23 research outputs found
Fluorescent Protein Voltage Probes Derived from ArcLight that Respond to Membrane Voltage Changes with Fast Kinetics
<div><p>We previously reported the discovery of a fluorescent protein voltage probe, ArcLight, and its derivatives that exhibit large changes in fluorescence intensity in response to changes of plasma membrane voltage. ArcLight allows the reliable detection of single action potentials and sub-threshold activities in individual neurons and dendrites. The response kinetics of ArcLight (Ď„1-on ~10 ms, Ď„2-on ~ 50 ms) are comparable with most published genetically-encoded voltage probes. However, probes using voltage-sensing domains other than that from the <i>Ciona intestinalis</i> voltage sensitive phosphatase exhibit faster kinetics. Here we report new versions of ArcLight, in which the <i>Ciona</i> voltage-sensing domain was replaced with those from chicken, zebrafish, frog, mouse or human. We found that the chicken and zebrafish-based ArcLight exhibit faster kinetics, with a time constant (Ď„) less than 6ms for a 100 mV depolarization. Although the response amplitude of these two probes (8-9%) is not as large as the <i>Ciona</i>-based ArcLight (~35%), they are better at reporting action potentials from cultured neurons at higher frequency. In contrast, probes based on frog, mouse and human voltage sensing domains were either slower than the <i>Ciona</i>-based ArcLight or had very small signals.</p> </div
A visibility overshoot index for interventional x-ray image quality assessment
Dose reduction remains an important goal in interventional x-ray. We propose an image quality (IQ) measure called the visibility overshoot index. Given a patient image and a specified clinical task, the index quantifies the maximum acceptable dose reduction. The dose control system can then use this information to deliver the minimum dose necessary for detection of clinical signals, reducing unnecessary radiation exposure. We developed an experimental visual model to estimate signal detectability as a function of image features such as noise and signal contrast. The model is used to find a feature’s threshold–the maximum change in noise or signal contrast where signal detectability remains possible. An automated algorithm measures the magnitudes of these features on a frame. Visibility overshoot is expressed in terms of the image features: the noise overshoot and contrast overshoot indices are the ratio of the threshold to measured noise/contrast. The indices demonstrate good agreement with detector dose, channelized hotelling observer results, and clinicians’ judgments. In our study of a cylindrical object phantom acquired at seven dose levels, we found that the noise overshoot index is linearly related to the square root of detector dose and the CHO detectability index, with Pearson correlation 0.995–1.0 for signals 1-4 mm diameter. For interventional cardiology and neurology sequences acquired at standard and 25–30% dose, the index and clinicians rank IQ similarly. Results on the phantom suggest at least 15% dose reduction could be achieved in fluoroscopy mode. Our patient-specific IQ approach could bring additional dose savings to clinical practice
Chicken Arclight-A173 resolves 100 Hz, 2 ms depolarization pulses.
<p>A). Two HEK 293 cells transfected with chicken ArcLight-A173 imaged with the fast CCD camera. The patched cell is in the top-right of this image. The ROI is shown in red. B). The red trace is –ΔF/F of the optical recording. The blue trace is the command voltage pulses. Overlapping the traces shows the temporal relationship between the optical recording and the command voltage pulses. It is most likely that the membrane was not fully charged during the pulse. We estimated that the actual stepped potential to be on the order of 70mV [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081295#B17" target="_blank">17</a>].</p
Single action potentials were resolved with chicken ArcLight in cultured cortical neurons.
<p>A). Top left panel: Confocal image of chicken ArcLight-A173 expressed in an acutely cultured mouse cortical neuron. Scale bar = 20 μm. Bottom left panel: An 80x80 fast CCD camera image of a neuron, from which the action potentials in the right panel were recorded. The ROIs were shaded with the matching colors to the traces. Right panel: Single trial recordings of evoked action potentials using chicken ArcLight-A173. Both the optical recording around the soma and electrode recordings were low-pass filtered with a 95 Hz Gaussian filter. The processes’ optical signals were low-pass filtered with a 50 Hz Gaussian filter. The un-filtered traces are shown as the light-colored lines. Bleaching was corrected with exponential subtraction. B). Top left panel: Confocal image of chicken ArcLight-Q175 expressed in an acutely cultured mouse cortical neuron. Scale bar = 20 μm. Bottom left panel: An 80x80 fast CCD camera image of a neuron, from which the action potentials in the right panel were recorded. The ROIs were shaded with the matching colors to the traces. Right panel: Single trial recordings of evoked action potentials using chicken ArcLight-Q175. Both the optical recording around the soma and electrode recordings were low-pass filtered with a 108 Hz Gaussian filter. The processes’ optical signals were low-pass filtered with a 50 Hz Gaussian filter.</p
Fluorescence changes of a probe based on the chicken voltage-sensing domain and the Mermaid FRET pair (mUKG-mKOk).
<p>A). Averaged optical traces of ten trials of this probe from a HEK 293 cell in response to voltage clamp depolarization steps. The fusion site was at chicken voltage sensitive domain S188, corresponding to <i>Ciona</i> ArcLight-S249 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081295#pone.0081295.s001" target="_blank">Figure S1</a>). The data is corrected by exponential subtraction and Gaussian low-pass filter of 210 Hz. B). The ΔF/F <i>vs</i> V plot of this probe. Averaged data from multiple cells were used in the analysis. -20mV: n=2; 30mV: n=4; 80mV: n=2; 130mV: n=3; 180mV: n=2. The data did not fit well with the Boltzmann equation, but were best fit with the area version of the Gaussian function.</p