End-to-End PCA-ICA¶
This tool uses 2.0 compute credits per hour.
The PCA-ICA workflow can be used to perform automated source extraction from calcium imaging movies. The workflow consists of several processing steps that aim to retrieve the spatial location and temporal dynamics of neurons in a calcium imaging movie along with the corresponding neural events.
Inputs¶
| Parameter | Required? | Default | Description |
|---|---|---|---|
| Input Movie Files | True | N/A | path to the input isxd movie files |
| Temporal Downsample Factor | True | 2 | Factor by which the movie is to be temporally downsampled |
| Spatial Downsample Factor | True | 2 | Factor by which the movie is to be spatially downsampled |
| Cropping Vertices | False | N/A | A list of 4 values representing the coordinates of the top-left corner, width, and height of the area to crop: [top_left_x, top_left_y, width, height]. |
| Fix Defective Pixels | True | True | If True, check for defective pixels and correct them |
| Trim Early Frames | True | True | If True, remove early frames that are usually dark or dim |
| Low Cutoff | False | 0.005 | Low cutoff frequency of the spatial filter. If left blank, no low cutoff frequency is used |
| High Cutoff | False | 0.5 | High cutoff frequency of the spatial filter. If left blank, no high cutoff frequency is used |
| Retain Mean | True | False | If True, retain the mean pixel intensity for each frame (the DC component) |
| Subtract Global Minimum | True | True | If True, each frame is subtracted from the global minimum pixel intensity computed across the entire movie. By doing this, all pixel intensities will stay positive valued |
| Max Translation | True | 20 | The maximum translation allowed by motion correction in pixels. |
| Low Bandpass Cutoff | False | N/A | Boolean values; If not None, the low cutoff of the spatial filter is applied to each frame prior to motion estimation |
| High Bandpass Cutoff | False | N/A | If not None, the high cutoff of the spatial filter is applied to each frame prior to motion estimation |
| ROI | False | N/A | ROI vertices for motion estimation. If input is empty, the algorithm will use the entire frame |
| Global Registration Weight | True | 1 | When set to 1, only the reference frame is used for motion estimation. When less than 1, the previous frame is also used for motion estimation. The closer to 0, the more the previous frame is used and the less the reference frame is used. |
| Preserve Input Dimensions | True | False | If true, the output movie will be padded along the edges to match the dimensions of the input movie. The padding value will be set to the 5th percentile of the pixel value distribution collected from 10 evenly sampled frames from the input movie. |
| Ref ΔF/F Image | True | mean | The reference image or baseline image used to compute ΔF/F |
| Number of PCs | True | 150 | The number of principal components (PCs) to estimate. |
| Number of ICs | True | 120 | The number of independent components (ICs) to estimate. |
| Unmixing Type | True | spatial | Type of unmixing that needs to be applied. Can be either “temporal”, “spatial” or “spatio-temporal” |
| ICA Temporal Weight | True | 0 | The temporal weighting factor used for ICA. |
| Maximum Iterations | True | 100 | The maximum number of iterations for ICA |
| Convergence Threshold | True | 1e-05 | The convergence threshold for ICA. |
| Block Size | True | 1000 | The size of the blocks for the PCA step. The larger the block size, the more memory that will be used. |
| Auto Estimate Number of ICs | True | True | If True the number of ICs will be automatically estimated during processing. The number of PCs will be overriden and set to 1.2 times the number of estimated ICs. |
| Average Cell Diameter | True | 10 | Average cell diameter in pixels (only used when auto_estimate_num_ics is set to True) |
| Threshold | True | 5 | The threshold in median-absolute-deviations that the trace has to cross to be considered an event. |
| Tau | True | 0.3 | The minimum time in seconds that an event has to last in order to be considered. |
| Event Time Reference | True | beginning | The temporal reference that defines the event time. |
| Ignore Negative Transients | True | True | A boolean parameter that dictates whether to include or exclude negative transients for event detection |
File Inputs¶
| Source Parameter | File Type | File Format |
|---|---|---|
| Input Movie Files | miniscope_movie, miniscope_movie | isxd, isxc |
Recommended Workflow¶
Below is a summary of the processing stages used in the PCA-ICA workflow along with recommended processing settings.
Step 1: Preprocess¶
Spatial and temporal downsampling can help run PCA/ICA quicker.
Step 2: Spatial Bandpass Filter¶
We recommend that you apply our usual recommendation of spatial bandpass filtering with global mean subtraction.
Step 3: Motion Correct¶
No changes to the typical settings are necessary for motion correction when using PCA/ICA.
Step 4: ΔF/F¶
The ΔF/F algorithm normalizes each pixel value in the movie, so that it represents a deviation or change from a baseline. This should be applied to motion corrected microscope movies to remove any remaining spatial variation in the intensity of your signal.