Metabolic Cofactor Autofluorescence

Symptom

Endogenous fluorescence from NADH, FAD, and other metabolic cofactors in high-metabolism tissues such as kidney, liver, pancreas, and spleen. Complex extracellular matrices contribute additional background.

1. 1 Abundant NADH and FAD in metabolically active organs
2. 2 Complex extracellular matrix composition
3. 3 High baseline metabolic activity
4. 4 Overlapping emission with common fluorophores

1. 1 Assign low-abundance targets to far-red or near-IR channels
2. 2 Reserve shorter wavelength channels for abundant targets only
3. 3 Test Sudan Black B and cupric sulfate side-by-side to find optimal quencher
4. 4 Include no-primary/no-secondary controls to quantify baseline autofluorescence
5. 5 Use spectral unmixing when available to separate metabolic background

Related Video (2)

Current Protocols ★ 92

Deciding on an Approach for Mitigating Autofluorescence

"Directly addresses mitigation strategies for autofluorescence in fluorescence microscopy, core to solving this metabolic cofactor background problem"

JoVE (Open Access) ★ 78

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

"Demonstrates use of sFLIM to manage native autofluorescence in tissue samples, directly applicable to NADH/FAD background in high-metabolism tissues"