SCYM Fluorochrome Selection 3 — Questions and Answers
Question 1: Which fluorochrome combination is contraindicated in the same panel due to direct spectral overlap on the 488 nm laser?
- FITC and PE (Correct answer)
- PE and PE-Cy7
- FITC and PerCP-Cy5.5
- APC and APC-Cy7
Correct answer: FITC and PE
FITC and PE cannot easily coexist on the 488 nm laser in the same panel without very high compensation due to overlapping emission spectra.
Question 2: What is the primary limitation of tandem dyes such as PE-Cy7 compared to pure fluorochromes?
- They cannot be excited by common lasers
- They have low extinction coefficients
- Incomplete energy transfer can produce anomalous emission peaks (Correct answer)
- They require UV excitation
Correct answer: Incomplete energy transfer can produce anomalous emission peaks
Tandem dyes can suffer from incomplete FRET energy transfer, resulting in donor (e.g., PE) emission appearing in unexpected channels.
Question 3: For a marker present on only 0.1% of lymphocytes, which fluorochrome brightness tier is most appropriate?
- Dim (e.g., PerCP)
- Intermediate (e.g., FITC)
- Bright (e.g., PE or BV421) (Correct answer)
- Any brightness is equally suitable
Correct answer: Bright (e.g., PE or BV421)
Rare or dim populations require bright fluorochromes to generate sufficient signal above background noise.
Question 4: Which of the following fluorochromes is classified as a 'large Stokes shift' dye, useful for simplifying panel design?
- PE
- FITC
- Pacific Orange (Correct answer)
- BV421
Correct answer: Pacific Orange
Pacific Orange has a large Stokes shift, absorbing around 400 nm and emitting around 551 nm, allowing flexible panel design.
Question 5: APC has its excitation maximum at approximately which wavelength?
- 488 nm
- 532 nm
- 633–638 nm (Correct answer)
- 405 nm
Correct answer: 633–638 nm
APC (Allophycocyanin) has its excitation maximum near 650 nm, making it ideal for the 633 or 638 nm red laser.
Question 6: Which factor most directly affects compensation matrix accuracy when switching lot numbers of a tandem dye?
- Laser power stability
- Lot-to-lot variation in FRET efficiency altering spectral characteristics (Correct answer)
- Buffer composition of the sample
- Cell viability
Correct answer: Lot-to-lot variation in FRET efficiency altering spectral characteristics
Tandem dyes can vary between lots due to differences in FRET efficiency, altering their emission spectrum and requiring recalculation of compensation values.
Question 7: When selecting fluorochromes for surface and intracellular markers in the same panel, which consideration is unique to intracellular staining?
- Intracellular markers always require brighter dyes
- Fixation and permeabilization can quench or shift certain fluorochrome emissions (Correct answer)
- Intracellular dyes must be excited by UV
- Compensation is unnecessary for intracellular staining
Correct answer: Fixation and permeabilization can quench or shift certain fluorochrome emissions
Fixation and permeabilization reagents can quench or spectrally shift certain fluorochromes (especially tandems), altering their emission profiles.
SCYM Fluorochrome Selection 3
Which fluorochrome combination is contraindicated in the same panel due to direct spectral overlap on the 488 nm laser?