SCYM Fluorochrome Selection 2 — Questions and Answers
Question 1: Which fluorochrome has the highest quantum yield among commonly used dyes, making it ideal for detecting low-abundance antigens?
- Allophycocyanin (APC)
- Peridinin-Chlorophyll-Protein (PerCP)
- R-Phycoerythrin (PE) (Correct answer)
- Fluorescein Isothiocyanate (FITC)
Correct answer: R-Phycoerythrin (PE)
PE has one of the highest quantum yields (~0.98) of commonly used fluorochromes, making it excellent for detecting low-abundance antigens.
Question 2: When designing a multicolor panel, a marker with very high expression should be paired with a fluorochrome that has:
- High brightness
- Low brightness (Correct answer)
- Wide emission spectrum
- Long Stokes shift
Correct answer: Low brightness
Highly expressed antigens should be paired with dim fluorochromes to prevent spillover from oversaturation overwhelming adjacent detector channels.
Question 3: Which property of a fluorochrome directly determines the amount of spectral spillover into adjacent channels?
- Excitation maximum
- Stokes shift magnitude
- Emission spectrum width and tail (Correct answer)
- Molecular weight
Correct answer: Emission spectrum width and tail
The width and tail of the emission spectrum determine how much signal from one fluorochrome bleeds into adjacent detector channels.
Question 4: BV421 is best excited by which laser line commonly found on flow cytometers?
- 488 nm blue laser
- 532 nm yellow-green laser
- 405 nm violet laser (Correct answer)
- 638 nm red laser
Correct answer: 405 nm violet laser
BV421 (Brilliant Violet 421) is optimally excited by the 405 nm violet laser and emits around 421 nm.
Question 5: Which fluorochrome is most susceptible to photobleaching during extended acquisition times?
- APC-Cy7
- PE-Cy7
- FITC (Correct answer)
- BV605
Correct answer: FITC
FITC is highly susceptible to photobleaching compared to more photostable alternatives like BV dyes or tandem conjugates.
Question 6: In a panel using a UV laser (355 nm), which fluorochrome would be an appropriate choice for UV excitation?
- FITC
- PE
- BUV395 (Correct answer)
- BV711
Correct answer: BUV395
BUV395 (Brilliant Ultraviolet 395) is specifically designed for excitation with the 355 nm UV laser.
Question 7: When multiplexing fluorochromes on the same laser, which parameter is most important to minimize spectral overlap?
- Maximizing emission peak distance between dyes (Correct answer)
- Matching extinction coefficients
- Using dyes with identical quantum yields
- Selecting dyes with the same molecular weight
Correct answer: Maximizing emission peak distance between dyes
Maximizing the distance between emission peaks of co-excited fluorochromes minimizes spectral overlap and reduces the need for high compensation.
SCYM Fluorochrome Selection 2
Which fluorochrome has the highest quantum yield among commonly used dyes, making it ideal for detecting low-abundance antigens?