A MINiscope 1-photon-based Calcium Imaging Signal Extraction PIPEline.

MIN1PIPE is a fully automatic, Matlab-based toolbox, solving the full range problems in 1-photon calcium imaging in one package: data enhancement → movement morrection → signal extraction. It requires minimal parameter-tuning and integrates the semi-auto options. Each inidividual module can also be easily adapted for the 2-photon imaging setting.

1. Updates
2. Introduction and Features
3. Dependencies
4. Usage
5. Dataset
6. Custom Data
7. Practical Suggestions
8. References
9. Questions

3/25/2022 New version released (v3.1 & v4.0.1 (beta)): v4.0.1: MIN1PIPE now automatically detects se and spatialr. Please refer to the release notes for syntax. v3.1: latest stable version, with relatively liberal standards of selecting ROIs. Feedbacks regarding the bugs and/or suggestions are welcome.

11/19/2019 New version released (v2-alpha): new neural enhancing module with noise suppression: reduce the effect of sharp background structures. Add new output variable dff for dF/F. Feedbacks regarding the bugs and/or suggestions are welcome.

11/01/2018 New version released: updated movement correction module - balanced the running time for extremely large or shading videos; updated neural enhancing module - introduced dirt-cleaning function for dirty videos (potentially with dirts on the imaging sensor); updated seeds cleansing module - better seeds cleansing filters for seeds selection. Feedbacks regarding the bugs and/or suggestions are welcome.

09/26/2018 Updated data loading interface, for Doric scope videos with bit depth of 16.

07/16/2018 Patch version released. The program auto-detects available memory and processes data in chunk. Integrate fast read&write and memory mapping at key steps. The toolbox is undergoing some beta tests, so please expect frequent updates recently.

08/02/2018 Added guide for manual_seeds_select and real_neuron_select under the secion Usage.

08/16/2018 Added reading interface for .mat file format.

08/23/2018 Fixed bug of manual_seeds_select.

MIN1PIPE contains the following three modules:

• Neural Enhancing: remove spatial noise and then adaptively remove non-neural background in the field of view in a framewise manner.
• Movement Correction: remove field of view movement with a specially designed hierarchical nonrigid movement correction module (integrating KLT Tracker and LogDemons deformation registration method), which is free of assumption about movement type and amplitude.
• Neural Signal Extraction: identify neuronal ROIs and corresponding calcium traces with minimal false positive rates (incorporating GMM, LSTM as true neuron selector and modified CNMF as spatiotemporal calcium signal identifier)

Additional Features

• Semi-auto options: we also provide semi-auto options, including
  • Automated manual seeds selection module: for users who want to manually select seeds of neuron ROIs that will result in ZERO false positives
  • Post-process exclusion of "bad" neural components
• RNN-LSTM classifier training module: we provide a module of Recursive Neural Network (RNN) with Long-short Term Memory (LSTM) structure fully implemented in Matlab with which the users can train their specific calcium-dynamical classifier.

This Matlab implementation has the following dependencies (included under utilities):

• Modified CNMF
  • CVX: for OS other than Windows, users should download correpsonding CVX toolbox and replace the folder CVX in the codes. the package automatically determines the platform, then downloads and installs the appropriate version.

Additional Matlab toolboxes:

• Computer Vision System Toolbox
• Curve Fitting Toolbox
• Fuzzy Logic Toolbox
• Image Processing Toolbox
• Optimization Toolbox
• Parallel Computing Toolbox
• Signal Processing Toolbox
• Statistics and Machine Learning Toolbox
• Symbolic Math Toolbox
• Wavelet Toolbox

Other modified functions adapted from others are credited the original sources in code documentations.

• Do not add the package to the Matlab path before running, the package will add everything automatically.
• Download/clone the git repository of the codes
• Open Matlab and set the MIN1PIPE folder as the current folder in Matlab
• Run min1pipe.m.
  • The code automatically sets the package to the path, and processes the data the user specifies.
  • For argument options, please see min1pipe.m
• The only manual intervention is to select the data for processing.
  • A modal dialogue is popped up right after the execution of the code, and the users can select the folder containing the data.
  • For multi-video datasets automatically divided by the data acquisition softwares, only the first (or a random video of the session) needs choosing.
  • The algorithm will automatically judge the format of the datasets.
  • Currently support: .avi, .tif and .tiff.

Manual Options

• manual_seeds_select

  • A figure will pop up, including: max progection of the video after the neural enhancing and movement correction.
  • The user needs to click on the center of potentail neurons once
  • A red dot appears at the same position indicating that the seed has been successfully selected.
  • Press 'Enter' if all seeds are selected.

