SCYM Instrument Operation 4 — Questions and Answers
Question 1: Which instrument parameter should be adjusted to move a dim population from the edge of the FSC axis into a better-resolved position on the plot?
- Increase FSC PMT voltage or gain (Correct answer)
- Reduce sheath pressure
- Decrease SSC voltage
- Apply a compensation offset to FSC
Correct answer: Increase FSC PMT voltage or gain
Increasing FSC voltage or gain amplifies the signal so that dim populations shift away from the axis noise and become better resolved.
Question 2: A spectral cytometer uses how many detectors compared to a conventional cytometer of similar channel count?
- Fewer detectors covering broad bandpass regions
- The same number of detectors with narrower bandpass filters
- Many more detectors (often 30–80+) capturing the full emission spectrum (Correct answer)
- Exactly two detectors regardless of panel size
Correct answer: Many more detectors (often 30–80+) capturing the full emission spectrum
Spectral cytometers employ an array of many narrow-band detectors to capture the full emission spectrum of each fluorochrome rather than isolated bandpass windows.
Question 3: What is the purpose of setting a trigger (threshold) in flow cytometry?
- To set the voltage on the primary detector
- To eliminate electronic noise and debris by only processing events above a defined signal level (Correct answer)
- To compensate for spectral spillover
- To define the acquisition stop gate
Correct answer: To eliminate electronic noise and debris by only processing events above a defined signal level
The trigger threshold tells the instrument to only acquire and process events when a signal (usually FSC) exceeds a set value, eliminating noise and irrelevant debris.
Question 4: When setting up a compensation matrix, which control best represents the maximum possible spillover from a fluorochrome?
- FMO control
- Single-color positive control using the same fluorochrome on beads or cells (Correct answer)
- Isotype control
- Unstained sample
Correct answer: Single-color positive control using the same fluorochrome on beads or cells
A single-color positive control contains only one fluorochrome at maximum brightness, providing the most accurate measurement of how much it spills into adjacent channels.
Question 5: If a flow cytometer's waste container is not emptied and becomes full, which operational problem will most likely occur?
- Laser power will decrease automatically
- Backpressure will increase, disrupting sheath fluid flow and data quality (Correct answer)
- PMT voltages will drift upward
- The sort deflection plates will deactivate
Correct answer: Backpressure will increase, disrupting sheath fluid flow and data quality
A full waste container creates backpressure in the fluidics system, disrupting sheath flow uniformity and causing aberrant scatter and fluorescence signals.
Question 6: Which of the following describes the correct sequence for performing compensation on a multi-color panel?
- Acquire all samples, then set compensation, then re-acquire
- Set PMT voltages → acquire single-color controls → calculate and apply compensation matrix → acquire full panel (Correct answer)
- Apply compensation matrix first, then acquire single-color controls to verify
- Acquire unstained sample first, then acquire all single-color and experimental samples together
Correct answer: Set PMT voltages → acquire single-color controls → calculate and apply compensation matrix → acquire full panel
Proper compensation workflow requires finalizing voltages first, then acquiring single-color controls to calculate the matrix, which is then applied before acquiring the experimental samples.
Question 7: What does the term 'sort purity' refer to in cell sorting?
- The percentage of sorted cells that are viable after sorting
- The proportion of collected events that are actually the target population (Correct answer)
- The number of cells sorted per minute
- The degree to which the sort gate overlaps with the parent population
Correct answer: The proportion of collected events that are actually the target population
Sort purity measures the percentage of cells in the collection tube that truly belong to the target population defined by the sort gate.
SCYM Instrument Operation 4
Which instrument parameter should be adjusted to move a dim population from the edge of the FSC axis into a better-resolved position on the plot?