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 strategies for mitigating autofluorescence, the core symptom of this failure case."

JoVE (Open Access) ★ 65

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

"Spleen tissue confocal imaging is a context where this endogenous autofluorescence background occurs and must be handled."