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Cytometry

Noise

Overview of Noise

  • Definition: Noise refers to unwanted random electrical fluctuations or disturbances in an electronic system that can obscure or distort the desired signal
  • Impact in Flow Cytometry: Noise reduces the sensitivity and resolution of the instrument, making it difficult to detect weak signals and distinguish between closely spaced cell populations
  • Key Characteristics:
    • Randomness: Noise is unpredictable and varies randomly over time
    • Amplitude: Noise can have a range of amplitudes, from very small to relatively large
    • Frequency: Noise can occur at a range of frequencies, from low to high
  • Types of Noise in Flow Cytometry:
    • Thermal Noise (Johnson Noise)
    • Shot Noise
    • Flicker Noise (1/f Noise)
    • Electronic Interference
    • Optical Noise
    • Reagent Noise

Thermal Noise (Johnson Noise)

  • Definition: Thermal noise is generated by the random motion of electrons in a conductor due to thermal energy
  • Characteristics:
    • Ubiquitous: Present in all electronic components
    • Temperature Dependent: Increases with temperature
    • Frequency Independent: Uniformly distributed across all frequencies (white noise)
  • Mitigation Strategies:
    • Reduce Temperature: Cooling electronic components can reduce thermal noise, but this is not always practical
    • Use Low-Resistance Components: Resistors with lower resistance generate less thermal noise
    • Minimize Bandwidth: Reducing the bandwidth of the measurement system can reduce the amount of thermal noise that is detected
    • Shielding: Protect the detector from external interferences
  • Mathematical formula:
    • VRMS = √(4kBTRΔf)
      • VRMS: Root mean square voltage (level of noise)
      • kB: Boltzmann’s constant
      • T: Temperature (in Kelvin)
      • R: Resistance (Ohms)
      • Δf: Bandwidth (Hz)

Shot Noise

  • Definition: Shot noise arises from the discrete nature of electric charge and the random arrival of electrons or photons at a detector
  • Characteristics:
    • Quantum Phenomenon: Related to the quantized nature of light and charge
    • Signal Dependent: Increases with the average signal level
    • Frequency Independent: Uniformly distributed across all frequencies (white noise)
  • Mitigation Strategies:
    • Increase Signal Strength: Increasing the signal level can reduce the relative impact of shot noise
    • Use Detectors with High Quantum Efficiency: Detectors with higher quantum efficiency convert more photons into electrons, reducing shot noise
    • Averaging: Averaging multiple measurements can reduce shot noise
  • Mathematical formula:
    • iRMS = √(2qIΔf)
      • iRMS: Root mean square (RMS) current fluctuation
      • q: elementary charge
      • I: average current
      • Δf: bandwidth

Flicker Noise (1/f Noise)

  • Definition: Flicker noise is a type of electronic noise that exhibits a power spectral density that is inversely proportional to the frequency
  • Characteristics:
    • Low-Frequency Dominance: More prominent at lower frequencies
    • Origin Obscure: Exact physical mechanisms are complex and not fully understood
    • Device Dependent: Varies depending on the type of electronic component
  • Mitigation Strategies:
    • Use Low-Noise Components: Select electronic components that are designed for low flicker noise
    • Modulation Techniques: Modulate the signal to a higher frequency range where flicker noise is less dominant
    • Signal Averaging: Averaging multiple measurements can reduce flicker noise
  • Mathematical formula:
    • S(f) ∝ 1/fα
      • S(f): power spectral density
      • f: frequency
      • α: Constant near 1

Electronic Interference

  • Definition: Electronic interference is noise caused by external electromagnetic radiation or other electrical signals interfering with the flow cytometer’s electronic circuits
  • Sources:
    • Power lines
    • Radio transmitters
    • Cell phones
    • Other electronic devices
  • Characteristics:
    • External Origin: Comes from outside the flow cytometer
    • Specific Frequencies: Often occurs at specific frequencies related to the interfering source
    • Time Dependent: Can vary depending on the activity of the interfering source
  • Mitigation Strategies:
    • Shielding: Use shielded cables and enclosures to block electromagnetic radiation
    • Grounding: Ensure proper grounding of all electronic components
    • Filtering: Use power line filters and signal filters to remove unwanted frequencies
    • Isolation: Isolate the flow cytometer from other electronic devices
  • Other considerations:
    • Minimize electronic equipment around the flow cytometer
    • Use a dedicated power line to the instrument

