SCYM Data Acquisition & Analysis 2 — Questions and Answers
Question 1: What is the primary purpose of using a threshold (discriminator) during flow cytometry data acquisition?
- To amplify fluorescence signals
- To exclude events below a set signal level from data collection (Correct answer)
- To normalize scatter parameters
- To set the photomultiplier tube voltage
Correct answer: To exclude events below a set signal level from data collection
A threshold eliminates low-level electronic noise and debris by only collecting events whose signal exceeds the defined minimum value.
Question 2: When setting up a multicolor panel, the process of measuring how much fluorochrome signal appears in unintended detector channels is called:
- Optical filtering
- Spectral overlap (Correct answer)
- Photobleaching
- Backgating
Correct answer: Spectral overlap
Spectral overlap occurs because fluorochrome emission spectra are broad and extend into adjacent detector channels.
Question 3: Which compensation control provides the most accurate matrix for correcting spectral spillover?
- Unstained cells mixed with all fluorochromes
- Single-stained beads or cells at the same brightness as the experimental sample (Correct answer)
- Isotype controls for each antibody
- FMO controls
Correct answer: Single-stained beads or cells at the same brightness as the experimental sample
Compensation controls must match the brightness of the actual sample; single-stained controls at equivalent intensity yield the correct spillover coefficients.
Question 4: In flow cytometry, what does the term 'coincidence' (or 'doublet') refer to during acquisition?
- Two lasers exciting the same cell simultaneously
- Two or more cells passing through the interrogation point at the same time (Correct answer)
- A fluorochrome emitting at two wavelengths
- Simultaneous triggering of two photomultiplier tubes
Correct answer: Two or more cells passing through the interrogation point at the same time
Coincidence events occur when multiple cells pass through the laser beam together, creating artificially large or complex signals.
Question 5: A histogram displaying a broad, flat peak for a single fluorescent population most likely indicates:
- Optimal PMT voltage
- High coefficient of variation (CV) suggesting instrument or staining problems (Correct answer)
- Correct threshold setting
- Proper compensation
Correct answer: High coefficient of variation (CV) suggesting instrument or staining problems
A high CV (wide peak) reflects poor resolution, often caused by suboptimal PMT voltage, aggregated cells, or inconsistent staining.
Question 6: Fluorescence Minus One (FMO) controls are used in flow cytometry primarily to:
- Compensate for spectral spillover
- Define positive/negative gates in the presence of spillover from other fluorochromes (Correct answer)
- Set PMT voltages
- Establish instrument baseline noise
Correct answer: Define positive/negative gates in the presence of spillover from other fluorochromes
FMO controls contain all fluorochromes except the one being gated, revealing where the negative population sits after spillover fills the channel.
Question 7: During acquisition, what is the recommended approach when a sample shows an unexpected shift in bead position on quality control plots?
- Increase the sample flow rate
- Stop acquisition, investigate instrument alignment and laser stability, then recalibrate (Correct answer)
- Reduce PMT voltage by 50V
- Apply additional compensation
Correct answer: Stop acquisition, investigate instrument alignment and laser stability, then recalibrate
A QC bead shift indicates an instrument performance change; data collected during that period may be unreliable and the cause must be identified before continuing.
SCYM Data Acquisition & Analysis 2
What is the primary purpose of using a threshold (discriminator) during flow cytometry data acquisition?