STARDUST example

Example data and analysis for the STARDUST pipeline. Example data information from README: Slice is in aCSF + TTX perfusion, 200nM norepinephrine is added to the perfusion before start of recording, and the drug perfused to the recording chamber at around 80 frames. Recorded for 10 min total (635 frames) and acquired from Bruker. Note that the STARDUST pipeline is now adapted to three versions (STARDUST ROA-based analysis, STARDUST cell-based analysis, and STARDUST cell-assigned ROA analysis) as described in the protocol. Since the STARDUST cell-assigned ROA analysis is an extension of the STARDUST ROA-based analysis and therefore have mostly overlapping steps and outputs, in the example data, we provide the original and analysis files for (1) STARDUST cell-based ROA analysis using raw GCaMP recording, (2) STARDUST cell-based analysis using raw GCaMP recording, (3) STARDUST cell-based ROA analysis using motion-corrected GCaMP recording, and (4) STARDUST cell-based analysis using motion-corrected GCaMP recording. Raw videos: example_GCaMP.tif: raw GCaMP recording. example_tdTomato.tif: raw tdTomato recording. Motion corrected videos: example_GCaMP_corrected.tif: GCaMP recording, motion corrected using NoRMCorre. example_tdTomato_corrected.tif: tdTomato recording, motion corrected using NoRMCorre (applied same correction from the example_GCaMP_corrected.tif). 1. AQuA a) Process file in AQuA. Input parameters: - Data type: GCaMPExVivoLck - Temporal resolution: 0.945 seconds per frame (note that this parameter doesn’t affect outcome in STARDUST) - Spatial resolution: 0.884 μm per pixel - Exclude pixels shorter than this distance to border: 0 for the raw video, 2 for the corrected video Detection parameters: - intensity threshold scaling factor: 2.5 - Smoothing (sigma): 0.5 - Minimum size (pixels): 10 - Skip step 2,3,4 box checked. Run time: ~20 minutes (on a 16GB RAM computer). AQuA identified 37658 events. 2. Obtain output file from AQuA. Filtered by area range: 10-12 µm2. 3. Identify ROA from FijiAnalyze particle function identifies 1302 ROAs for original video, and 1364 ROAs for motion-corrected video. 4. Cell segmentation in Fiji: 79 cells. 5. STARDUST Jupyter notebook interactive code inputs: - Enter the frame number of drug application (enter 0 if no drug was applied): 80 - Enter the frame rate (in Hz) of the recording (e.g. 1): 1.058 - Enter the spatial resolution of the recording (in µm/pixel, if one pixel size is 0.884 x 0.884 µm^2, enter 0.884): 0.884