame rates as only a limited number of photons – if any– can be collected per time point. As an alternative to these camera approaches, photodiode arrays can be used but they are also limited in the number of spatial data points [13–15]. Because of the restriction in spatial resolution of the currently available systems, it remains extremely challenging to accurately record fast events in smaller cellular components such as neuronal processes.
In this study we report the successful development of a microscopy configuration based on a widefield approach with a dual camera system that allows recording images both at fast (up to 2000 Hz) and standard (2-10 Hz) frame rates with sufficient spatial resolution. With this setup it is possible to resolve individual neurotransmitter release sites and record their activity as they operate in a neuronal network.
In combination with our custom image processing tools we are able to detect the chronology of firing using the calcium indicator Fluo4 AM in neuronal circuits both in cell culture and tissue preparations. Furthermore, the approach yields high resolution feedback from sub-millisecond stimuli and allows calculating the propagation speed of the calcium wave front.
2.1 Microscopy setup
A Zeiss Examiner Z1 upright microscope was used as the core of our experimental physiology setup. A cooled CCD camera (PCO; Kelheim, Germany) mounted on the top port was used to record up to 2 Hz and a CMOS camera (Focuscope SV200-i, Photron; Tokyo, Japan) was attached to the side port [Fig. 1(a)] and allowed us to record images at a maximum speed of 2 kHz. This camera has a CMOS sensor of 512 by 512 pixels equipped with a GaAsP photocathode surface. It incorporates a manually adjustable image intensifier allowing full frame high speed recordings up to 2000 Hz, even for low light fluorescence. A custom-made automated mirror switches mechanically between both cameras in about 220 ms. Images were acquired through a water dipping lens (20x, 1.0 NA) and excitation light for fluorescence imaging was generated by a Poly V monochromator (Till Vision; Gräfelfing, Germany). Furthermore, an electric Grass S88 stimulator (Grass Technologies; Warwick, USA), a picospritzer (General Valve Corporation; Fairfield, USA) and a gravity-fed perfusion system (1 ml/min) were used to provide physiological stimuli and buffered solutions to the studied cells and tissues. All components (switching mirror included) were automatically controlled via a control unit and protocol editor available in the Till Vision software (Gräfelfing, Germany). The electrical stimuli (300 µs) were delivered via a local Pt/Ir electrode (Ø 50 µm) to the interconnectives of the nerve network either as single or train (20 Hz) pulses.
Fig. 1 System configuration and noise assessment. (a) Widefield upright microscope equipped with perfusion system, xenon lamp, spritz and electric stimulators, high spatial resolution CCD camera and fast CMOS camera; all automatically controlled through a programmable PC. (b) Average counts over 50 dark frames per image intensifier voltage in steps of 50 only show a sharp increase above 750 of image intensifier voltage. (c) Single dark frame at 1 ms exposure and 850 image intensifier voltage shows (shot) noise very localized in space and random in time. (d) Total noise as standard deviation depends on image intensifier voltage and size of the region of interest. (e) Myenteric ganglion in a preparation dissected from mouse large intestine. Single frame acquired with 1 ms exposure showing high signal-to-noise allowing individual varicosities of neuronal fibers to be resolved in live imaging experiments.
2.2 kHz camera performance
To test the performance of the Photron CMOS camera, 50 bias frames were recorded with a minimal exposure time of 3.7 µs for each intensifier voltage, increasing the image intensifier up to 850 (Δ = 50). All these frames had zero counts as a consequence of the preset threshold of the camera. It also shows that read noise, at least up to this threshold, is not influenced by the image intensifier. Secondly, 50 dark frames were recorded with an exposure time of about 1 ms for each intensifier voltage, increasing the image intensifier up to 850 (Δ = 50). For most pixels the counts were again zero, except for some noise that is only present at higher intensifier voltages [Fig. 1(b)]. This noise is single pixel limited as 98% of the spot noise population over all 850 dark frames appears in one single pixel only [Fig. 1(c)]. Possibly this is caused by cosmic radiation that is able to penetrate the camera shielding during dark frame acquisition and is then enhanced by the image intensifier. In order to have a good estimate of the overall noise levels we set out the standard deviation versus size of region of interest (spatial signal integration) and intensifier voltage measured on a fixed sample of comparable fluorescence intensity to our live tissue samples [Fig. 1(d)].
We recorded at 1000 Hz for all experiments, except for a specific set of calcium wave speed measurements that were recorded at the maximum of 2000 Hz. Thus, we benefit from the increased exposure time and reduced (shot) noise due to a lower intensifier voltage, while still retaining 2 extra orders of magnitude compared to standard (~10 Hz) acquisition rates. The A/D converter's 10 bit stacks were sampled down to 8 bit to reduce file size and to facilitate software handling. Recordings in live tissues proved that our system has sufficient resolution and sensitivity to resolve individual varicosities and their responses with high signal-to-noise ratio [Fig. 1(e)] at kHz recording rates.
2.3 Tissue and cell culture preparation
For live experiments we used enteric neurons, which reside in two specific layers within the intestinal wall: the submucosal and myenteric plexus [16]. These plexus consist of a complex network of neurons and glia and especially the myenteric plexus is organized in a cellular layer, which is ideally suited for widefield imaging [17]. These nerves communicate via classic synaptic contacts but many processes also have en route varicose release sites, which are referred to as boutons or varicosities in the remainder of the manuscript. For our recordings, we used both enteric nerve cell cultures (small intestine) and whole mount myenteric plexus preparations, taken from the mouse large intestine.
Mice (C57/Bl6) were killed by cervical dislocation and the large intestine was removed and placed in Krebs solution (in mM: 120.9 NaCl, 5.9 KCl, 1.2 MgCl2, 2.5 CaCl2, 1.2 NaH2PO4, 14.4 NaHCO3, 11.5 Glucose). All animal procedures were approved by the ethical committee of the University of Leuven (Leuven, Belgium). The myenteric plexus from the large intestine was loaded for 20 minutes with the fluorescent calcium indicator Fluo4 AM (1E-6 M). Cremophor (0.01% v/v) was added to enhance uniform loading. After washing (10 minutes in Krebs solution) the tissue was mounted on the microscope stage while continuously perfused with carbogenated (95% O2 −5% CO2) Krebs (22°C). Apart from tissue primary neuronal cell cultures were also used for some tests. These were made from mouse (C57/Bl6) intestine and grown for 3 days at 37°C and 5% CO2. They were then loaded with Fluo4 AM (5E-6 M), washed for 10 minutes and kept under constant perfusion with HEPES buffered solution during the experiment (22°C) [17,18] [Fig. 2(a) and 2(b)].
Fig. 2 Localization and identification of varicose release sites in a myenteric ganglion dissected from the mouse large intestine. Dashed red lines indicate the region shown in (c) (63x objective). (a) Snapshot images taken with a standard CCD camera (20x objective) yield highly detailed information. Inset Image recorded (standard CCD) with 5x objective shows the ganglionic network and the position of the stimulation electrode on the tissue. (b) Single original frame (20x objective) acquired with 1 ms exposure has sufficient signal-to-noise to resolve individual varicosities of neuronal fibers in live imaging experiments. Arrows indicate active varicosities. (c) Immunohistochemical staining (63x objective) to identify varicosities –synaptotagmin (red arrows), neuronal processes-peripherin (green arrowheads) and neuronal soma-HuC/D (white asterisks). (d-f) Images resulting from our autocorrelation software tool. Output of the autocorrelation tool in a time window of 500 ms before (d) and after (e) the stimulation. (f) Image that results from the subtraction of (d) from (e), which selectively shows those parts of the network that respond within 500 ms after stimulation, clearly highlighting responding varicosities.
After live experiments, some tissues were fixed at room temperature using 4% PFA (30 min) and processed for immunohistochemistry to identify the cellular components that were recorded from: rabbit anti-synaptotagmin (gift from Dr. R. Jahn, Göttingen, Germany) was used to identify varicosities, mouse anti-HuC/D (Invitrogen, Merelbeke, Belgium) for neuronal cell soma and goat anti-peripherin (Santa Cruz, Heidelberg, Germany) to label neuronal fibers. Secondary antibodies were coupled to AMCA (Jackson ImmunoResearch, West Grove, USA), alexa594 and alexa488 (Invitrogen, Merelbeke, Belgium) [Fig. 2(c)]. These tissues were then imaged with a scanning 2-photon/confocal microscope (LSM510 META, Zeiss, Germany) in the Cell Imaging Core (CIC, University of Leuven).
3.1 Autocorrelation of high frame rate images allows accurate localization and visualization of individual varicosities
Even though spatial resolution in our setup is significantly higher (512x512) than fast CCD (80x80) based systems, not all varicosities can easily be discerned from a single (1 ms exposure) frame. Therefore we designed a software tool based on an autocorrelation approach, which due to the high temporal resolution performs well to assist and facilitate varicosity identification. The algorithm calculates the arithmetic mean (V) of the autocorrelation [Eq. (1)] over a user-defined time window to construct a pixel by pixel autocorrelation image. V is calculated for each pixel with f(t) the fluorescence over time in this pixel, N the user-defined time window and M the total number of points in the autocorrelation function. By subtracting autocorrelation images produced before, from images at the time of stimulation [Fig. 2(d) and 2(e)], high-contrast images are generated that highlight the responding varicosities with a strongly reduced background [Fig. 2(f)].
(1)V= ∑T=0M−1(∑t=0N−1(f(t).f(t+T)) M
3.2 Detection of fast calcium events
Calcium responses could be measured in individual varicosities from Fluo4 loaded tissue using 3 different types of stimuli. The response to a single electric pulse was typically characterized by a sharp upstroke reaching its maximum in less than 10 ms (3 mice, 4 ganglia, max F/F0 = 2.2 ± 0.45) [Fig. 3(b)]. Using a 20 Hz pulse train stimulus, we could show that our detection is fast enough to resolve individual calcium steps at a frequency that matches the stimulation frequency (5 mice, 8 ganglia, max F/F0 = 3.2 ± 0.59) [Fig. 3(c)]. This response frequency was confirmed using fast Fourier transformations that reveal a ~20Hz dominant peak in the spectra [Fig. 3(e) and 3(f)]. A stepwise calcium response was also observed when the neurotransmitter serotonin (5-HT) was locally applied to the tissue using a pressure spritzer (5 mice, 5 ganglia, max F/F0 = 1.7 ± 0.19) [Fig. 3(a)]. To improve signal to noise, a low pass filter can be applied either directly or after taking the derivative of the signal [Fig. 3(d)], which facilitates automatic step detection as the derivative signal clearly divulges the individual calcium step location.
Fig. 3 Intracellular calcium responses in individual varicosities (top) and transformed signals (bottom). (a) Calcium steps evoked by a serotonin puff. (b) Calcium response evoked by a single electric pulse. (c) Calcium steps evoked by a 20 Hz electric pulse train. (d) Low pass filtered serotonin evoked calcium response (red) and low pass filtered derivative (blue) that is used to automatically detect the stepwise increase. (e) Fourier transform of (b). (f) Fourier transform of (c) confirming a frequency component around 20 Hz.
Next, we tested whether the improved temporal and spatial resolution could be used to assess calcium signal propagation in neuronal fibers. To address this question, interganglionic fiber tracts were stimulated with a single electrical pulse, classically used to elicit fast excitatory postsynaptic potentials in the enteric nervous system [8]. By recording at 2 kHz, we were able to continuously monitor the calcium signal as it travelled through ganglia and connecting fibers at a calculated propagation speed of about 80 mm/s (7 ganglia, 4 mice). Remarkably this speed is in the same order of magnitude as the one extrapolated from recordings of calcium waves in the intestinal muscles layers [19].
3.3 Computation and visualization of neuronal activity spread using dynamic intensity processing (DIP)
To highlight the importance of timing in the sequence of biological events such as communication in neuronal networks, we sought to further improve the temporal contrast of our recordings by designing an algorithm that comprises image thresholding, peak detection and color mapping as schematized in Fig. 4.
