ISAC Equipment Operation and Maintenance 3 — Questions and Answers
Question 1: What is the primary function of a photomultiplier tube (PMT) in a flow cytometer?
- To generate laser excitation light
- To convert photons into an amplified electrical signal (Correct answer)
- To focus the sample stream into the flow cell
- To filter unwanted scatter signals
Correct answer: To convert photons into an amplified electrical signal
PMTs convert incoming photons into electrons through the photoelectric effect and then amplify the signal via a series of dynodes.
Question 2: During a sort purity check, the collected fraction contains 85% target cells instead of the expected >95%. Which instrument parameter should be investigated FIRST?
- Laser power setting
- Sort gate placement and drop delay calibration (Correct answer)
- Sheath fluid pH
- PMT voltage for FSC
Correct answer: Sort gate placement and drop delay calibration
Sort purity is most directly affected by incorrect gate placement or an inaccurate drop delay, which causes non-target cells to be deflected into the collection tube.
Question 3: A researcher notices that the MFI of a dim population shifts significantly between the beginning and end of a long acquisition run. What is the MOST likely explanation?
- Antibody degradation during the run
- Laser power drift or instrument warm-up stabilization (Correct answer)
- Changes in the cell biology during acquisition
- Software miscalculation of MFI
Correct answer: Laser power drift or instrument warm-up stabilization
Laser power can drift until the system fully stabilizes, causing systematic shifts in fluorescence intensity readings over time.
Question 4: What is the recommended approach when a flow cytometer exhibits high background fluorescence in a no-stain control?
- Increase PMT voltage to improve signal-to-noise ratio
- Check for autofluorescence, contaminated sheath fluid, or dirty flow cell (Correct answer)
- Add more antibody to overwhelm background signal
- Switch to a different fluorochrome panel
Correct answer: Check for autofluorescence, contaminated sheath fluid, or dirty flow cell
High background in unstained controls typically results from cell autofluorescence, contaminated reagents, or residual fluorescent material in the fluidics.
Question 5: Which maintenance procedure is recommended to prevent biohazard aerosol exposure during a cell sorter maintenance session?
- Run ethanol at 30% concentration through the system
- Run a bleach solution through the system and allow contact time before opening the sort chamber (Correct answer)
- Disconnect the high-voltage supply before opening any panels
- Use UV light to sterilize the sort collection area
Correct answer: Run a bleach solution through the system and allow contact time before opening the sort chamber
Running 10% bleach through the sort stream decontaminates the fluidics and minimizes aerosol biohazard risk before technicians open the sort chamber.
Question 6: What does a decrease in the break-off point during electrostatic cell sorting indicate?
- Increased sheath pressure causing faster flow rate
- The nozzle tip may be partially clogged or vibration frequency needs adjustment (Correct answer)
- The sample concentration is too low
- The sort gate is set incorrectly
Correct answer: The nozzle tip may be partially clogged or vibration frequency needs adjustment
The break-off point is controlled by acoustic vibration frequency and nozzle condition; a change indicates a clog or need to retune the frequency.
Question 7: In flow cytometry, what is the purpose of running single-color controls?
- To verify that all antibodies bind to their targets
- To calculate compensation values for spectral spillover between channels (Correct answer)
- To establish the cell viability threshold
- To calibrate the laser alignment
Correct answer: To calculate compensation values for spectral spillover between channels
Single-color controls contain only one fluorochrome and are used to measure spillover of that fluorochrome into other detector channels for compensation matrix calculation.
ISAC Equipment Operation and Maintenance 3
What is the primary function of a photomultiplier tube (PMT) in a flow cytometer?