IF Signal-To-Noise-Ratios

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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.”

Biological 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

Structural formula of flavin adenine dinucleotide (FAD), showing absorption at 450 nm and emission at 515 nm.

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Structural formula of Nicotinamide adenine dinucleotide (NADH), showing absorption at 340 nm and emission at 460 nm.

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Fixative-induced Autofluorescence

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.

Most Commonly Used Fixative For IF Staining
 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.