Fig. 4 Dynamic intensity processing (DIP) visualized by input (a,b,c), processing (d,e,f) and output (g,h,i). The input (a) time lapse stack of Fluo4 loaded neuronal cell culture. (b) Time lapse stack of Fluo4 loaded live neuronal tissue. (c) Calcium peaks in these recordings differ only by a few ms, while they are quite distant in space. Therefore, while interpreting the raw data, it is impossible to distinguish when these events occur compared to others. (d) Creating a threshold mask (red) with user defined threshold value. (e) For each non-thresholded pixel: Starting from the time trace of each pixel (upper trace), differentiate (middle trace) and low-pass filter (lower trace) the differential trace. (f) The square of this trace is ideally suited for Igor Pro's peak detection algorithm (Maximum peaks = 1; Minimum peak percent = 20) that finds the time coordinate of the peak's maximum. This value is then color coded (using a rainbow color map spread over a user defined time window: from stimulus start until the moment the final responses occur). Pixels with values outside the user defined window are color mapped in black. (g,h) Output of the resulting color coded peak position time value in a new image using the pixel coordinates of the original pixel for either cell culture (g) (Media 1) or nerve tissue (h). Arrows indicate varicosities. Thresholded pixels are not processed and receive a value of 0. (i) The color coded result resolves the spatiotemporal interpretation difficulties of the raw data.
The initial thresholding [Fig. 4(d)] is introduced to discard background pixels and reduce computation time. In a following step (b) the signal is differentiated to transform responses into peaks. An increase in signal-to-noise results from low-pass filtering [Fig. 3(d)] (blue) and Fig. 4(e) (lower trace) and squaring [Fig. 4(f)]. Next, Igor Pro's peak detection algorithm based on smoothed derivatives is called to define the peak location. Once the new image is calculated the rainbow color scale can be adjusted at any time to color code a specific time window [Fig. 4(f)]. This allows discriminating both very fast calcium rises appearing within ms after the stimulus as well as cells or fibers that respond within a few tens of ms after the stimulus. By using this millisecond time gating, specific structures and responses can be isolated, even for samples with ample background fluorescence as is the case in Fluo4 loaded myenteric plexus tissues. This image processing tool, optimized for fast recordings, is similar in readout to the time-to-maximum images earlier described by our group for standard recordings [17]. DIP generates MB size images from GB size time lapse stacks clearly summarizing the temporal aspects of activity spread in individual varicosities and neuronal networks in both cell culture (g) and tissue (h). This reveals that fibers, even though they are physically close to the stimulated cell are not necessarily the first responders. Figure 4(g) shows that fibers are responding in the direction towards the electrode (bottom right), rather than away from it (Media 1). This suggests that these processes belong to neurons that are among the last to respond in a cascade of responders that starts at the electrode, with the connecting circuitry mainly present outside the field of view. Alternatively we are not sure whether some signals remain under the detection limit. These color maps allow fast interpretation of large data volumes and introduce time with ms resolution as a practical scientific research parameter.
Specific color map selections [Fig. 5] will give a better contrast to the human eye and can be used to highlight different aspects (varicosities [Fig. 5(c)]; overall flow [Fig. 5(d)]) of the information flow in a neuronal network. In order to cover the full time window of interest and to increase contrast for local and consecutive physiological events a follow-up procedure was created that produces either a movie that expands (Media 1 and Media 2) or one that shifts the color scale over the values from the DIP image [Fig. 5(a) and 5(d)-5(f)]. Both movies clearly show directionality of intracellular calcium rises in the millisecond range.
Fig. 5 (a) Principle of the expanding rainbow mapping approach. (b) Original single frame (1 ms exposure) in grayscale. (c) Millisecond time gating: selecting individual varicosities from within a complex tissue nerve network that respond in synchrony. (d,e,f) Images selected from an expanding rainbow movie (Media 2). (d) The initial 'fast' intracellular calcium wave (arrows) in the ganglion is visualized (electrode bottom right). (e) Expanding the rainbow mapping generates improved color contrast, which emphasizes the newly changed areas while the initial 'fast' flux is switched to background blue colors. Arrows indicate varicosities. (f) An image from the end of the response, when the color map is fully stretched, reveals that the 'late' fibers run predominantly along the sides of an interganglionic connective (white arrows). Also, varicosities with different timing can be detected (red arrows).
3.4 Pre- and postsynaptic intracellular calcium responses can be temporally resolved
To further address the synchronicity in fiber and varicosity responses in a network, we used a cell culture model of enteric neurons and took advantage of our newly developed DIP to distinguish between timing of responsive elements. Even though some network elements are spatially close together, it is still possible to detect a ~20 ms delay between some of their responses [Fig. 6]. We used the nicotinic receptor blocker hexamethonium to silence cholinergic transmission, the dominant component of fast neurotransmission in the ENS, and found that the slower fibers [Fig. 6(a)] were delayed even further [Fig. 6(b)]. Upon washout of the drug, this effect was reverted [Fig. 6(c)]. This indicates that cholinergic transmission is implicated in the complex wiring most likely via autaptic communication (as present in the artificial conditions of the cell culture system). We found that the effect of hexamethonium can be predicted from the maximum response delay in the first recording, as only in those recordings with substantial delays (17 ± 1.4 ms) blocking of nicotinic receptors was able to amplify the delay (27.5 ± 3.5 ms), while in other recordings with synchronous responses (10.2 ± 2.5 ms) hexamethonium did not have any effect (10.6 ± 2.4 ms). Therefore, we expect that the observed distinct delays reflect the length of the network element before it reappears in the field of view.
Fig. 6 (a) DIP image of a neuronal network in cell culture responding to a single electric pulse. (b) DIP image of the same neuronal network in cell culture responding to a single electric pulse after 2.5 minutes of hexamethonium perfusion. (c) DIP image of the same neuronal network in cell culture responding to a single electric pulse after hexamethonium washout. (d) Schematic representation as a model for (a) of direct activation of one circuit (blue) and activation of a consecutive circuit (green) connected via a cholinergic synapse. (e) Schematic representation as a model for (b) of direct activation of one circuit (blue) and delayed activation of a consecutive circuit (red) with blocked cholinergic synapses. (f) Schematic representation as a model for (c) of direct activation of one circuit (blue) and restored activation of a consecutive circuit (green) again connected via cholinergic synapses.
Instead of focusing on different response elements in a network, we also analyzed individual varicosities to examine whether circuitry information could be deduced from a single spot [Fig. 7]. For this set of experiments we used isolated tissues, which have an advantage in that they contain the native wiring of the network. The responses evoked by a single electrical stimulus were highly repeatable over sequential trials (3 repeats, 258 varicosities from 2 mice). The majority of varicosities displayed one single sharp upstroke in response to the single stimulus [Fig. 3(b)], however in a select number of cases (~10%) a biphasic response could be detected [Fig. 7(a)] suggestive of a recurrent or delayed secondary activation. These secondary responses seemed to be hard wired as they were also consistent in repeated recordings. To investigate the nature of this second peak, we again used hexamethonium and imaged the varicosities (330 varicosities from 6 mice) first in control and secondly in the presence of hexamethonium (1E-4 M, 2.5 min). In over 40 varicosities, secondary responses were observed. Hexamethonium completely abolished the secondary response in 14 varicosities [Fig. 7(b)] while the double response in 26 varicosities was not affected by hexamethonium. After washout (2.5 min) all secondary responses reappeared [Fig. 7(c)], clearly proving the involvement of an intermediate cholinergic connection.
Fig. 7 (a) Double calcium response from an individual varicosity from a myenteric neuron triggered by a single electric pulse. Blue dotted lines indicate automatic step detection for fast analysis. (b) Response from the same varicosity after 2.5 minutes of hexamethonium perfusion. The secondary activation is abolished. (c) The secondary activation reappears after hexamethonium washout. (d) Schematic representation of direct activation and secondary synaptic activation with extended circuit in between. (e) Schematic representation of primary activation and blocked secondary synaptic activation. (f) Schematic representation of primary activation and recovered secondary synaptic activation after hexamethonium washout.
We conclude that with our microscopy setup it is possible to simultaneously record and discriminate between pre- (direct/antidromic) and postsynaptic activity in an all optical way.
Live fluorescence microscopy is a powerful and heavily used tool in biomedical research mainly because it yields an optical readout from many cells simultaneously, which creates the possibility to visualize cellular interactions and circuits. The main disadvantage of these techniques is the lack of speed at which recordings can be made in comparison to electrophysiological approaches [20]. To overcome this, strategies have been designed to improve camera capture speed or – in case of scanning microscopy – enhance the (resonating) speed of the scanner.
Confocal measurements on the millisecond time scale are an option but only by limiting the recorded area to single lines or single points. In this case, the increase in temporal resolution is at the expense of a general overview. As an alternative approach to increasing temporal resolution resonant scanners have been implemented [21,22], which sweep the excitation beam over the field of view much faster than classic linear galvanometric scanning mirrors. Here the disadvantage is that pixel dwell times are often too short to collect a sufficient amount of photons. Alternatively acousto-optic deflectors can be used to change the beam direction in a non-mechanical way [23].
Unlike point or line scanning systems, also fast (kHz) cameras have been elegantly used in cardiac [24], peripheral [8,9] and central nervous system [10] research to improve recording speed. However, to maximize readout speed, sensitivity has to be raised, very often accomplished by increasing pixel size and therefore lowering spatial resolution. Here we report a microscopy configuration with an additional high spatial resolution fast camera (512 by 512 readout), which makes it possible to record both fast and slow events in small (close to diffraction limited) objects, such as varicosities in a neuronal network. A custom-made mechanical sliding mirror allows switching (220 ms delay) back and forth from fast to standard recordings automatically. Our hardware setup, combined with custom made analysis tools, close an important gap in the available microscopy techniques because of the obvious speed advantage while still having the possibility to simultaneously record from different components in an integrated network. Another advantage relates to the flexibility of the technique as it can easily be integrated in existing microscopy setups.
Our system (hard- and software) is unique in terms of temporal and spatial resolution as it reveals events that could not be detected before in an optical way. The distinct intracellular calcium steps that we recorded in individual varicosities during serotonin application are a good example. Most likely each step is the consequence of a unitary event like a single action potential or the opening of a 5HT3 receptor (cluster). This is in line with the stepwise intracellular calcium rise that was measured for nicotinic receptor stimulation [8] of enteric neurons, which was indeed associated with action potential firing. We further show that we can resolve distinct intracellular calcium steps in varicosities using action potential pulse trains (20 Hz), thus yielding much more detail about the response upstroke compared to classic CCD cameras.
Our image capture speed proved fast enough to deduce calcium propagation speed (~80 mm/s) in unmyelinated neuronal fibers of the enteric nervous system, which is remarkably similar to what was extrapolated from muscle recordings [19]. It remains to be elucidated why these high speeds are necessary to control physiological behaviour (intestinal motility, mixing) that is far slower. It could be argued that this conduction speed is that high to allow signal integration over larger areas generating concerted actions of smooth muscle units in the gut wall. Muscle twitch indeed happens simultaneously for groups of cells spread over 100-200 µm, a distance that is bridged in ~2 ms at the measured calcium propagation speed. Compared to action potential propagation in unmyelinated fibers the calcium propagation speed is still low. This may be due to the fact that the experiments were performed at room temperature or caused by a true mismatch between Ca2+ signal and action potential propagation. The latter could be explained if Na+ and Ca2+ channels are not evenly distributed along the fiber or if the volume of the process is not constant. We do not expect that delays are due to the properties of the indicator, as Fluo4 has a sufficiently high affinity (Kd = 345 nM), S/N ratio and Ca2+ binding kinetics [25,26] to generate enough photons during the short (ms) integration times. The temperature dependency of Fluo4 [27] is most likely negligible in light of the effect temperature will have on the physiology of the network. Higher conduction velocities as present in myelinated (motor) neurons cannot be measured at the moment, since this would require imaging larger fields of view with lower magnification lenses and therefore less detail.
