What causes autofluorescence?

• Intrinsic cellular components: Molecules like NADH, flavins, and porphyrins emit fluorescence when excited.

• Cellular structures: Granules, mitochondria, and extracellular matrix components contribute to background signal.

• Sample preparation: Fixation (especially aldehyde-based), permeabilization, and certain buffers can increase autofluorescence.

• Tissue samples: Solid tissues exhibit higher autofluorescence than PBMCs due to structural complexity and pigments.

• Cell activation: Activated cells often show increased autofluorescence due to metabolic changes.

Cell types with high autofluorescence

• Monocytes and macrophages: Rich in granules and metabolic cofactors.

• Dendritic cells: High metabolic activity and granularity.

• Epithelial cells: Contain structural proteins and pigments.

• Tumor cells: Often exhibit elevated autofluorescence due to metabolic changes.

• Fixed cells: Aldehyde-based fixation amplifies autofluorescence.

Dyes most affected by autofluorescence

• FITC (green channel): Highly susceptible due to overlap with NADH and flavin emissions.

• PE (orange channel): Can be influenced by cellular pigments.

• Brilliant violet and UV dyes: Less affected but interference can occur in complex samples.

• Low-brightness dyes: Dim fluorophores are easily masked by autofluorescence.

Impact on lasers

• UV (355 nm) and blue (488 nm): Autofluorescence is strongest here, affecting UV dyes (BUV series) and FITC.

• Violet (405 nm): Moderate interference.

• Red/far-red (633 nm and beyond): Least affected, ideal for critical markers.

Strategies to mitigate autofluorescence

1. Use spectral flow cytometry

• Spectral unmixing can model and subtract autofluorescence signatures.
• Include an unstained control for autofluorescence reference.

2. Choose alternative fluorophores

• Select dyes with emissions outside the autofluorescence range (eg far-red or infrared dyes like APC or Alexa Fluor 700).

3. Sample preparation

• Minimize fixation or use fixatives that reduce autofluorescence.
• Wash samples thoroughly to remove residual reagents.

4. Include proper controls

• Unstained controls are essential for identifying autofluorescence.
• FMO controls help distinguish true signal from background.

5. Gate strategically

• Use additional markers to confirm population identity when autofluorescence complicates gating.

Troubleshooting autofluorescence

Problem 1: High background signal

• Cause: Autofluorescent cell types or tissue samples
• Fix: Use far-red dyes, include unstained controls, consider spectral cytometry

Problem 2: Poor resolution of dim markers

• Cause: Dim fluorophores masked by autofluorescence
• Fix: Assign bright dyes (PE, BV421) to low-expression markers; avoid FITC for critical markers

Problem 3: Autofluorescence in tissue samples

• Cause: Aldehyde-based fixatives enhance autofluorescence
• Fix: Minimize fixation time, wash thoroughly, use far-red dyes for key markers

Problem 4: Autofluorescence in tissue samples

• Cause: Structural proteins and pigments in solid tissues
• Fix: Use spectral cytometry, choose dyes outside autofluorescence range, include unstained tissue controls

Problem 5: Autofluorescence interfering with viability dye

• Cause: Overlap with viability dye emission
• Fix: Select viability dyes in far-red channels; validate gating with FMO controls

Extra tip

If available, spectral unmixing is the most effective way to handle autofluorescence. Always include an unstained control for autofluorescence reference.