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Cytometry

Pulse Measurement

Overview of Pulse Measurement

  • Definition: Pulse measurement involves analyzing the shape and characteristics of the electrical pulses generated as cells or particles pass through the detection zone in a flow cytometer
  • Purpose in Flow Cytometry:
    • Extract Information: To obtain information about the size, shape, and fluorescence intensity of cells
    • Discriminate Events: To distinguish between single cells, cell aggregates, and debris
    • Improve Data Quality: To reduce noise and artifacts
  • Key Parameters:
    • Time Delay
    • Window Extension
    • Area (Integral)
    • Width (Duration)
    • Height (Peak)
    • Coulter Impedance

Time Delay

  • Definition: The time between the trigger and the start of pulse processing; refers to how long the instrument waits before beginning to analyze the signal
  • Purpose:
    • Optimize signal capture: Allows the instrument to synchronize with the arrival of the signal
    • Reduce noise: By waiting until the signal is stable, it can minimize the influence of the background noise
  • Setting the correct time delay:
    • The time delay needs to be carefully calibrated
    • Too short a time delay can cause the loss of initial parts of the signal, reducing accuracy
    • Too long a time delay can delay processing and reduces throughput
  • Troubleshooting:
    • If signals are weak, it can be useful to adjust the time delay
    • Unstable signals may suggest an incorrectly set delay.

Window Extension

  • Definition: A feature in pulse processing that extends the time window of signal integration beyond the trigger point
  • Purpose:
    • Capture Complete Signal: Ensures that the entire signal from a cell passing through the detection zone is captured, especially for cells with longer transit times
    • Improve Accuracy: Enhances the accuracy of pulse area and height measurements by accounting for the entire signal
  • Considerations:
    • Proper Calibration: The window extension must be properly calibrated to match the transit time of cells through the laser beam
    • Avoid Overlap: The window extension should be short enough to avoid overlapping signals from multiple cells
  • Troubleshooting:
    • Incomplete signal capture: Increasing the window extension may help capture the entire signal
    • Signal overlap: Reducing the window extension to prevent overlap

Area (Integral)

  • Definition: The area under the pulse curve, representing the total amount of fluorescence emitted by a cell
  • Calculation: Calculated by integrating the signal amplitude over the duration of the pulse
  • Purpose:
    • Quantify Fluorescence Intensity: Provides a measure of the total amount of fluorophore bound to the cell
    • Reduce Sensitivity to Variations in Cell Speed: Less sensitive to variations in cell speed compared to pulse height
    • Discriminate Aggregates: Helps differentiate between single cells and cell aggregates, as aggregates typically have a larger pulse area
  • Applications:
    • Quantifying protein expression levels
    • Measuring cell viability
    • Analyzing cell cycle distribution

Width (Duration)

  • Definition: The duration of the pulse, representing the time it takes for a cell to pass through the detection zone
  • Measurement: Measured as the time between the start and end of the pulse, typically at a defined threshold
  • Purpose:
    • Estimate Cell Size: Provides an estimate of cell size, as larger cells typically have longer transit times
    • Discriminate Aggregates: Helps differentiate between single cells and cell aggregates, as aggregates typically have a longer pulse width
    • Identify Irregular Events: Can be used to identify irregular events such as cell doublets or debris
  • Applications:
    • Cell sizing
    • Doublet discrimination
    • Quality control

Height (Peak)

  • Definition: The maximum amplitude of the pulse, representing the peak fluorescence intensity of a cell
  • Measurement: Measured as the maximum value of the signal during the pulse
  • Purpose:
    • Quantify Fluorescence Intensity: Provides a measure of the maximum amount of fluorophore bound to the cell
    • Sensitive to Variations in Cell Speed: More sensitive to variations in cell speed compared to pulse area
    • Identify Bright Events: Can be used to identify cells with high levels of fluorescence
  • Applications:
    • Quantifying protein expression levels
    • Measuring cell activation
    • Identifying rare cell populations