The first of our processing tools allows summarizing a GB time lapse stack into MB images that are easy and fast to interpret. They either enhance spatial contrast (time correlation) or temporal contrast (DIP). Despite these obvious advantages, it is important to realize that this type of computation takes time, for instance a time lapse stack reduced to 350 frames around the event of interest requires typically around 10 min to be processed because of the pixel by pixel multi-step analysis. This process can be sped up by adjusting the threshold value, which is especially useful in recordings from cell cultures as more background pixels can be discarded from the calculations.
A second tool, DIP, also allows time gating at ms resolution, which can be used to identify varicosities that fire in synchrony. This technique can be employed to separate single active fibers within complex neuronal circuits from background information, even using widefield microscopy. Furthermore, it allows determining signaling order within neuronal networks as well as distinguishing between direct and synaptic or long-distance communication. Indeed, using hexamethonium, a drug that blocks the most important fast component of neurotransmission in the enteric nervous system [28], we were able to delay responses by forcing the response propagation over longer (non-cholinergic) connections.
In tissue experiments, where neuronal wiring is still intact, single fast responses to fiber tract stimulation dominate the response spectrum of varicosities in the entire field of view. Recurring post-synaptic activity within the circuitry is limited to a stochastic minimum. Although altered intracellular calcium buffering, summation of inputs or paracrine factors may influence the response in a single varicosity at any given moment, most varicosities in which a secondary intracellular calcium step occurred, were shown to faithfully repeat that response in up to 3 consecutive recordings. Only in the presence of hexamethonium, the secondary response was completely abolished in a subgroup of varicosities. These data indicate that at least one cholinergic release site is involved in the generation of the secondary response, which proves the potential of fast imaging to distinguish between direct and circuit related activation. Synaptic contact redundancy or involvement of a neurotransmitter other than acetylcholine may explain the presence of hexamethonium resistant responses.
In summary, we show that high resolution full frame kHz imaging further expands the application spectrum of calcium imaging, since calcium propagation speed and stepwise calcium increases associated with single action potentials in individual varicosities can be detected. Our image processing tools assist in the discrimination of neuronal network activity on the millisecond timescale.
We would like to thank the members of LENS for their critical comments and skilled technical assistance. WB, is a postdoctoral fellow of the Fonds voor Wetenschappelijk Onderzoek (FWO). This work was funded by BOF, University of Leuven (OT ZKC1808) to PVB and FWO (G0501.10) to PVB; KN 1.5.135.06 to WB. Recordings were made on the equipment of the Cell Imaging Core supported by Hercules foundation grants (to PVB).
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8. K. Michel, M. Michaelis, G. Mazzuoli, K. Mueller, P. Vanden Berghe, and M. Schemann, "Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge," J. Physiol. 589(Pt 24), 5941–5947 (2011). [PubMed]
9. A. L. Obaid, M. E. Nelson, J. Lindstrom, and B. M. Salzberg, "Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system," J. Exp. Biol. 208(15), 2981–3001 (2005). [CrossRef] [PubMed]
10. K. Holthoff, D. Zecevic, and A. Konnerth, "Rapid time course of action potentials in spines and remote dendrites of mouse visual cortex neurons," J. Physiol. 588(7), 1085–1096 (2010). [CrossRef] [PubMed]
11. G. Li, R. Stewart, M. Canepari, and M. Capogna, "Firing of hippocampal neurogliaform cells induces suppression of synaptic inhibition," J. Neurosci. 34(4), 1280–1292 (2014). [CrossRef] [PubMed]
12. R. Davies, J. Graham, and M. Canepari, "Light sources and cameras for standard in vitro membrane potential and high-speed ion imaging," J. Microsc. 251(1), 5–13 (2013). [CrossRef] [PubMed]
13. M. Neunlist, S. Peters, and M. Schemann, "Multisite optical recording of excitability in the enteric nervous system," Neurogastroenterol. Motil. 11(5), 393–402 (1999). [CrossRef] [PubMed]
14. D. Zecević, J. Y. Wu, L. B. Cohen, J. A. London, H. P. Höpp, and C. X. Falk, "Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill-withdrawal reflex," J. Neurosci. 9(10), 3681–3689 (1989). [PubMed]
15. A. L. Obaid, T. Koyano, J. Lindstrom, T. Sakai, and B. M. Salzberg, "Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus," J. Neurosci. 19(8), 3073–3093 (1999). [PubMed]
16. J. B. Furness, The Enteric Nervous System (Blackwell Pub., Malden, Mass., 2006), pp. xiii, 274 p.
17. W. Boesmans, M. A. Martens, N. Weltens, M. M. Hao, J. Tack, C. Cirillo, and P. Vanden Berghe, "Imaging neuron-glia interactions in the enteric nervous system," Front Cell Neurosci. 7, 183 (2013). [CrossRef] [PubMed]
18. P. Vanden Berghe, J. Tack, and W. Boesmans, "Highlighting synaptic communication in the enteric nervous system," Gastroenterology 135(1), 20–23 (2008). [CrossRef] [PubMed]
19. R. J. Stevens, N. G. Publicover, and T. K. Smith, "Propagation and neural regulation of calcium waves in longitudinal and circular muscle layers of guinea pig small intestine," Gastroenterology 118(5), 892–904 (2000). [CrossRef] [PubMed]
20. F. Helmchen and A. Konnerth, Imaging in Neuroscience: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2011), 1084 p.
21. S. Schwartz, "Real-Time Laser-Scanning Confocal Ratio Imaging," Am. Lab. 25, 53–57 (1993).
22. R. P. Aylward, "The advances & technologies of galvanometer-based optical scanners," Opt. Scanning: Design Appl. 3787, 158–164 (1999). [CrossRef]
23. G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, "Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity," Nat. Neurosci. 11(6), 713–720 (2008). [CrossRef] [PubMed]
24. G. Antoons, K. Mubagwa, I. Nevelsteen, and K. R. Sipido, "Mechanisms underlying the frequency dependence of contraction and [Ca2+](i) transients in mouse ventricular myocytes," J. Physiol. 543(3), 889–898 (2002). [CrossRef] [PubMed]
25. K. R. Gee, K. A. Brown, W. N. Chen, J. Bishop-Stewart, D. Gray, and I. Johnson, "Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes," Cell Calcium 27(2), 97–106 (2000). [CrossRef] [PubMed]
26. A. Takahashi, P. Camacho, J. D. Lechleiter, and B. Herman, "Measurement of intracellular calcium," Physiol. Rev. 79(4), 1089–1125 (1999). [PubMed]
27. M. L. Woodruff, A. P. Sampath, H. R. Matthews, N. V. Krasnoperova, J. Lem, and G. L. Fain, "Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice," J. Physiol. 542(3), 843–854 (2002). [CrossRef] [PubMed]
28. J. J. Galligan, K. J. LePard, D. A. Schneider, and X. Zhou, "Multiple mechanisms of fast excitatory synaptic transmission in the enteric nervous system," J. Auton. Nerv. Syst. 81(1-3), 97–103 (2000). [CrossRef] [PubMed]
B. Sakmann and E. Neher, "Patch clamp techniques for studying ionic channels in excitable membranes," Annu. Rev. Physiol. 46(1), 455–472 (1984).
G. Kim and K. Kandler, "Paired recordings from distant inhibitory neuron pairs by a sequential scanning approach," J. Neurosci. Methods 200(2), 185–189 (2011).
J. R. P. Geiger, J. Lübke, A. Roth, M. Frotscher, and P. Jonas, "Submillisecond AMPA receptor-mediated signaling at a principal neuron-interneuron synapse," Neuron 18(6), 1009–1023 (1997).
Y. Y. Ma and D. A. Prince, "Functional alterations in GABAergic fast-spiking interneurons in chronically injured epileptogenic neocortex," Neurobiol. Dis. 47(1), 102–113 (2012).
J. B. Pawley, Handbook of Biological Confocal Microscopy, 2nd ed., The language of science (Plenum Press, New York, 1995), pp. xxiii, 632, 634 p. of plates.
R. Gräf, J. Rietdorf, and T. Zimmermann, "Live cell spinning disk microscopy," Adv. Biochem. Eng. Biotechnol. 95, 57–75 (2005).
E. Wang, C. M. Babbey, and K. W. Dunn, "Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems," J. Microsc. 218(2), 148–159 (2005).
K. Michel, M. Michaelis, G. Mazzuoli, K. Mueller, P. Vanden Berghe, and M. Schemann, "Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge," J. Physiol. 589(Pt 24), 5941–5947 (2011).
A. L. Obaid, M. E. Nelson, J. Lindstrom, and B. M. Salzberg, "Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system," J. Exp. Biol. 208(15), 2981–3001 (2005).
K. Holthoff, D. Zecevic, and A. Konnerth, "Rapid time course of action potentials in spines and remote dendrites of mouse visual cortex neurons," J. Physiol. 588(7), 1085–1096 (2010).
G. Li, R. Stewart, M. Canepari, and M. Capogna, "Firing of hippocampal neurogliaform cells induces suppression of synaptic inhibition," J. Neurosci. 34(4), 1280–1292 (2014).
R. Davies, J. Graham, and M. Canepari, "Light sources and cameras for standard in vitro membrane potential and high-speed ion imaging," J. Microsc. 251(1), 5–13 (2013).
M. Neunlist, S. Peters, and M. Schemann, "Multisite optical recording of excitability in the enteric nervous system," Neurogastroenterol. Motil. 11(5), 393–402 (1999).
D. Zecević, J. Y. Wu, L. B. Cohen, J. A. London, H. P. Höpp, and C. X. Falk, "Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill-withdrawal reflex," J. Neurosci. 9(10), 3681–3689 (1989).
A. L. Obaid, T. Koyano, J. Lindstrom, T. Sakai, and B. M. Salzberg, "Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus," J. Neurosci. 19(8), 3073–3093 (1999).
J. B. Furness, The Enteric Nervous System (Blackwell Pub., Malden, Mass., 2006), pp. xiii, 274 p.
W. Boesmans, M. A. Martens, N. Weltens, M. M. Hao, J. Tack, C. Cirillo, and P. Vanden Berghe, "Imaging neuron-glia interactions in the enteric nervous system," Front Cell Neurosci. 7, 183 (2013).
P. Vanden Berghe, J. Tack, and W. Boesmans, "Highlighting synaptic communication in the enteric nervous system," Gastroenterology 135(1), 20–23 (2008).
R. J. Stevens, N. G. Publicover, and T. K. Smith, "Propagation and neural regulation of calcium waves in longitudinal and circular muscle layers of guinea pig small intestine," Gastroenterology 118(5), 892–904 (2000).
F. Helmchen and A. Konnerth, Imaging in Neuroscience: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2011), 1084 p.
S. Schwartz, "Real-Time Laser-Scanning Confocal Ratio Imaging," Am. Lab. 25, 53–57 (1993).
R. P. Aylward, "The advances & technologies of galvanometer-based optical scanners," Opt. Scanning: Design Appl. 3787, 158–164 (1999).
G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, "Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity," Nat. Neurosci. 11(6), 713–720 (2008).
G. Antoons, K. Mubagwa, I. Nevelsteen, and K. R. Sipido, "Mechanisms underlying the frequency dependence of contraction and [Ca2+](i) transients in mouse ventricular myocytes," J. Physiol. 543(3), 889–898 (2002).
K. R. Gee, K. A. Brown, W. N. Chen, J. Bishop-Stewart, D. Gray, and I. Johnson, "Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes," Cell Calcium 27(2), 97–106 (2000).
A. Takahashi, P. Camacho, J. D. Lechleiter, and B. Herman, "Measurement of intracellular calcium," Physiol. Rev. 79(4), 1089–1125 (1999).
M. L. Woodruff, A. P. Sampath, H. R. Matthews, N. V. Krasnoperova, J. Lem, and G. L. Fain, "Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice," J. Physiol. 542(3), 843–854 (2002).
J. J. Galligan, K. J. LePard, D. A. Schneider, and X. Zhou, "Multiple mechanisms of fast excitatory synaptic transmission in the enteric nervous system," J. Auton. Nerv. Syst. 81(1-3), 97–103 (2000).
Antoons, G.
Aylward, R. P.
Babbey, C. M.
Bishop-Stewart, J.
Boesmans, W.
Brown, K. A.
Camacho, P.
Canepari, M.
Capogna, M.
Chen, W. N.
Cirillo, C.
Cohen, L. B.
Davies, R.
Duemani Reddy, G.
Dunn, K. W.
Fain, G. L.
Falk, C. X.
Fink, R.
Frotscher, M.
Galligan, J. J.
Gee, K. R.
Geiger, J. R. P.
Gräf, R.
Graham, J.
Gray, D.
Hao, M. M.
Herman, B.
Holthoff, K.
Höpp, H. P.
Johnson, I.
Jonas, P.
Kandler, K.
Kelleher, K.
Kim, G.
Konnerth, A.
Koyano, T.
Krasnoperova, N. V.
Lechleiter, J. D.
Lem, J.
LePard, K. J.
Li, G.
Lindstrom, J.
London, J. A.
Lübke, J.
Ma, Y. Y.
Martens, M. A.
Matthews, H. R.
Mazzuoli, G.
Michaelis, M.
Michel, K.
Mubagwa, K.
Mueller, K.
Neher, E.
Nelson, M. E.
Neunlist, M.
Nevelsteen, I.
Obaid, A. L.
Peters, S.
Prince, D. A.
Publicover, N. G.
Rietdorf, J.
Roth, A.
Saggau, P.
Sakai, T.
Sakmann, B.
Salzberg, B. M.
Sampath, A. P.
Schemann, M.
Schneider, D. A.
Schwartz, S.
Sipido, K. R.
Smith, T. K.
Stevens, R. J.
Stewart, R.
Tack, J.
Takahashi, A.
Vanden Berghe, P.
Wang, E.
Weltens, N.
Woodruff, M. L.
Wu, J. Y.
Zecevic, D.
Zhou, X.
Zimmermann, T.
Adv. Biochem. Eng. Biotechnol. (1)
Am. Lab. (1)
Annu. Rev. Physiol. (1)
Cell Calcium (1)
Front Cell Neurosci. (1)
J. Auton. Nerv. Syst. (1)
J. Exp. Biol. (1)
J. Microsc. (2)
J. Neurosci. (3)
J. Neurosci. Methods (1)
J. Physiol. (4)
Neurobiol. Dis. (1)
Neurogastroenterol. Motil. (1)
Opt. Scanning: Design Appl. (1)
Physiol. Rev. (1)
» Media 1: AVI (3188 KB)
(1) V= ∑ T=0 M−1 ( ∑ t=0 N−1 (f( t ).f( t+T )) M


