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Autofluorescence complicates the analysis of IF stainings. In general, the spectra of autofluorescence are very broad, compared to those of fluorescent probes. Thus, it is challenging to distinguish between autofluorescence and the actual fluorescence of interest, leading to misunderstanding of the image analysis. To overcome the problem of autofluorescence, it is important to have an idea of its source before starting to optimize the protocol or playing around with the filter sets. Autofluorescence is often used as a collective term for “biological autofluorescence” or so-called “fixativeinduced autofluorescence.”
Cells contain components that show fluorescent when excited by a suitable wavelength. This biological autofluorescence has its origin in endogenous fluorophores. Biological autofluorescence mainly comes from mitochondria, lysosomes, and aromatic amino acid components. The most important components causing intrinsic fluorescence are Flavin coenzymes (FAD, FMN) (Figure 1) or pyridine nucleotides (NADH) (Figure 2).
|Figure 1||Figure 2|
When fixing with aldehydes, these aldehydes can react with cellular amines or proteins, resulting in a fluorescent product. These issues can be diminished by reducing the aldehyde group to a hydroxyl group (e.g., by sodium borohydride).
Please note: These days, several commercial reagents are available to optimize and reduce background signal and autofluorescence. There are ready-to-use drop-in solutions on the market that suppress background signal and cell culture media suitable for live cell imaging. In case of a background suppressor that chemically reduces autofluorescence, overdosage has to be avoided as it is always combined with a loss of the actual signal.
|Type of fixative||Name||Advantage||Disadvantage|
|Organic solvent||Methanol||Cellular architecture is conserved.||Damaging to several epitopes. Lipid components lost|
|Organic solvent||Acetone||Gentle for epitopes.||Lipid components lost.|
|Chemical Cross-linker||Paraformaldehyde||Cellular morphology conserved.||Cross-linking of epitopes, autofluorescence.|