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@@ -75,7 +75,7 @@ These packages detect calcium dynamics and use individual neuron statistics to p
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Furthermore, they are optimized for two-photon microscopy as opposed to wide-field microscopy.
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`SNAzzy` provides a series of automated analyses and quantifications to analyze global calcium levels in time-series acquired with widefield microscopes.
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![Schematic of the SNAzzy pipeline.
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![**Schematic of the SNAzzy pipeline.**
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Time-lapse taken from fluorescent widefield microscopes (raw data) enters the processing stage (green).
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The processing stage outputs two types of CSV files: time series of signal intensities from each recorded channel and ROI length.
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CSV files enter the analysis stage (blue) to generate normalized fluorescent traces and detect peaks along with other signal processing metrics.
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From these adaptive ROI, the average signal intensity for both channels is extracted.
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The results are saved as CSV files and are the basis for downstream analysis.
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![ROI length measurement algorithm and validation.
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![**ROI length measurement algorithm and validation.**
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A) Steps to calculate the ROI length.
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The ROI length is calculated by estimating the centerline (red line) using points of maximum (dots) in the distance transform, followed by RANSAC to ignore outliers (orange dots).
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B) Validation of the method as relative error (measured - annotated) / annotated.
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To obtain a robust centerline estimate that can ignore outliers, we use RANSAC [@fischler:1981] over the local maxima points and measure the overlap between the fitted line and the binary image.
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CNS length is also detected frame by frame and exported as a CSV file \autoref{fig:fig1}.
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![Peak detection algorithm.
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![**Peak detection algorithm.**
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A low-pass filter (orange line) is applied to the ∆F/F signal (black line) to remove fast transients.
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The peak in the filtered signal (orange dot) is then ported back to the ∆F/F (blue dot) signal by selecting the leftmost peak within a search window (blue lines).\label{fig:fig3}](figures/snazzy-fig3.png)
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Finally, a large number of different metrics and representations derived from ∆F/F, CNS length, and peaks can be visualized and plotted using the GUI.
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These include SNA onset, burst duration and spectrograms, among others.
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![GUI for data validation, curation, visualization and plotting.
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![**GUI for data validation, curation, visualization and plotting.**
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Initial GUI screen.
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A ∆F/F trace (white) and the corresponding peaks (magenta dots) are shown.
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The low-passed signal (green line) is used as a reference to determine peaks.
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# Acknowledgments
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We acknowledge Newt PenkoffLidbeck and D. Berfin Azizoglu for feedback on the manuscript.
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We acknowledge Newton PenkoffLidbeck and D. Berfin Azizoglu for feedback on the manuscript.
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This work was partially funded by NINDS and the BRAIN initiative (R00NS119295).
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# References

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