text:
Thu, Oct 16, 14
The Moody Blues' Justin Hayward Says Touring Solo Has "Become a Kind of Drug for Me"
972thevan.com
It's been just over a month since The Moody Blues wrapped up their latest North American tour, but now frontman Justin Hayward is set to embark on a new U.S. solo trek . The monthlong outing kicks off Friday in Durham, North Carolina, and is plotted out through a November 17 concert in Chicago.
This tour leg is the third that the 68-year-old singer/guitarist has mounted without his band since the early-2013 release of his latest solo album, Spirits in the Western Sky , and he tells ABC News Radio that he's sort of getting addicted to playing these stripped-down shows.
"It's become a kind of drug for me," says Hayward. "I'm enjoying it, and it's the perfect balance to The Moody Blues."
Justin, who has been alternating solo tours and Moody Blues treks since the summer of 2013, notes that he's "very pleased that one allows the other."
He adds, "You know, the Moodies, the big production...if I just did that I'm not sure I'd be satisfied anymore. So, to have this, to bring my own acoustic guitars out and to enjoy them and to just play the role of [an] acoustic guitar player and singer again is very refreshing."
The new trek will see Hayward and his small backup group -- virtuoso guitarist Mike Dawes and multi-instrumentalist/backing vocalist Julie Ragins -- visiting a variety of East Coast cities, as well as some Midwest sites. Several of the shows will take place at the same venues where Justin performed during his August 2013 tour in support of Spirits , including The Concert Hall in New York City and the Buckhead Theatre in Atlanta.
In fact, Hayward's 2013 show at the latter theater was the one that was captured for his recently released Spirits…Live CD, DVD and Blu-ray.
Meanwhile, Hayward promises that fans who come out to his new concerts will get to experience "a different show" from his previous outings.
"I'm introducing quite a few different numbers to what we did on the DVD, and it's kind of evolving, this stage show," he explains.
Among the tunes Justin says he's adding to his set are two Moody Blues gems -- "Watching and Waiting" from 1969's To Our Children's Children's Children album and "You Can Never Go Home" from 1971's Every Good Boy Deserves Favour . Hayward points out that these numbers hail from a time he refers to as his "blue period."
"I went through very disturbed time, I think, in…the late '60s and early '70s," he explains, "and I wanted to revisit some of those songs as I kind of cathartic exercise for myself, to try and figure out what the hell was going on."
Hayward tells ABC News Radio that following the solo trek, he'll rejoin The Moody Blues for another road trip. He says that although nothing's confirmed, the band is eyeing a 2015 U.S. tour leg that would run from the middle of March through May.
Here are all of Hayward's solo U.S. tour dates:
10/17 — Durham, NC, Carolina Theatre
10/18 — Newberry, SC, Newberry Opera House
10/20 — Clearwater, FL, Capitol Theater
10/22 — Jacksonville, FL, Florida Theatre
10/23 — Orlando, FL, The Plaza LIVE
10/24 — Ft Lauderdale, FL, Parker Playhouse
10/26 — Atlanta, GA, Buckhead Theatre
10/28 — York, PA, The Pullo Center
10/29 — Sellersville, PA, Sellersville Theatre
10/31 — Atlantic City, NJ, Borgata
11/1 — New York, NY, The Concert Hall
11/2 — Tarrytown, NY, Music Hall Theatre
11/4 — Ridgefield, CT, Ridgefield Playhouse
11/6 — Vienna, VA, Wolf Trap for the Performing Arts
11/8 — Munhall, PA, Carnegie of Homestead Music Hall
11/11 — Nashville, TN, City Winery – Nashville
11/12 — Paducah, KY, Carson Center
11/13 — St. Louis, MO, River City Casino and Hotel
11/14 — Bloomington, IL, The Castle Theater
11/16 — Milwaukee, WI, Vogel Hall Marcus Center
11/17 — Chicago, IL, City Winery – Chicago

