Fluorochrome Selection Flashcards

Which fluorochrome combination is contraindicated in the same panel due to direct spectral overlap on the 488 nm laser?

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.

What is the primary limitation of tandem dyes such as PE-Cy7 compared to pure fluorochromes?

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.

For a marker present on only 0.1% of lymphocytes, which fluorochrome brightness tier is most appropriate?

Answer: Bright (e.g., PE or BV421)

Rare or dim populations require bright fluorochromes to generate sufficient signal above background noise.

Which of the following fluorochromes is classified as a 'large Stokes shift' dye, useful for simplifying panel design?

Answer: Pacific Orange

Pacific Orange has a large Stokes shift, absorbing around 400 nm and emitting around 551 nm, allowing flexible panel design.

APC has its excitation maximum at approximately which wavelength?

Answer: 633–638 nm

APC (Allophycocyanin) has its excitation maximum near 650 nm, making it ideal for the 633 or 638 nm red laser.

Which factor most directly affects compensation matrix accuracy when switching lot numbers of a tandem dye?

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.

When selecting fluorochromes for surface and intracellular markers in the same panel, which consideration is unique to intracellular staining?

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.