Autofluorescence

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File:PaperAutofluorescence.jpg
Micrograph of paper autofluorescing under ultraviolet illumination. The individual fibres in this sample are around 10 μm in diameter.

Autofluorescence is the natural fluorescence of biological structures such as mitochondria and lysosomes, in contrast to fluorescence originating from artificially added fluorescent markers (fluorophores).[1]

The most commonly observed autofluorescencing molecules are NADPH and flavins; the extracellular matrix can also contribute to autofluorescence because of the intrinsic properties of collagen and elastin.[1]

Generally, proteins containing an increased amount of the amino acids tryptophan, tyrosine, and phenylalanine show some degree of autofluorescence.[2]

Autofluorescence also occurs in non-biological materials found in many papers and textiles. Autofluorescence from U.S. paper money has been demonstrated as a means for discerning counterfeit currency from authentic currency.[3]

Microscopy

File:Unmixed Autofluorescence.gif
A multispectral image of tissue from a mouse intestine, showing how autofluoresce can obscure several fluorescence signals.

Autofluorescence can be problematic in fluorescence microscopy. Light-emitting stains (such as fluorescently labelled antibodies) are applied to samples to enable visualisation of specific structures.

Autofluorescence interferes with detection of specific fluorescent signals, especially when the signals of interest are very dim — it causes structures other than those of interest to become visible.

In some microscopes (mainly confocal microscopes), it is possible to make use of different lifetime of the excited states of the added fluorescent markers and the endogenous molecules to exclude most of the autofluorescence.

File:Label-free Localisation Microscopy SPDM - Super Resolution Microscopy Christoph Cremer.jpg
Autofluorescence super resolution microscopy/optical nanoscopy image of cellular structures that are invisible with confocal light microscopy

In a few cases, autofluorescence may actually illuminate the structures of interest, or serve as a useful diagnostic indicator.[1]

For example, cellular autofluorescence can be used as an indicator of cytotoxicity without the need to add fluorescent markers.[4]

The autofluorescence of human skin can be used to measure the level of advanced glycation end-products (AGEs), which are present in higher quantities during several human diseases.[5]

File:BananaSkin40X Fluorescence.tif
Autofluorescence in banana skin under different light conditions.

Optical imaging systems that utilize multispectral imaging can reduce signal degradation caused by autofluorescence while adding enhanced multiplexing capabilities.[6]

The super resolution microscopy SPDM revealed autofluorescent cellular objects which are not detectable under conventional fluorescence imaging conditions.[7]

Autofluorescent molecules

Molecule Excitation
(nm) 		Fluorescence
(nm) Peak 		Template:Vertical header Template:Vertical header Template:Vertical header Template:Vertical header
NAD(P)H 340 450 Template:Sc Template:Sc Template:Sc [8]
Chlorophyll 465–665 673–726 Template:Sc
Collagen 270–370 305–450 Template:Sc [8]
Retinol 500 Template:Sc Template:Sc Template:Sc [9]
Riboflavin 550 Template:Sc Template:Sc Template:Sc [9]
Cholecalciferol 380–460 Template:Sc [9]
Folic acid 450 Template:Sc Template:Sc Template:Sc [9]
Pyridoxine 400 Template:Sc Template:Sc Template:Sc [9]
Tyrosine 270 305 Template:Sc Template:Sc Template:Sc [2]
Dityrosine 325 400 Template:Sc [2]
Excimer-like
aggregate
(collagen) 		270 360 Template:Sc [2]
Glycation adduct 370 450 Template:Sc [2]
Indolamine Template:Sc
Lipofuscin 410–470 500–695 Template:Sc Template:Sc Template:Sc [10]
Lignin
(a polyphenol) 		335–488 455–535 Template:Sc [11]
Tryptophan 280 300–350 Template:Sc Template:Sc Template:Sc
Flavin 380–490 520–560 Template:Sc Template:Sc Template:Sc
Melanin 340–400 360–560 Template:Sc Template:Sc Template:Sc [12]
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See also

References

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