redpajama_set_name: RedPajamaCommonCrawl


As it is specified by the name all the things that we do within our website like as Meta title, Meta description, Unique and quality content, image, keyword stuffing, internal linking, etc. On-page technique refers structuring or building your website to be search engine friendly or a website which can be crawl by the Google crawlers.
Other tactics to get your business popular among the targeted audience is Off-page SEO and it will help make your website popular on the internet, so you can get more visibility. With the on-page techniques we can get visibility in search engines. It is the only off-page SEO techniques which help improving your website position or keyword ranking in SERP. Now take a look at the list of top 10 best off-page SEO techniques now in trend.
A must known Off-page SEO technique is social media engagement. If you want to make your business, website or blog popular, engage with people on multiple social media platforms and spread awareness about your product or services on social media. Social media presence will help grow your business and also help you get more back links and brand awareness with in the specific age groups.
Social bookmarking are one of the best and major platforms to promote your website and to get maximum quality back-links. When you bookmark your webpage URL's or blog post on popular social bookmarking websites, you gain high traffic to your webpage or blog and it helps improving your website ranking.
Search forums which are related to your website and business and make a connection with that community related to your business. Reply to threads, answer people's questions and give your suggestions and advice. Make use of "Do-Follow" forums.
Directory Submission is constantly working to build quality back links but now a days trend for directory is low but it is use full for generating back-links for your website . Choose an effective directory and select a proper category. It takes quite some time to deliver good results, but these results stand out over a longer time period.
Article or blog submission is one of the major part for website promotion. Submit your articles in a high PR article submission directory and get quality traffic. You can also give links to your website. Make sure your content is unique and of high quality. Low quality content and content that has more keyword stuffing might get rejected. Choose the correct category and give a good title to your content.
Q & A helps creating awareness about the brand and helps you to improve your services after getting people engagement over the questions. One of the best ways you can get high traffic is from question and answer websites. Join high PR question and answer sites and search for questions related to your business, blog or website and give clear answers to these questions. Give a link to your website which will help in bringing you more visibility.
Do you want to make your videos popular, head to popular video submission sites. Give a proper title, description, tags and reference links. It is one of the more popular ways to get quality back links because all video submission websites have high PR.
Sharing your photos on popular image submission websites is the good way for generating back-links and traffic to your website and spread awareness among the people. Before submitting your images please optimize them with the correct URL and title tag. Before submitting your images, check if they have a proper title, description and tags.
Create and attractive documents related to your business or blog are key for your business. The documents should have unique content and should be in either PDF or PPT formats. Submit these documents in the document sharing websites.
Proper On-page and Off-page optimization work will give better position to your website and keywords which are related to your business in SERP. The above websites are listed based on the website's PA (Page Authority), DA (Domain Authority) and PR (Page Rank). Please comment here if you know any other qualities that we can add to this list which we might have forgotten.
Note: Google Page Rank (PR) is no longer a valid quality factor for any webpage URL or Blog, hence you can ignore that column and above mentioned PA, DA may vary a little bit with the current data. We will keep updating other Digital Marketing blog which helps you to get more engagement.


Chalet hotel "Les Campanules" welcomes you at the very heart of the Mont-Blanc mountain range, in the village of Les Houches, right next to Chamonix. We invite you to spend some truly quality time in a relaxing natural environment of greenery and forest.
You will have the most amazing and unforgettable views of the Mont-Blanc mountain range, the Bossons and Taconnaz glaciers as well as the "needle peak of the South" also known as the Aiguille du Midi... This family orientated hotel, which embodies the region insures the promise of a joyful stay thanks to its tangible warm atmosphere and friendly service.
Comprised of 43, one to 4 person rooms, which are all contemporary, yet true to the region, integrally equipped with satellite TV, free Wi-Fi and ensuite bathrooms and toilets. Enjoy our 6-person apartment, our restaurant and a choice of several reception rooms to relax in.
Our in-house restaurant offers a menu highlighting fresh seasonal products as well as specialities of the Savoie region. All our dishes are home-made, ensuring an exquisite taste experience. We exclusively cater to our in-house guests during the evening service.
Catering to all your professional needs, why not use our 100m² plenary room (which is easily converted into a theatre, catwalk or seminar room...) and is equipped with all the latest technical equipment (screen, video projector, blue tooth microphone, speaker system, flip charts.
We look forward to welcoming car and motorbike enthusiasts in a charming setting for a well-earned rest during your trip across the Alps or your Mont-Blanc round trip for example.
Free parking at the hotel, undercover parking for motorbikes.
The view to the Mont-Blanc summit, the forest environment and that high-altitude meeting with nature are all yours to enjoy.
We are open all year around, so come and stay with us any time of the year to enjoy each of the four seasons!
Do you need to unwind? Then enjoy our dedicated relaxation space complete with sauna, hammam and fitness equipment.
We offer personalised massage therapies, on request.
Yoga and Pilates sessions are offered by experienced and fully certified instructors. Be inspired to discover these unique relaxation techniques.
In winter: free shuttles are provided for skiers (from the hotel to the ski pistes of Les Houches), lift passes can be purchased at the reception, ski/equipment can be rented from partnering businesses, we can provide information about winter hikes, snow shooing... we are committed to make your stay hassle-free and very comfortable.
In summer: hikes start from the hotel, we will happily provide information and assist with reservations to visit the unique local sites of: Aiguille du Midi, Mer de Glace, tramway du Mont-Blanc, parc de Merlet... We are eager to share our love for our amazing region and point you towards the most cost-effective summer package deals to enjoy the valley.
In summer and winter, enjoy the numerous high-altitude domains of the valley, such as the: Aiguille du Midi, Montenvers train, Mer de Glace, Brévent, Flégère, Mont-Blanc tramway, Le Tour, Col de Balme, etc.
Recharge your batteries, either on your own, as a couple or as a family, with a mountain hike on the numerous suggested itineraries.
The children will be absolutely delighted to discover Parc de Merlet, located just a few bends of the road above us, with more than 80 mountain animals displayed roaming freely in their natural environment.
Be exhilarated by mountaineering all year round, from climbing to ski-touring the Mont-Blanc loop, all is possible with high mountain guides and trek leaders.
Relax and let us tempt you with numerous offerings: massages, manicures, pedicures and facials... All you need to do is ask!
Enjoy yourself at the Chamonix Casino, the bowling, the Alpine museum, the amusement parc at Planards or the Giannada foundation art gallery (Martigny - Switzerland).
Please don't hesitate to ask us for more details on any number of these activities, we will very gladly provide you with all the information.
Traditional restaurant and local specialties, service in the pure tradition of the French hotel industry.
Free parking cars and motorcycles.
Free shuttle from the hotel to Les Houches ski slopes.
Living room fireplace, billiard room and bar area.
Sauna, fitness area and yoga class.


