Instrument Optimization

Overview of Instrument Optimization

Definition: Instrument optimization is the process of adjusting the settings of a flow cytometer to achieve the best possible performance for a specific application
Purpose:
- Maximize Signal Intensity: To maximize the signal from target cells
- Minimize Background Noise: To reduce background noise and improve the signal-to-noise ratio
- Improve Resolution: To improve the ability to distinguish between different cell populations
- Standardize Performance: To ensure consistent performance of the flow cytometer over time
Key Aspects of Instrument Optimization:
- Laser Power Adjustment
- PMT Voltage Optimization
- Compensation Settings
- Threshold Settings
- Area Scaling

Definition: Adjusting the power output of the lasers used for excitation
Considerations:
- High Laser Power:
  - Advantages: Increases signal intensity, allowing for the detection of weakly expressed markers
  - Disadvantages: Can increase photobleaching, increase background noise, and damage cells
- Low Laser Power:
  - Advantages: Reduces photobleaching, reduces background noise, and is gentler on cells
  - Disadvantages: Decreases signal intensity, making it difficult to detect weakly expressed markers
Optimization Steps:
- Start with a low laser power setting
- Gradually increase the laser power while monitoring the signal intensity and background noise
- Choose the laser power setting that provides the best balance between signal intensity and background noise
When to adjust laser power
- When the signal is too low for a particular probe
- When there is extensive photo bleaching
- This is a delicate balance!

Definition: Adjusting the voltage applied to the photomultiplier tubes (PMTs) or avalanche photodiodes (APDs) used for light detection
Considerations:
- High Voltage:
  - Advantages: Increases signal intensity, allowing for the detection of weakly expressed markers
  - Disadvantages: Increases background noise, saturates PMTs and shortens lifespan
- Low Voltage:
  - Advantages: Reduces background noise and extends PMT lifespan
  - Disadvantages: Decreases signal intensity, making it difficult to detect weakly expressed markers
Optimization Steps:
1. Start with a low PMT voltage setting
2. Gradually increase the PMT voltage while monitoring the signal intensity and background noise
3. Choose the PMT voltage setting that provides the best balance between signal intensity and background noise
4. When you set up the voltages, review your controls to ensure you are not missing data from either the high or low expressing cells

Definition: Adjusting the compensation settings to correct for spectral overlap between fluorochromes
Considerations:
- Proper compensation is essential for accurate quantification of multiple markers in a single sample
- Overcompensation can lead to negative values or distortion of the data
- Undercompensation can lead to inaccurate results and misinterpretation of the data
Optimization Steps:
1. Use single-stain controls to accurately calculate compensation values
2. Apply the compensation matrix to the experimental data
3. Visually inspect the data to ensure that the compensation is correct
4. Adjust the compensation values as needed to achieve optimal compensation
Note the appropriate controls
- Always be mindful of setting the compensation with proper controls, such as the sample that was stained only with that reagent

Definition: Setting the threshold levels to exclude unwanted events from the analysis
Considerations:
- Low Threshold: Captures a larger population
- High Threshold: Ignores a significant portion of cells
Optimization Steps:
1. Begin with a low threshold
2. Check the events that are not the target and adjust appropriately
3. Use controls for optimal threshold settings

Definition: The data is represented on the display using set parameters
Linear Scale:
- Pros: Displaying the samples on a linear scale is an accurate way to analyze the data and easily compare different cell populations
- Cons: These can be difficult to assess when the range of the data is vast
Logarithmic Scale:
- Pros: The values are easily displayed and the resolution is improved
- Cons: Does not show the differences in populations that fall within the higher populations
Biexponential Scale:
- Pros: This can visualize the low and high populations
- Cons: It can be difficult to analyze and interpret the data

Standardized Protocols:
- Use standardized protocols for instrument optimization
- Document all settings and procedures
Regular Monitoring:
- Monitor instrument performance regularly using control samples and metrics
- Track changes in instrument settings over time
Training and Education:
- Provide adequate training and education to flow cytometry operators
Scheduled Maintenance:
- Use this time to test the system
- Replace worn or damaged parts

Weak Signals:
- Possible Causes:
  - Low laser power
  - Incorrect PMT voltages
  - Misaligned optics
- Troubleshooting Steps:
  - Check laser power
  - Optimize PMT voltages
  - Align optical system
High Background Noise:
- Possible Causes:
  - Excessive PMT voltages
  - Stray light
  - Contaminated reagents
- Troubleshooting Steps:
  - Reduce PMT voltages
  - Shield stray light
  - Use clean reagents
Compensation Errors:
- Possible Causes:
  - Incorrect compensation values
  - Poor single-stain controls
- Troubleshooting Steps:
  - Verify compensation values
  - Prepare high-quality single-stain controls

Instrument Optimization: Adjusting the settings of a flow cytometer to achieve the best possible performance
Laser Power: The power output of the lasers used for excitation
PMT Voltage: The voltage applied to the photomultiplier tubes
Compensation: Correcting for spectral overlap between fluorochromes
Threshold: A setting that is used to exclude unwanted events from the analysis
Area Scaling: A method for scaling the area of the gates used to select cell populations