SCYM Flow Cytometry Principles 3 — Questions and Answers
Question 1: Which type of laser is most commonly used in clinical flow cytometers for exciting FITC and PE simultaneously?
- Red diode laser (640 nm)
- Blue argon-ion or solid-state laser (488 nm) (Correct answer)
- Violet laser (405 nm)
- UV laser (355 nm)
Correct answer: Blue argon-ion or solid-state laser (488 nm)
The 488 nm blue laser efficiently excites both FITC and PE, which is why they are among the most widely used fluorochromes in clinical cytometry.
Question 2: What is the Stokes shift in the context of fluorescence?
- The time delay between photon absorption and emission
- The difference in wavelength between the excitation and emission peaks of a fluorochrome (Correct answer)
- The shift in scatter angle caused by cell membrane irregularities
- The change in laser wavelength due to thermal drift
Correct answer: The difference in wavelength between the excitation and emission peaks of a fluorochrome
Stokes shift refers to the longer emission wavelength compared to the excitation wavelength, which allows emitted light to be separated from excitation light.
Question 3: In a flow cytometer, bandpass filters are used to:
- Block all light from reaching the detector
- Transmit only a specific range of wavelengths to the appropriate detector (Correct answer)
- Amplify fluorescence signals before detection
- Separate cells based on size before laser interrogation
Correct answer: Transmit only a specific range of wavelengths to the appropriate detector
Bandpass filters allow only a defined range of wavelengths to pass through to the detector, enabling detection of specific fluorochrome emissions.
Question 4: What is 'doublet discrimination' in flow cytometry?
- Using two lasers to improve fluorochrome excitation
- A gating strategy to exclude cell aggregates from analysis (Correct answer)
- Measuring two scatter parameters simultaneously
- Applying two rounds of compensation to improve accuracy
Correct answer: A gating strategy to exclude cell aggregates from analysis
Doublet discrimination uses pulse width or area versus height plots to gate out cell aggregates that could be mistaken for single large cells.
Question 5: Which PMT (photomultiplier tube) voltage setting principle is correct?
- Higher voltage decreases signal amplification
- Higher voltage increases signal amplification and sensitivity (Correct answer)
- Voltage settings do not affect resolution
- PMT voltage only affects scatter channels, not fluorescence
Correct answer: Higher voltage increases signal amplification and sensitivity
Increasing PMT voltage amplifies the electrical signal from detected photons, improving sensitivity for dim populations but also amplifying noise.
Question 6: What is the significance of the coefficient of variation (CV) in flow cytometry QC?
- It measures the percentage of positive cells in a sample
- It measures the spread or variability of a fluorescent bead peak, reflecting instrument performance (Correct answer)
- It quantifies the number of cells acquired per second
- It indicates the ratio of FSC to SSC for a population
Correct answer: It measures the spread or variability of a fluorescent bead peak, reflecting instrument performance
A low CV for calibration bead peaks indicates good instrument performance and consistent laser alignment and fluidics.
Question 7: Tandem dyes like PE-Cy7 are used in flow cytometry primarily to:
- Replace scatter parameters for size discrimination
- Extend the number of colors detectable from a single laser (Correct answer)
- Improve compensation accuracy in two-color panels
- Increase the excitation efficiency of UV lasers
Correct answer: Extend the number of colors detectable from a single laser
Tandem dyes use energy transfer to shift emission far from the excitation wavelength, allowing detection of additional colors from one laser.
SCYM Flow Cytometry Principles 3
Which type of laser is most commonly used in clinical flow cytometers for exciting FITC and PE simultaneously?