Poor Resolution of Dim Markers Masked by Autofluorescence (Flow Cytometry (Autofluorescence))

Question

Accepted Answer

Low-expression markers become indistinguishable from background. Positive and negative populations show poor separation, with dim fluorophores completely masked by cellular autofluorescence. Common causes: 1) Dim fluorophores (low-brightness dyes) easily masked by autofluorescence signal 2) FITC highly susceptible due to overlap with NADH and flavin emissions 3) UV (355 nm) and blue (488 nm) lasers induce strongest autofluorescence affecting FITC and BUV dyes 4) Low-expression antigens generating weak signal compared to background Solutions: 1) Assign bright dyes (PE, BV421) to low-expression markers instead of FITC 2) Avoid FITC for critical markers; use dyes with emissions outside autofluorescence range 3) Use red/far-red lasers (633 nm and beyond) which are least affected by autofluorescence 4) Include FMO (Fluorescence Minus One) controls to distinguish true signal from background 5) Consider spectral unmixing to separate dim marker signal from autofluorescence