• real_neuron_select

  • The goal of this function is to collect the set of false-positive ROIs that the user selects.
  • A figure will pop up, including: 2 X max progection of the video after the neural enhancing and movement correction 10 calcium traces/session.
  • The user can freely click on the calcium traces, or the max projection map (bottom max map) to indicate the false positive that should be removed.
  • To do this, move the mouse cursor at the corresponding position, until a red contour of that ROI is shown on the map (bottom max map).
  • Click to select that ROI, either on the calcium trace area or the max map area.
  • A red dot of the center of the selected ROI is shown on the top max map.
  • To terminate the current session, press 'Enter'.
  • The function will automatically open the next session, until all sessions are looped.
  • A .mat file with 'filename_refined.mat' will be created in the same folder with refined key output variables.

Key Parameters:

• Fsi: frame rate of original video
• Fsi_new: frame rate of temporally downsampled video
• spatialr: spatial downsampling scalar, e.g. 0.5 for a spatial downsampling rate of 2 (now optional)
• se: structure element size, estimated from typical half-neuron size after spatial downsampling (now optional)

Procedure Parameters:

• ismc: whether use movement correction module
• flag: whether use automatic or semi_automatic seeds selection
• isvis: whether visualize after processing, including results of each step
• ifpost: whether use post-process

Key Outputs:

• roifn: processed vectorized ROI footprints; contains single cell in each column (vectorized spatial map)
• sigfn: processed calcium traces of corresponding ROIs; contains single cell in each row (calcium trace)
• spkfn: spike train inferred from refine_sig.m
• roifnr: processed vectorized ROI footprints without calcium deconvolution
• sigfnr: processed calcium traces without calcium deconvolution, meaning "no artificial cleaning"
• seedsfn: ROI centers in pixel coordinates; indices of all ROIs and be converted to (h, w) position using ind2sub
• Params: used parameters
• reg: data after neural enhancing (and movement correction), saved for reprocessing

Other fixed preset parameters can be found in min1pipe.m, and the table in the paper.

As a demo, we demonstrate the use of 1-photon calcium imaging video recorded with UCLA miniscope:

demo_min1pipe.m

The same code can also be adapted to custom scripts for the processing.

Here are the visualization of the demo dataset processing:

To use the code on a custom dataset, no specific requirements are needed. The processed data and the data after movement correction are saved in the same folder of the raw data in ".mat" format, with "_data_processed" and "_reg" as endings separately.

If post-process is selected, there will be an additional ".mat" file created with "_data_processed_refined".

• Data tips
  • Data should be arranged in sessions.
  • Each session contains multiple videos automatically divided by the recording softwares.
  • For Inscopix data, data are divided and renamed with a pattern of adding "-" "indices". We suggest sticking to this format for .tif and .tiff data.
  • For UCLA miniscope, data are named with "msCam"/letter(s) "indices", and we suggest sticking to this format for .avi data.
  • For best practice, remove apparent artifects such as bright edges of the grin lenses, even though the algorithm can handle these conditions.
  • For sessions with only a few neurons and possibly huge artifects/contaminations, semi-auto seeds selection can be considered at first hand;
  • Usually it poses more difficulties (more time spent/less accuracy) on the movement correction module under such (few neurons/key information) circumstances.
  • For sessions with small translational movement, other traditional rigid correction methods can also be used. The option of rigid correction would also be integrated in future releases.
• Software
  • Matlab R2017 and later.
• Hardware
  • Better hardwares are always preferred, for professional data analysis such as in the regular lab environment, even though the algorithms can be adapted to personal computers.
  • Typically, ~4 times of the size of a single session dataset (after downsampling) of memory is recommanded for processing.
  • Memory mapping will be integrated in future Future arrived.
  • Matlab parallel computing only supports NVIDIA graphic cards.
  • Patch version requires sufficient space in local hard disk (especially the disk containing the dataset currently running).

Updates

Please cite the MIN1PIPE journal paper if it helps your research.

@article{lu2018MIN1PIPE,
  title={MIN1PIPE: A Miniscope 1-photon-based Calcium Imaging Signal Extraction Pipeline.},
  author={Lu, J., Li, C., Singh-Alvarado, J., Zhou, Z., Fröhlich, F., Mooney, R., & Wang, F.},
  journal={Cell reports},
  volume={23},
  number={12},
  pages={3673--3684},
  year={2018},
  publisher={Elsevier}
}

Archives

The paper is now accepted by Cell Reports.

Please cite the MIN1PIPE paper if it helps your research.

@article{lu2018MIN1PIPE,
  title={MIN1PIPE: A Miniscope 1-photon-based Calcium Imaging Signal Extraction Pipeline.},
  author={Lu, J., Li, C., Singh-Alvarado, J., Zhou, Z., Fröhlich, F., Mooney, R., & Wang, F.},
  journal={bioRxiv, 311548},
  year={2018}
}

or the related arXiv version:

@article{lu2017seeds,
  title={Seeds Cleansing CNMF for Spatiotemporal Neural Signals Extraction of Miniscope Imaging Data},
  author={Lu, Jinghao and Li, Chunyuan and Wang, Fan},
  journal={arXiv preprint arXiv:1704.00793},
  year={2017}
}

Please email to [email protected] for additional questions.