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User Guide

This guide explains how to use Rodrigolab's Flow Cytometry Analyzer to filter and analyze your FCS files.

Getting Started

Loading an FCS File

  1. Launch the application
  2. Click the "Load FCS File" button
  3. Select your FCS file from the file dialog
  4. The file will be loaded and channels will be displayed

Understanding the Interface

The main interface consists of: - Header: Application title and theme selector - Controls Panel: File operations and export options - Channel List: Available channels from your FCS file - Plot Area: Interactive scatter plot visualization - Controls: Zoom, pan, and scale options

Basic Workflow

Step 1: Load Your Data

Click "Load FCS File" and select your FCS file. Once loaded, you'll see: - A list of available channels - The first two channels automatically selected for plotting - A scatter plot showing the data

Step 2: Select Channels

  • Click on channel names to select them for plotting
  • The first selected channel becomes the X-axis
  • The second selected channel becomes the Y-axis
  • The plot updates automatically when you change selections

Step 3: Create a Filter

  1. Draw a Selection:
  2. Left-click and drag on the plot to draw a lasso selection
  3. The selection automatically closes when you release the mouse

  4. Points inside the selection will be highlighted in green

  5. Confirm Selection:

  6. Click "Confirm Selection" to save the filter
  7. The filter is saved and you can see how many points were selected
  8. You can now create additional filters on different channel combinations

Step 4: Create Additional Filters (Optional)

  1. Select different channels
  2. Draw a new selection on the new plot
  3. Click "Confirm Selection" to save another filter
  4. All filters are automatically combined (intersection)

Step 5: Export Filtered Data

  1. Click "Export Filtered Data"
  2. Choose your export format:
  3. CSV: Comma-separated values
  4. Excel: XLSX with metadata and data sheets
  5. Parquet: Compressed columnar format
  6. Select the output location
  7. The filtered data (points passing all filters) will be exported

Plot Controls

  • Pan: Right-click and drag to move around the plot
  • Zoom: Use the mouse wheel to zoom in/out
  • Reset Zoom: Click the "Reset" button to return to default view
  • Zoom Buttons: Use "+" and "−" buttons for precise zoom control

Scale Options

  • Log Scale: Check "X-axis: Log scale" or "Y-axis: Log scale" to use logarithmic scaling
  • Linear Scale: Uncheck to use linear scaling
  • Useful for channels with wide value ranges

Selection Tools

  • Lasso Selection: Left-click and drag to draw a freehand selection
  • Clear Selection: Click "Clear Selection" to remove the current selection
  • Reset Filter: If you return to a channel pair with an existing filter, you can reset it

Advanced Features

Multiple Filters

  • Create filters on different channel combinations
  • All filters are automatically combined (only points passing ALL filters are kept)
  • View filter summary showing total events, passing events, and excluded events

Showing Excluded Points

  • Check "Show excluded points from past filters" to visualize points excluded by other filters
  • Excluded points appear in orange/brown color
  • Helps you understand how filters interact

Themes

  • Select from Light, Dim, or Dark themes
  • "System" option follows your system preference
  • Theme preference is saved between sessions

Filter Management

  • Filter Summary: Shows statistics about your filters
  • Reset Filter: Allows you to reset a filter for a specific channel pair
  • Existing Filter Warning: Alerts you when you return to a channel pair with a saved filter

Export Formats

CSV

  • Simple comma-separated values
  • Can be opened in Excel, Google Sheets, or any text editor
  • Contains only the filtered data

Excel (XLSX)

  • Two sheets: "metadata" and "data"
  • Metadata sheet contains FCS file header and text segment information
  • Data sheet contains the filtered data
  • Preserves data types and formatting

Parquet

  • Compressed columnar format
  • Efficient for large datasets
  • Can be read by Python (pandas), R, and other data analysis tools

Tips and Best Practices

  1. Start with Common Channels: Begin with FSC-A vs SSC-A or other standard channel pairs
  2. Use Log Scale: Enable log scale for channels with wide ranges (fluorescence channels)
  3. Zoom for Precision: Zoom in when making precise selections
  4. Check Filter Summary: Monitor how many events pass your filters
  5. Save Your Work: Export frequently to avoid losing your filtering work
  6. Multiple Filters: Use multiple filters to progressively refine your population

Keyboard Shortcuts

Currently, the application is primarily mouse-driven. Keyboard shortcuts may be added in future versions.

Troubleshooting

If you encounter issues, see the Troubleshooting Guide.