Pulse Processing Relationships and Gating Strategies

  • Area vs. Height:
    • Single cells will typically have a linear relationship between pulse area and pulse height
    • Aggregates or doublets will have a higher pulse area relative to pulse height
    • Gating on area vs. height can be used to exclude aggregates and improve data quality
  • Area vs. Width:
    • Single cells will typically have a consistent relationship between pulse area and pulse width
    • Aggregates or doublets will have a wider pulse width relative to pulse area
    • Gating on area vs. width can be used to exclude aggregates and improve data quality
  • Height vs. Width:
    • Single cells will typically have a consistent relationship between pulse height and pulse width
    • Gating on height vs. width can be used to exclude debris and improve data quality
  • Gating Strategy:
    • Create a gate on FSC-A vs. FSC-H to exclude doublets based on the relationship between pulse area and pulse height
    • Create a gate on SSC-A vs. SSC-H to further exclude doublets based on side scatter characteristics
    • Adjust gate positions based on the specific cell types and experimental conditions
    • Confirm the effectiveness of the gating strategy by visualizing the gated populations on other parameter plots

Coulter Impedance

  • Principle: A technique used for counting and sizing cells based on changes in electrical impedance as cells pass through a small aperture
  • Mechanism:
    • Cells are suspended in an electrolyte solution and pass through a small aperture
    • Electrodes on either side of the aperture measure the electrical impedance (resistance to the flow of electrical current)
    • When a cell passes through the aperture, it displaces some of the electrolyte, increasing the impedance
    • The magnitude of the impedance change is proportional to the cell volume
  • Purpose:
    • Cell Counting: Counting the number of impedance changes provides an accurate cell count
    • Cell Sizing: Measuring the magnitude of the impedance changes provides information about cell volume
  • Applications:
    • Cell Counting: Accurate counting of cells in a sample
    • Cell Sizing: Determining the size distribution of cells
    • Hematology Analyzers: Used in automated hematology analyzers for complete blood counts (CBCs)
  • Advantages:
    • Accurate cell counting
    • Provides information about cell volume
    • Relatively simple and inexpensive
  • Disadvantages:
    • Does not provide information about cell surface markers or internal complexity
    • Can be affected by cell aggregates and debris
  • Related pulse measurements:
    • The pulse height is proportional to cell volume, whereas the pulse width is a constant value
  • Troubleshooting:
    • High background counts: Filtering the electrolyte to remove any debris
    • Inaccurate cell counts: Verifying proper maintenance, cleaning, and callibration

Troubleshooting Pulse Measurement Issues

  • Inaccurate Cell Counts:
    • Possible Causes:
      • Cell aggregates
      • Debris
      • Incorrect threshold settings
    • Troubleshooting Steps:
      • Optimize sample preparation to minimize cell aggregates and debris
      • Adjust threshold settings to exclude unwanted events
      • Use pulse processing parameters (area, width, height) to discriminate single cells from aggregates
  • Poor Resolution:
    • Possible Causes:
      • Excessive noise
      • Incorrect amplifier settings
      • Poor optical alignment
    • Troubleshooting Steps:
      • Reduce noise using the strategies described in the “Noise” study guide
      • Optimize amplifier settings
      • Check and align the optical system
  • Unexpected Doublet Populations:
    • Possible Causes:
      • High cell concentration
      • Poor sample preparation
      • Incorrect pulse processing settings
    • Troubleshooting Steps:
      • Reduce cell concentration
      • Optimize sample preparation to minimize cell aggregation
      • Adjust pulse processing settings to discriminate doublets

Key Terms

  • Pulse Measurement: Analyzing the shape and characteristics of the electrical pulses generated as cells pass through the detection zone
  • Time Delay: The time between the trigger and the start of pulse processing
  • Window Extension: The feature extends the time window of signal integration beyond the trigger point
  • Area (Integral): The area under the pulse curve, representing the total amount of fluorescence emitted by a cell
  • Width (Duration): The duration of the pulse, representing the time it takes for a cell to pass through the detection zone
  • Height (Peak): The maximum amplitude of the pulse, representing the peak fluorescence intensity of a cell
  • Coulter Impedance: A technique used for counting and sizing cells based on changes in electrical impedance