text:
Metal reclamation and recycling of electronic waste
Revision as of 18:39, 24 November 2008 by Robinwilkes (talk | Contributions)
(Difference) ← Older revision | Latest revision (Difference) | Newer revision → (Difference)
Hi! This page is currently under construction. Check back later!
This page was part of a project for MECH370, a Queen's University class on materials processing. It is now open edit.
Please leave comments using the discussion tab.
Currently, the majority of electronic waste is disposed of in a landfill. The remainder is either processed in energy inefficient method that only recovers a small amount of the available metals or burned to recover saleable metals such as copper, aluminum and iron. This is a problem for a number of reasons.
This waste contains recoverable trace amounts of precious metals, and larger quantities of a variety of other metals and alloys, especially copper, aluminum and steel.
Electronic waste contains high concentrations of heavy metals, brominated flame retardants and other plastic additives that have proven adverse effects on humans. Note: more information can be found at Electronic waste.
The volume of electronic waste produced is high, and growing fast [1]. In 2005, the United States alone disposed of was at least 1.5 million tons of electronic waste, and at most one quarter of that was recycled [2]. The situation is similar in Canada, with more than 71 000 tonnes of waste being disposed of in 2005, with just 26% being recycled [3]. In the province of Ontario, Canada, the percentage recycled in 2004 was just 2% [4].
Discarding recoverable metals is energy inefficient. The energy savings possible by recovering metals are high compared to mining new material. The energy savings for aluminum is 95%, copper 85%, steel 74%, lead 65%, and zinc 60%. Recovering other materials such and paper and plastics can reduce energy usages by 64% and 80%, respectively [5]. The worst case scenario eliminates only the ore extraction and transportation steps, which are both energy intensive.
1 Materials involved
1.2 Surface mounted components
1.3 Casings
1.4 CRT and LCD screens
1.5 Wire
1.6 Batteries
1.7 Precious metals
2 Current solutions
2.1 Shredding
2.2 Municipal incineration
2.3 Pyrometallurgical recovery
2.4 Exhaust gas scrubbing
3 Progress towards a sustainable future
3.1 Alternative materials and manufacturing
3.2 Comprehensive processing plan
Materials involved
There are a wide variety of materials involved in the multitude of items that can be classified as electronic waste. Each component adds to the complexity of any recycling effort. Below is an exploration of a number of the most common components, by weight.
A printed circuit board, or PCB, is the piece of hardware acts as a base and provides electrical connections to the mounted components. They are present in many types of electronic waste, including cellphones, computers, TVs, and printers. A PCB is made of a number of components. Each step of the productions process is outlined below, to give an idea of the variety of materials involved.
picture of a circuit board here
FR-4 is the most common base material [6] for printed FR-4 is an abbreviation of Flame Retardant 4, referring to its flame resistance and self-extinguishing properties. It is a brittle material formed by hardening a woven fiberglass sheet with an epoxy resin, usually created from ethylene clorohydrin and bisphenol-A [7]. To give it the aforementioned self extinguishing properties, a brominated flame retardant is incorporated in the epoxy. Some flame retardants can be incorporated at the molecular level, like TBBPADEPRECATED TEMPLATE - PLEASE USE {{W}} INSTEAD. [6]. It can be easily coloured, with common colours including green, blue, red, and black.
The type glass used to create the fiberglass sheets is S-glass. Content ranges, by weight are, 52-56% silicon dioxide, 16-25% calcium oxide, 12-16% aluminum oxide, 5-10% boron oxide, 0-2% sodium oxide or potassium oxide, 0-5% magnesium oxide, 0.05-0.4% iron oxide, 0-0.8% titanium oxide, and 0-1% fluorides [6].
The FR-4 is layered with copper traces, and bonded with subsequent layers. Computer motherboards today have as many as eight layers [8].
Surface mounted components
A wide variety of components are soldered to printed circuit boards. A resistor is comprised of copper leads attached to a painted ceramic or carbon core [9]. Microchips are composed of small amounts of silicon, aluminum, and copper, [10] with plastic coatings. CPUs today have aluminum heatsinks as well.
Most consumer electronic devices have plastic casings, such as a TV or a cell phone. Household appliances also have aluminum or steel cases. Other products, such as computer cases have both metallic and plastic components to them.
CRT and LCD screens
CRT, or cathode ray tubeDEPRECATED TEMPLATE - PLEASE USE {{W}} INSTEAD., monitors are currently being phased out in favour of LCDDEPRECATED TEMPLATE - PLEASE USE {{W}} INSTEAD. monitors.
A CRT monitor consists of the electron gun and focusing equipment, a plastic casing and leaded glass. An analysis of the glass resulted in the following results, by weight, are 49.61% silicon dioxide, 24.17% lead oxide, 7.79% potasium oxide, 5.32% sodium oxide, 3.63% aluminum oxide, 2.99% strontium oxide, 2.30% calcium oxide, 1.96% barium oxide, 1.49% magnesium oxide, 0.58% zirconium oxide, 0.07% iron oxide and 0.07% phosphorous oxide [11]. The lead and phosphorous oxide [12] are most difficult to deal with. Most of the lead (up to 98.6%) can be recovered through a pyrovacuum process, where high heat and low pressure allow the lead to be vaporized and later recovered, with the aid of carbon powder[11].
Wire is a common element in electronic waste, as it has a method of connecting to a household wall socket, either through a power cord or charger. Internal wiring is common especially in desktop computers and older audio and video equipment.
Batteries are a staple for portable electronic devices, whether in lithium ionDEPRECATED TEMPLATE - PLEASE USE {{W}} INSTEAD., rechargable NiCdDEPRECATED TEMPLATE - PLEASE USE {{W}} INSTEAD. or NiMHDEPRECATED TEMPLATE - PLEASE USE {{W}} INSTEAD., AAA or AA alkaline cellDEPRECATED TEMPLATE - PLEASE USE {{W}} INSTEAD. form. While batteries are electronic waste, they will not be covered in this analysis. There are numerous battery recycling programs; see [1][2][3].
Precious metals are used in electronics for their superior conductivity and resistance to oxidation. They are used as A study undertaken by Cui, J. et Al., at the Norwegian University of Science and Technology showed that there are recoverable amounts of precious metals, such as silver, gold and palladium. For typical electronic scrap, they found and average of 2000 ppm silver, 1000 ppm gold and 50 ppm palladium. Outliers include printed circuit boards with 3300 pm silver and cell phones with 210 ppm palladium[13]. This is significant, as this means that in the United States, using 2005 data [14], these numbers show that there was up to 1500 tons of gold and 3000 tons of silver in the electronic waste that was disposed of in the U.S.A. in 2005.
Current solutions
Solutions is a misnomer for this category. Electronic waste processed in a manor shown below either does not recover usable materials, is selective of feedstock, or processes the electronic waste in a manner that has measured environmental effects.
It should be noted that the bromine in brominated flame retarded plastics and printed circuit boards were found to mainly produce a gas made of two dangerous compounds: hydrogen bromideDEPRECATED TEMPLATE - PLEASE USE {{W}} INSTEAD. and bromobenzeneDEPRECATED TEMPLATE - PLEASE USE {{W}} INSTEAD..[15]
In this process, commonly known as mechanical e-waste recycling, electronic waste is shredded by specialized equipment. An example of a shredder used in Kansas, U.S.A.[16] may be found here. This waste is then sorted mechanically, by magnetic fieldDEPRECATED TEMPLATE - PLEASE USE {{W}} INSTEAD. or eddy currentDEPRECATED TEMPLATE - PLEASE USE {{W}} INSTEAD. separators, or novel means such as vertical vibration separation[17]. This last method has demonstrated effective for separating metals from plastics, especially copper[17].
Another product of this method is fine dust. Research has been conducted into possible uses of this dust. In a study conducted by Kakimoto, K et Al., it can be successfully incorporated into Portland cement, without the loss of strength, up to 30% by weight. An analysis of this dust found to that it was mainly silicon dioxide, calcium oxide and aluminum oxide, with trace amounts of lead and copper.
This process is effective at separating metals from plastics, but fails to address the separation of low concentration metals from printed circuit boards.
Municipal incineration
Municipal incineration is simply just incinerating electronic waste with other household and business waste in a municipal incinerator. While many components of electronic waste have high usable energy content, this dilutes metal concentrations down even further.
Stewart, E. et Al. studied the emissions of incinerated computer components and monitored it for VOCs, SVOCs, dioxins, halogens, and metals. With a mass flow rate of 1.944 kg/h, their findings are as follows, with all weights listed being per dry standard cubic meter. The halogen chlorine at 7.9 mg/dscm, dichlorine 0.3 mg/dscm, bromine 13.1 mg/dscm, dibromine 17.8 mg/dscm. The metal concentrations were copper at 4600-7950 µg/dscm, lead 3790-4840 µg/dscm, antimony 790-1540 µg/dscm, cadmium 55-81 µg/dscm, manganese 19-52 µg/dscm, nickel 8-17 µg/dscm, barium 6-28 µg/dscm, arsenic 3-6 µg/dscm, chromium 3-4 µg/dscm, cobalt 0.7-2.3 µg/dscm, and beryllium 0.1-0.3 µg/dscm. Volatile organic compounds were present at bromobenzene 830-920 µg/dscm, tribromomethane 40-70 µg/dscm, bromomethane 20-190 µg/dscm, benzene 25-35 µg/dscm, dibromomethane 0-20 µg/dscm. Semi-volatile organic components were measured at naphtalene 1.2-3.3 µg/dscm, acetophenone 1.0-2.0 µg/dscm, chlorobenzene 0.4-1.4 µg/dscm, and dibenzofuran 0.3-0.8 µg/dscm [18]. These levels are all extremely small. It would be easy to dismiss them as insignificant, except for the scale of the trial - at less than 2 kg per hour, scaling up to multiple tonnes as hour would cause a proportionate increase in emissions. An output of such a wide range of harmful substances would then disqualify this method as a sustainable way of dealing with electronic waste.
Pyrometallurgical recovery
This process is conducted by adding shredded electronic waste with high copper contents to copper ore concentrate and is then refined through the use of heat. Two examples of this, each recycling about 100 000 tonnes of electronic waste per year, are shown below.
At a copper smelter, in Quebec, Canada, the Noranda process is used. Copper ore concentrates at about 24% copper and shredded electronic waste are immersed in a liquid metal bath, at 1250 C. Oxygen enriched air (39% oxygen) is added. This converts iron, lead and zinc into oxides, which are removed with the silicon dioxide based slag. It is refined in an anode furnace, resulting in an alloy of 99.1% copper, with the remainder made up of silver, gold, platinum, palladium, selenium, tellurium and nickel [13].
At the Ronnskar smelter in Sweden, a similar process is undertaken. Shredded electronic waste with low metal concentrations is added at the start of the process, while higher concentration e-waste is added later. Zinc is removed at an earlier step with slag and then separately refined. During the refining process, precious metals such as selenium, gold, silver and palladium, are removed at separate steps than lead, and nickel. This leaves a high purity copper as the final product Cite error: Closing </ref> missing for <ref> tag. One type of bacteria, C. violaceum, was able to leach gold from larger pieces of electronic waste (5 x 10 mm). It dissolved 14.9% of the approximately 10 mg of gold present as dicyanoaurate [19].
The conditions required for the organisms to survive and leach these metals dictates that the electronic waste piece sizes are extremely small and have a low spacial density. This means that this process would be useful only for recovering metals from the dust generated by shredding.
Exhaust gas scrubbing
There have been a number of demonstrations of bromine recovery, either through the use of acidic and base gas scrubbers[20], or just a sodium hydroxide scrubber [21].
Progress towards a sustainable future
Alternative materials and manufacturing
A number of alternatives have been proposed to the materials and processes currently used. These initiatives are detailed below.
An alternative to FR-4 is a circuit board composed of chicken feathers with a soy-based epoxy. If this technology becomes commonplace, this would have a massive effect on the composition of the average electronic waste.
Lead free solder. The RoHSDEPRECATED TEMPLATE - PLEASE USE {{W}} INSTEAD. Directive of the European Union currently specifies that products that are sold in their jurisdiction must contain no more than trace amounts of lead. Fortunately, this has had an impact on products available in North America, with the RoHS logo adorning many of the relevant electronic products in N.A. as well. This, along with the move to LCD based monitors and TVs will cause a large reduction in percentage lead composition of electronic waste.
Elimination of halogens. Simply by reading this page, the complications that halogens such as bromine cause are made apparent. An alternative to brominated flame retardants would have a very positive effect on the environmental release of halogens due to electronic waste.
Comprehensive processing plan
From all of the processes outlined so far, an attempt at a comprehensive plan for the recovery of metals and ultimately the recycling of electronic waste can be made.
When the waste is collected, batteries and CRT monitors are diverted to specialist recyclers.
The remaining waste is shredded, with care that the dust is collected.
The dust then undergoes a bioleaching process to remove metals.
The waste is subjected to magnetic separation, removing iron and steel. This is sent to a steel mill.
Then waste then undergoes eddy current separation, which removes the majority of the remaining metal.
The remaining, mainly plastic, waste undergoes either a thermal depolymerization or plasma arc gassification process to use as much of the internal energy present, either in the production of electricity or hyrdrocarbons.
This process increases efficiency over single processes in a number of ways.
↑ http://www.epa.gov/epawaste/conserve/materials/ecycling/manage.htm Accessed Nov. 11, 2008
↑ http://www.epa.gov/epawaste/conserve/materials/ecycling/docs/fact7-08.pdf Accessed Nov. 11, 2008
↑ http://www.ec.gc.ca/wmd-dgd/default.asp?lang=En&n=F3852FB1-1 Accessed Nov. 11, 2008.
↑ http://www.ene.gov.on.ca/en/news/2007/061201.php
↑ Nnorom, I. et Al., 2007, "Overview of electronic waste (e-waste) management practices and legislations, and their poor applications in the developing countries," Resources, Conservation and Recycling, 2008, (52), p. 5.
↑ 6.0 6.1 6.2 Coombs, C., 2001, Printed Circuits Handbook Fifth Edition, McGraw-Hill, New York, section 6.
↑ http://www.p-m-services.co.uk/how%27s_fr4_made_.htm Accessed Nov. 7, 2008.
↑ http://www.fudzilla.com/index.php?Itemid=37&id=7256&option=com_content&task=view Accessed Nov. 7, 2008.
↑ http://www.ecawa.asn.au/home/jfuller/electronics/resistors.htm Accessed Nov. 11, 2008.
↑ http://www.intel.com/education/makingchips/index.htm Accessed Nov. 11, 2008.
↑ 11.0 11.1 Chen, M. et Al., 2008, "Lead recovery and the feasibility of foam glass production from funnel glass of dismantled cathode ray tube through pyrovacuum process," Journal of Hazardous Materials, currently unprinted, p. 2.
↑ http://avogadro.chem.iastate.edu/MSDS/P2O5.htm Accessed Nov. 12, 2008.
↑ 13.0 13.1 Cui, J. et Al., 2007, "Metallurgical recovery of metals from electronic waste: A review," Journal of Hazardous Materials, 2008, (158), p. 3.
↑ http://www.epa.gov/epawaste/conserve/materials/ecycling/docs/fact7-08.pdf p. 1, Accessed Nov. 11, 2008
↑ Chien, Y. et Al., 1999, "Fate of bromine in pyrolysis of printed circuit board wastes," Chemosphere, 40, (4).
↑ http://www.prlog.org/10046578-automated-waste-shredding-system-operational.html Accessed Nov. 12, 2008.
↑ 17.0 17.1 Mohabuth, N. et Al., 2006, "Investigating the use of vertical vibration to recover metal from electrical and electronic waste," Minerals Engineering, 2007, (20).
↑ Stuart, E. et Al., 2003, "Emissions from the Incineration of Electronics Industry Waste," IEEE, 2003, (03).
↑ Cui, J. et Al., 2007, "Metallurgical recovery of metals from electronic waste: A review," Journal of Hazardous Materials, 2008, (158), p. 18.
↑ Boerrigter, H. et Al., "Bromine Recovery From The Plastics Fraction of Waste of Electrical and Electronic Waste (WEEE) With Staged Gassification," European Brominated Flame Retardant Industry Panel, 2002.
↑ http://www.flameretardants.eu/Objects/2/Files/R-2002_Conference_Geneve.pdf Accessed November 20, 2008.
Retrieved from "https://www.appropedia.org/index.php?title=Metal_reclamation_and_recycling_of_electronic_waste&oldid=58394"
MECH370
Materials processing


