ISAC Panel Design and Fluorochrome Selection 2 — Questions and Answers
Question 1: What is the purpose of a fluorescence minus one (FMO) control in a multicolor flow cytometry panel?
- To measure laser power stability over time
- To define accurate gate boundaries by capturing spillover spread from all other fluorochromes (Correct answer)
- To calculate absolute cell counts from a known volume
- To assess cell viability independently of staining
Correct answer: To define accurate gate boundaries by capturing spillover spread from all other fluorochromes
FMO controls contain every fluorochrome except the one being gated, so the signal seen in that channel represents only the spread from other fluorochromes, enabling accurate gate placement.
Question 2: Which of the following best describes 'spreading error' in multicolor flow cytometry?
- Physical dispersal of cells across the laser interrogation point
- Increased variance in fluorescence measurements caused by spectral spillover and its mathematical compensation (Correct answer)
- Uneven conjugation of fluorochromes to antibodies
- Technical errors introduced during data file export
Correct answer: Increased variance in fluorescence measurements caused by spectral spillover and its mathematical compensation
Spreading error is the additional variance introduced when compensation is applied to correct for spillover; it increases noise and can reduce the ability to resolve closely spaced populations.
Question 3: When designing a panel on an instrument with limited laser lines, which strategy best maximizes the number of parameters measured?
- Using only the two brightest fluorochromes available
- Selecting fluorochromes with widely separated emission peaks on each laser (Correct answer)
- Using multiple antibodies labeled with the same fluorochrome
- Increasing laser power to resolve overlapping signals
Correct answer: Selecting fluorochromes with widely separated emission peaks on each laser
Choosing fluorochromes whose emission peaks are widely spaced minimizes spectral overlap, allowing more independent detection channels to be used per laser line.
Question 4: What characteristic makes PE (phycoerythrin) one of the most valuable fluorochromes in flow cytometry panels?
- Its narrow emission spectrum that eliminates spillover
- Its extremely high quantum yield and molar absorptivity resulting in exceptional brightness (Correct answer)
- Its ability to be excited simultaneously by all laser lines
- Its resistance to photobleaching during extended acquisitions
Correct answer: Its extremely high quantum yield and molar absorptivity resulting in exceptional brightness
PE's very high quantum yield combined with a large absorption cross-section makes it one of the brightest fluorochromes available, ideal for detecting low-abundance targets.
Question 5: Which of the following is a recognized disadvantage of using tandem dyes in flow cytometry experiments?
- They cannot be conjugated to antibodies
- They show lot-to-lot variability and can degrade, producing incomplete energy transfer artifacts (Correct answer)
- They require ultraviolet lasers not available on standard instruments
- They are incompatible with intracellular staining protocols
Correct answer: They show lot-to-lot variability and can degrade, producing incomplete energy transfer artifacts
Tandem dyes are susceptible to dye degradation and lot-to-lot spectral variability because partial breakdown of the FRET pair causes donor emission bleed-through, complicating compensation.
Question 6: In spectral flow cytometry, what replaces traditional bandpass filters for signal detection?
- Prisms or diffraction gratings that disperse light across an array of detectors (Correct answer)
- Higher-powered lasers that reduce spectral overlap
- Digital compensation matrices applied before acquisition
- Avalanche photodiodes replacing photomultiplier tubes
Correct answer: Prisms or diffraction gratings that disperse light across an array of detectors
Spectral cytometers use optical elements such as prisms or gratings to spread emission light across a detector array, capturing the complete emission spectrum of each event.
Question 7: What does 'unmixing' refer to in spectral flow cytometry data analysis?
- Physically separating cells into distinct collection tubes
- A mathematical process that deconvolves overlapping spectra to quantify each fluorochrome's individual contribution (Correct answer)
- Removing doublets and debris from the data file
- Normalizing laser power across acquisition runs
Correct answer: A mathematical process that deconvolves overlapping spectra to quantify each fluorochrome's individual contribution
Unmixing uses reference spectra for each fluorochrome and a least-squares algorithm to separate the contributions of all fluorochromes from the composite signal detected on each event.
ISAC Panel Design and Fluorochrome Selection 2
What is the purpose of a fluorescence minus one (FMO) control in a multicolor flow cytometry panel?