Optical Noise

  • Definition: Optical noise refers to unwanted light signals that interfere with the detection of fluorescence or scatter signals
  • Sources:
    • Stray Light: Light from the laser or other sources that reaches the detectors without passing through the sample
    • Autofluorescence: Natural fluorescence from cellular components or media
    • Light Scatter: Scattered light from particles or debris in the sample
  • Characteristics:
    • Light-Based: Involves unwanted light signals
    • Wavelength Dependent: Can vary depending on the wavelength of light
    • Sample Dependent: Can vary depending on the composition of the sample
  • Mitigation Strategies:
    • Optical Filters: Use appropriate optical filters to block unwanted wavelengths of light
    • Apertures and Baffles: Use apertures and baffles to block stray light
    • Proper Sample Preparation: Filter samples to remove particles and debris, and use appropriate blocking reagents to reduce autofluorescence
    • Optimized Instrument Settings: Adjust laser power and detector settings to minimize noise
  • Other considerations:
    • Be sure to run proper controls to identify any background noise

Reagent Noise

  • Definition: Noise that arises from the staining reagents used in flow cytometry, such as antibodies and fluorescent dyes
  • Sources:
    • Non-Specific Binding: Antibodies binding to unintended targets
    • Aggregated Antibodies: Antibodies forming aggregates that scatter light
    • Improperly Labeled Reagents: Inconsistent labeling of reagents
    • Dye Instability: Degradation or aggregation of fluorescent dyes
  • Characteristics:
    • Reagent-Specific: Varies depending on the type and quality of reagents used
    • Sample-Dependent: Can vary depending on the composition of the sample
    • Time-Dependent: Can change over time due to reagent degradation
  • Mitigation Strategies:
    • Use High-Quality Reagents: Select antibodies and dyes from reputable suppliers
    • Properly Titrate Antibodies: Determine the optimal concentration of antibodies to minimize non-specific binding
    • Filter Reagents: Filter reagents to remove aggregates
    • Use Appropriate Blocking Reagents: Block Fc receptors and other non-specific binding sites
    • Store Reagents Properly: Store reagents according to the manufacturer’s instructions to prevent degradation
    • Run proper controls (FMOs) to identify spread.

General Strategies for Noise Reduction

  • Optimize Instrument Settings: Adjust laser power, detector voltage, and amplifier gain to minimize noise while maximizing signal
  • Use Appropriate Filters: Select optical and electronic filters to block unwanted frequencies and wavelengths
  • Shielding and Grounding: Use shielded cables and proper grounding to reduce electronic interference
  • Cooling: Cool electronic components to reduce thermal noise (if practical)
  • Averaging: Average multiple measurements to reduce random noise
  • Filtering: Filter samples and reagents to remove particles and debris
  • Blocking: Use blocking reagents to reduce non-specific binding
  • Controls: Use proper controls to identify and subtract background noise
  • Good lab practices: Keep the cytometer clean, use validated procedures, and maintain a high degree of quality control.

Troubleshooting Noise Issues

  • High Background Noise:
    • Possible Causes:
      • High detector voltage
      • Stray light
      • Autofluorescence
      • Non-specific binding of reagents
      • Electronic interference
    • Troubleshooting Steps:
      • Reduce detector voltage
      • Shield from stray light
      • Optimize staining protocols
      • Use blocking reagents
      • Check for electronic interference
  • Weak Signals:
    • Possible Causes:
      • Low laser power
      • Misaligned optics
      • Low detector voltage
      • Excessive noise
    • Troubleshooting Steps:
      • Check laser power
      • Align optics
      • Increase detector voltage (but be mindful of noise)
      • Reduce noise using the strategies described above
  • Erratic Signals:
    • Possible Causes:
      • Electronic interference
      • Fluctuating laser power
      • Air bubbles in the fluidics system
    • Troubleshooting Steps:
      • Check for electronic interference
      • Stabilize laser power
      • Eliminate air bubbles in the fluidics system

Key Terms

  • Noise: Unwanted random electrical fluctuations or disturbances
  • Thermal Noise (Johnson Noise): Noise generated by the random motion of electrons in a conductor
  • Shot Noise: Noise arising from the discrete nature of electric charge and the random arrival of electrons or photons
  • Flicker Noise (1/f Noise): Noise with a power spectral density inversely proportional to frequency
  • Electronic Interference: Noise caused by external electromagnetic radiation or other electrical signals
  • Optical Noise: Unwanted light signals that interfere with the detection of fluorescence or scatter signals
  • Autofluorescence: Natural fluorescence from cellular components or media
  • Stray Light: Light that reaches the detectors without passing through the sample
  • Reagent Noise: Noise arising from the staining reagents used in flow cytometry