Hi Guys! Another Famous Fridays is here! And a very Happy Birthday to two famous people in my family--my sisters!!
Today we focus on the amazing Francois Payard, legendary French pastry chef and patisserie owner and I definitely hit the jackpot here! Guys,--Warm. Chocolate. Tart. Doesn't it sound like a dream come true?!! And at the risk of sounding presumptuous, I think this may be one of the simplest and best desserts I have ever made and anyone who knows me, knows what a bold and daring statement this is! Seriously, I know chocolate tart is not traditional for Thanksgiving, but you are going to want to include this on your dessert table. I know I am!
Payard was born in Nice and is a third generation pastry chef, not only famous for his gorgeous and intricately designed pastries but also because he adheres to the simple traditions of classic French pastry. In other words, he makes things that not only look good, but taste good too! My daughter, husband and I went to his flagship store on 74th & 3rd Ave for a special birthday brunch over the summer and we swooned over the delicious macarons and pastries. If you have a chance, stop in and treat yourself to a goodie!
This tart is quite simple to make and has the added ease of being able to be prepared in steps. it's a breeze to make the tart dough, which all gets mixed in the food processor and then chilled overnight.
The next day you roll it out and prebake it in a tart pan (I always use beans to weigh down my dough). It's very easy and forgiving and bakes up light and sweet and almost chewy.
And the most amazing thing is that you barely do anything to the chocolate other than adding a little milk, cream and one egg and the result tastes like you ordered dessert at the most fancy restaurant! Seriously it takes like 10 minutes to put it together and basically you're just heating milk and cream. The filling is warm, creamy and rich, but not overly so and the sweetness of the dough compliments it perfectly. And you can make it the day before, keep it chilled and then reheat it right before serving because it really is divine served warm!
The tart is great on its own but (as almost all things are) elevated to new heights with a scoop of vanilla ice cream cuddling up to it.
I have so many things to be thankful for and now Francois Payard and this amazing chocolate dessert are two more to add to the list!
Have a great weekend and look for more great Thanksgiving recipes next week!!
4. On a wooden board, or other surface, sprinkle some flour. Then sprinkle the dough lightly with flour as well as your rolling pin and roll out the dough into a 12 inch circle. Make sure that as you are rolling you lift the dough and reflour the surface a bit as it has a tendency to stick. Transfer it to the prepared pan and press it into the sides, removing any dough that hangs over the top of the pan. Prick the dough all over with a fork and chill the pan for about 20 minutes.
6. To make the filling: Preheat oven to 325ºF. Place the chopped chocolate in a medium bowl and set aside.
7. Pour the milk and cream into a medium sized pot and bring to a boil over medium-high heat. Pour the mixture over the chocolate and allow it to stand for 30 seconds. Then whisk it until the mixture is smooth and the chocolate is completely melted. Cool for 10 minutes.
8. Whisk the egg into the chocolate mixture and pour the filling into the tart shell. Bake for 10-13 minutes, until the edges of the filling are set, although the center will still be rather jiggly. Cool for 10 minutes on a wire rack and serve warm.
9. If making this a day ahead of serving, reheat in a 250ºF oven till warm.


text:
SportLocal Sport
sport, local-sport,
North Whyalla have proven to be West Whyalla's "kryptonite" this season once again, defeating the Dragons by five points in a round 12 thriller. The 6.5 (41) to 5.6 (36) win puts the Magpies at two wins and a draw over West this season. J. Trezise, T. Brown and W. Dare were best for North while I. Mills, A. Smith, S. Winders were best for West. North ended the Dragons' two-year undefeated streak in Round 2 with a six-point win. This was followed by a draw between the two sides in round 7. In other results South Whyalla returned to the winners' list with a much-needed 68-point win over Roopena on the back of a five-goal performance by N. Van Eekeren. Weeroona Bay continued their good form by accounting for Central Whyalla by 17 points 9.9 (63) to 6.10 (46).
July 17 2018 - 11:12AM
Magpies win tight contest in upset
Louis Mayfield
North Whyalla have proven to be West Whyalla's "kryptonite" this season once again, defeating the Dragons by five points in a round 12 thriller.
The 6.5 (41) to 5.6 (36) win puts the Magpies at two wins and a draw over West this season. J. Trezise, T. Brown and W. Dare were best for North while I. Mills, A. Smith, S. Winders were best for West.
North ended the Dragons' two-year undefeated streak in Round 2 with a six-point win. This was followed by a draw between the two sides in round 7.
In other results South Whyalla returned to the winners' list with a much-needed 68-point win over Roopena on the back of a five-goal performance by N. Van Eekeren.
Weeroona Bay continued their good form by accounting for Central Whyalla by 17 points 9.9 (63) to 6.10 (46).
ADRA's Christmas spirit


Snowflake Obsidian is black volcanic glass with inclusion of small, white, radially clustered crystals of cristobalite that produce a blotchy or snowflake pattern. Pendant stone is 1.5" (38.1mm) long x 8.4mm (0.32") wide. Earring stones are 0.73" (18.6mm) long x 0.33' (8.4mm) wide. All dimensions are approximate. Ear posts are stainless steel. Ask about available chains and cords!.


