Spectral Overlap

Overview of Spectral Overlap and Compensation

Definition:
- Spectral Overlap: Occurs when the emission spectra of two or more fluorochromes overlap, meaning that the light emitted by one fluorochrome can be detected in the channel intended for another fluorochrome. This can lead to inaccurate results if not corrected
- Compensation: A mathematical process used to correct for spectral overlap by subtracting the contribution of one fluorochrome from the signal of another. This ensures that the fluorescence signal in each channel accurately reflects the expression of the target antigen
Importance:
- Accurate Results: Compensation is essential for accurate quantification of multiple antigens in a single sample
- Multi-Color Analysis: Allows for the use of multiple fluorochromes in a single experiment, increasing the amount of information that can be obtained
- Panel Design: Understanding spectral overlap is crucial for designing flow cytometry panels that maximize resolution and minimize spillover
Key Concepts:
- Emission Spectra
- Spillover
- Compensation Controls
- Compensation Matrix
- Overcompensation and Undercompensation

Definition: The range of wavelengths of light emitted by a fluorochrome when excited by a specific wavelength of light
Importance:
- Understanding the emission spectra of fluorochromes is essential for predicting spectral overlap and designing flow cytometry panels
- Emission spectra are typically represented as graphs that show the relative intensity of light emitted at different wavelengths
Factors Affecting Emission Spectra:
- Fluorochrome Type: Different fluorochromes have different emission spectra
- Excitation Wavelength: The wavelength of light used to excite the fluorochrome can affect the emission spectrum
- Environment: The environment surrounding the fluorochrome can affect the emission spectrum
Spectral viewers can be used to select fluorochromes with minimal compensation values for optimal results.

Definition: The amount of fluorescence from one fluorochrome that is detected in the channel intended for another fluorochrome
Causes:
- Spectral Overlap: Overlapping emission spectra
- Imperfect Filters: Optical filters may not completely block all wavelengths of light
- Detector Sensitivity: Detectors may be sensitive to a wider range of wavelengths than intended
Consequences:
- Inaccurate Quantification: Can lead to inaccurate quantification of antigens
- False Positives: Can cause false-positive results if cells expressing one antigen are incorrectly identified as expressing another antigen
Example:
- FITC fluorescence being detected in the PE channel

Definition: Samples used to calculate the compensation values needed to correct for spectral overlap
Types:
- Single-Stain Controls: Cells or beads stained with a single fluorochrome
- “CompBeads”: Antibody-capture beads are widely used and are designed to bind to any antibody of the same species, thus allowing for the capture of the antibody of interest and fluorophore
- Autofluorescence Controls: Unstained cells used to measure the level of autofluorescence in the sample
Requirements:
- Bright Signal: Should have a bright signal in the channel being compensated
- Similar Autofluorescence: Should have similar autofluorescence characteristics as the experimental samples
- Sufficient Events: Should contain a sufficient number of events to accurately calculate compensation values
Compensation Strategy:
- Ideally, each fluorochrome in the panel should be accounted for through the creation of appropriate compensation controls
Compensation controls should be run with every experiment
Compensation controls should be treated exactly the same as your samples

Definition: A matrix that contains the compensation values needed to correct for spectral overlap
Calculation: Calculated using compensation controls
Application: Applied to the experimental data to remove the contribution of each fluorochrome from the other channels
Software: Most flow cytometry software packages have built-in tools for calculating and applying compensation matrices
Manual Compensation:
- Compensation can be performed manually by adjusting the compensation values until the spillover is eliminated
- However, automatic compensation is highly recommended

Overcompensation:
- Definition: Occurs when too much fluorescence is subtracted from a channel, resulting in negative values or distortion of the data
- Appearance: Populations may appear to be skewed or have negative values
- Causes: Incorrect compensation values or overestimation of spillover
Undercompensation:
- Definition: Occurs when not enough fluorescence is subtracted from a channel, resulting in residual spillover
- Appearance: Populations may appear to be smeared or have a broader distribution
- Causes: Incorrect compensation values or underestimation of spillover
Optimization:
- Proper compensation is essential for accurate results
- Carefully review the compensation controls and adjust the compensation values as needed to achieve optimal compensation
Troubleshooting:
- Be sure to adjust the compensation with proper controls to avoid any false positive data

Definition: Software algorithms that automatically calculate and apply compensation values
Advantages:
- Simplified Process: Simplifies the compensation process
- Increased Accuracy: Can improve the accuracy of compensation
- Reduced User Bias: Reduces the potential for user bias in the compensation process
Considerations:
- Quality Controls: Even with automated compensation tools, it is essential to review the compensation controls and verify that the compensation values are appropriate

Definition: Manual compensation involves adjusting compensation settings by eye
When To Use Manual Compensation:
- When automatic compensation tools are not available
- To double check automatic compensation results
Challenges:
- Time-consuming
- Requires experience
- Can be less accurate

Smeared Populations:
- Possible Cause: Undercompensation
- Troubleshooting Step: Increase compensation value
Populations That are Negative for All Markers:
- Possible Cause: Overcompensation
- Troubleshooting Step: Reduce compensation value
Compensation Artifacts:
- Possible Cause: Complex compensation settings
- Troubleshooting Step: Simplify panel design
Compensation Issues With Experimental Samples:
- Possible Cause: High auto-fluorescence
- Troubleshooting Step: Adjust technique to prevent it from happening

Spectral Overlap: When one channel reads a signal from more than one fluorophore
Emission Spectrum: A graph that shows the relative intensity of light emitted at different wavelengths for a particular fluorochrome
Spillover: When the emission spectrum of one fluorochrome overlaps into the detection range of another fluorochrome
Compensation: An adjustment to the raw data used to remove spillover
Under-Compensation: Not enough spillover is removed from the data
Over-Compensation: Too much spillover is removed from the data
Compensation Controls: Necessary for setting proper compensation
Unstained Control: Used to assess and remove background noise from the signals