SCYM Principles of Flow Cytometry & Instrumentation 3 — Questions and Answers
Question 1: What is spectral spillover in multicolor flow cytometry?
- Laser light that leaks into the wrong detector channel
- Fluorescence emission from one dye detected in a channel designated for another dye (Correct answer)
- Autofluorescence that saturates all detectors simultaneously
- Cross-contamination of samples in the flow cell
Correct answer: Fluorescence emission from one dye detected in a channel designated for another dye
Spectral spillover occurs because fluorochromes emit across a broad spectrum, causing a portion of one dye's emission to be detected in channels designed for other dyes.
Question 2: Which control is used to set the compensation matrix in multicolor flow cytometry?
- Fluorescence minus one (FMO) control
- Single-stain compensation control (Correct answer)
- Isotype control
- Unstained cell control
Correct answer: Single-stain compensation control
Single-stain compensation controls, containing only one fluorochrome at a time, are used to measure and mathematically subtract spillover from each fluorochrome into other channels.
Question 3: What does the side scatter (SSC) parameter primarily reflect in flow cytometry?
- Cell size and volume
- Internal complexity, granularity, and nuclear structure (Correct answer)
- Surface antigen density
- Membrane lipid content
Correct answer: Internal complexity, granularity, and nuclear structure
SSC is generated by light scattered at approximately 90° from the laser beam, reflecting internal complexity such as granularity, nuclear lobularity, and intracellular structures.
Question 4: In electrostatic cell sorting, what determines which droplets receive an electrical charge?
- The size of the droplet as it breaks from the stream
- Whether the cell in the droplet meets the sort gate criteria (Correct answer)
- The viscosity of the sheath fluid at the sort point
- The fluorescence emission wavelength of the labeled cell
Correct answer: Whether the cell in the droplet meets the sort gate criteria
The sorter's electronics evaluate whether the cell meets the pre-defined sort gate criteria and, if so, apply an electrical charge to the droplet at the moment it breaks from the stream.
Question 5: What is the typical laser power range used in most clinical and research flow cytometers?
- 0.1–1 milliwatt
- 5–100 milliwatts (Correct answer)
- 500 milliwatts–5 watts
- 10–50 watts
Correct answer: 5–100 milliwatts
Most flow cytometers use lasers in the 5–100 mW range, which provides sufficient power to excite fluorochromes without causing excessive photobleaching or cell damage.
Question 6: Which of the following best describes 'time-of-flight' in the context of flow cytometer pulse processing?
- The time it takes for a cell to travel from the sample tube to the flow cell
- The duration of the electronic pulse generated as a cell traverses the laser beam (Correct answer)
- The delay between laser excitation and fluorescence emission
- The time required to sort a single cell into a collection tube
Correct answer: The duration of the electronic pulse generated as a cell traverses the laser beam
Time-of-flight (pulse width) is the duration of the electronic pulse generated as a cell crosses the laser beam, which correlates with cell size and is used for doublet discrimination.
Question 7: What is the function of a neutral density (ND) filter in flow cytometry?
- To select specific emission wavelengths for detection
- To uniformly reduce light intensity across all wavelengths (Correct answer)
- To convert polarized light into unpolarized light
- To correct for chromatic aberration in the optical path
Correct answer: To uniformly reduce light intensity across all wavelengths
Neutral density filters attenuate light intensity uniformly across all wavelengths, used to reduce laser power or signal intensity without affecting spectral characteristics.
SCYM Principles of Flow Cytometry & Instrumentation 3
What is spectral spillover in multicolor flow cytometry?