Here goes my little boy again. He lost his pencil again and it's becoming a normal case now. I've talked with his teacher last month to help me with this problem and now it's happening again. Last week his classmate pointed me where his other pencils are and the boy who got it returned the pencils to him. Now my little boy told me that his classmate borrowed those pencils to him. Maybe he didn't want to embarrass his classmate. Oh boy! Hope with my discovery of that this frequent cases will stop because I don't want to make an issue out of this. Now his notebook is missing and it's supposed to be with his teacher. I'll go again to his school this afternoon to follow up his notebook and hope I'll get a positive answer. Their notebooks were bought from the school so it's not that easy to replace it. Anyway it's not his fault so I'm not forced to replace it. Meanwhile Josh brought temporary notebook to write on.


The empirical failure of the uncovered interest rate parity condition seems to be the reflection of risk premia on foreign currencies. After the formation of foreign currency portfolios according to interest rate differentials or forward discounts, recent studies suggest that either consumption- or currency return-based pricing factors explain the cross-section of foreign currency portfolio returns. The contribution of this paper is twofold. It shows that the returnbased explanation applies to foreign currency portfolios sorted from the perspective of a Euro Area investor. Secondly, the main results of this paper suggest that the decisive pricing factor, the so called carry trade premium, mirrors business cycle related risks. Times of relatively large amounts of uninsured Euro Area consumption growth risk are associated with an expected increase of the carry trade premium.


text:
'Women Talking's Sheila McCarthy Signs With Atlas Artists
Pete Buttigieg, In 'Ellen Show' Appearance, Again Responds To Rush Limbaugh
By Ted Johnson
Ted Johnson
@tedstew
More Stories By Ted
T.J. Holmes And Amy Robach To Exit 'GMA3,' Leave ABC News
Carlos Osorio/AP/Shutterstock
Pete Buttigieg is in Los Angeles today for campaign events and an evening fundraiser co-hosted by Seth MacFarlane and Lee Daniels, but he also squeezed in time for a guest spot on The Ellen Show.
Host Ellen DeGeneres asked him to respond to Rush Limbaugh's questioning a gay man debating President Donald Trump.
"How is this going to look? A 37-year-old gay guy kissing his husband on stage next to Mr. Man Donald Trump?" Limbaugh said on his show earlier this month.
Buttigieg told DeGeneres, "Look, I guess he just has a different idea of what makes a man than I do. Look, I'm not going to take lectures on family values from the likes of Rush Limbaugh or anybody who supports Donald Trump, frankly.
Montecito Resident Ellen DeGeneres, Sheltering In Place, Posts Video Of Raging Water Beside Her Home: "This Is Crazy!"
He added, "You know, when I was packing my bags for Afghanistan, Donald Trump was working on Season 7 of Celebrity Apprentice. I'm just done with that kind of… And since when is strength about the chest-pounding and the loud-mouthed guy at the end of the bar? The strongest people I know are not the loudest people; they're the ones who have the deepest sense of who they are and what they value and what they care about. And one of those people, by the way, one of the strongest people I know is my husband, Brad Pitt."
The reference to Pitt was an apparent joke Buttigieg has with his husband, Chasten, who tweeted a response and clip from DeGeneres' show.
This joke comes full circle tomorrow… https://t.co/4r0dNtOPeD pic.twitter.com/gVGQ5v8FT7
— Chasten Buttigieg (@Chas10Buttigieg) February 21, 2020
At a CNN town hall earlier this week, Buttigieg was asked to respond to Limbaugh's claim that Trump told him to "never apologize" for the remarks.
At the town hall, Buttigieg said, "The thing about my marriage is it has never involved me having to send hush money to a porn star for cheating on my spouse."
Buttigieg's Los Angeles visit on Thursday also included a town hall event sponsored by Fox 11 and USC Center for the Political Future. His fund-raising event ranged in price from $54 per person up to $2,800 per person.
The campaign is under the gun to raise millions in advance of Super Tuesday, when California and 13 other states hold primaries and caucuses. Buttigieg's campaign sent out an email to supporters saying that they needed to raise $13 million to stay competitive, after raising $6 million in January and $11 million so far this month.


I have never purchased online before. Is Melbourne Diamond Company a reputable retailer?
Our directors have been in the Australian diamond wholesale business since 1981 and saw the opportunity to introduce new and innovative ways for customers to purchase high quality diamonds and jewellery in the privacy of their own homes and at their own pace. We are also a registered company with ASIC and abide by all of the Australian Consumer Regulations.
Can we come and see you at a retail store?
We are an online retailer only and that is how we can offer such great pricing on our products ! We are available during business hours 7 days per week, and also 24 hours a day / 7 days per week via our online chat service.
Yes. We offer a Lifetime Warranty to protect against manufacturer defects in craftsmanship. The warranty does not include normal wear and tear or damages incurred due to trauma, negligence, lack of regular maintenance, loss or theft.
How do I safeguard my purchase against damage, theft, or loss?
We recommend that you contact an independent insurance company or make arrangements with your current home contents insurance provider. Try to always compare rates and coverage before choosing a provider and ask specific questions regarding potential situations (eg "What happens if my jewellery gets lost or stolen?" or "What happens if my wedding ring or diamond goes missing?"). Knowing your valuable jewellery is safely insured will give you peace of mind.


text:
The Cell and Tomb of Saint Melania the Younger in Jerusalem
Saint Melania the Younger was initially a wealthy matron from Rome, who came to Jerusalem in 417 to establish a convent near the Mount of Olives and live out the rest of her days in asceticism. When she arrived in Jerusalem with her mother and husband Pinianus, Melania with her mother took up her abode in a little cell of the common hospice for pilgrims, close to the Church of the Resurrection, while Pinianus, it seems, was separated from them, the men being lodged in a place apart from the women. Here, then, in the heart of Jerusalem, and flooded with the luminous rays from our Lord's Cross and Sepulchre, Melania's soul was consumed more and more with the fire of Divine Love. Buried in obscurity, and enjoying the most complete self-effacement, she found her delight in continual fasting, in unwearied prayer, in charity and assiduous study of the Holy Scriptures. Her brief rest was taken on the hard ground, covered only with little mats of rough hair-cloth. When the shadows of evening fell, and the custodians at the end of Vespers closed the gates of Constantine's Basilica, it was a beautiful and touching sight to behold Melania go forth alone from her little cell, and prostrate herself before the doors of the Sanctuary, there to pass the night in prayer and vigil until they were opened again at cock-crow in the morning. The severe and prolonged fasts, with other austerities, brought on an illness during which it was with great difficulty that the Saint was prevailed upon to accept a pillow upon which to rest her aching head.
In the area of this cell, after her repose, Cyril of Scythopolis relates how Patriarch Elias (494-516) brought together the ascetics associated with the Holy Sepulchre, who had earlier been living around the Tower of David, and built a monastery for them near the patriarchal residence. These ascetics were known as the Spoudaioi, for they served the Holy Sepulchre with haste and diligence, and the monastery came to be known as the Monastery of the Spoudaioi (or Spoudaeon Monastery). This monastery was dedicated to the Mother of God, because it was believed that here the Virgin Mother, upon seeing her Son crucified, cried out with a loud wail of sorrow. Later on it came to be known as the Hodegetria Monastery, due to an icon there of the Panagia Hodegetria, and eventually it was called Megale Panagia (Great Panagia), as it is known today. It is also called the Monastery of Saint Melania the Roman, because under the monastery is the crypt of Saint Melania where her cell is located, as well as her tomb. There is also a chapel there dedicated to Saint Melania. The monastery also treasures a Gospel Book copied by Saint Melania, and chains she wore hidden under her clothing to increase her ascetic exercises. Over the centuries the monastery was occupied by men and women at different times, but today it is occupied by nuns. In the 19th century there were about thirty nuns there, while today there are about a dozen.
Megale Panagia Monastery in Jerusalem
Icon of Panagia Hodegetria
Chapel of Saint Melania the Roman
Tomb of Saint Melania
Cell of Saint Melania
Chains of Saint Melnia
Labels: Orthodoxy In Holy Land, Saints of December, Saints of the Holy Land


With a depth of about 800m, a length of 1.7km and a cliff slope of 70-90o, Tu San is believed to be the deepest canyon in Vietnam and Southeast Asia.
Deep green Nho Que River. It flows from the area of Nghiem Son Mountain that is 1500 meters height. This river is not too long, about 46 kilometers, in which the upper area accounts for more than 5 kilometers is the border of two countries Vietnam - China, the rest part flows through Ha Giang and Cao Bang province.


After more than 30 years of working on environmental policy, I moved to within a few hundred feet of one of the Great Lakes. Given the opportunity to stroll along the shore as often as I wanted, I suddenly realized I didn't know what I could legally do when the water's edge traversed private property. I only knew the courts had been taking up disputes regarding this issue.
One local I consulted said you could walk the first 10 feet of the beach. Another said you had to keep one foot in the water at all times. I knew I couldn't assume anything.
Fortunately, Jim Olson was available.
FLOW's founder and president is one of the nation's leading authorities on the public trust doctrine, the tenet of common law that holds that our Great Lakes, their submerged lands and their shores are publicly owned — and that government has a responsibility to act as our trustee to protect them.
Jim has set forth the state of that doctrine as it applies to Michigan's Great Lakes shores. Simply put, the Michigan Supreme Court has upheld the right of the public to traverse the beach up to the ordinary high water mark. No private property owner can exclude the public from that strip of public land.
With that access comes responsibility. Not just the respect for our great waters and shores that should always apply, but also respect for shoreline private property owners. Shoreline access is not a license to litter, make noise, or otherwise disrupt the private property owner's enjoyment of his or her rights.
With that knowledge, I have trod the shores of the Great Lake I live near, savoring the sounds of swishing water and the panorama of sky and inland sea. It's a sacred gift. And the public trust doctrine protects it.
Read our beach walker's guide, and enjoy your Great Lakes shoreline.