Autofluorescence

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

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.

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]

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	Animals (Zoae)	Fungi	Plants	Reference
NAD(P)H	340	450	Z	F	P	[8]
Chlorophyll	465–665	673–726			P
Collagen	270–370	305–450	Z			[8]
Retinol		500	Z	F	P	[9]
Riboflavin		550	Z	F	P	[9]
Cholecalciferol		380–460	Z			[9]
Folic acid		450	Z	F	P	[9]
Pyridoxine		400	Z	F	P	[9]
Tyrosine	270	305	Z	F	P	[2]
Dityrosine	325	400	Z			[2]
Excimer-like aggregate (collagen)	270	360	Z			[2]
Glycation adduct	370	450	Z			[2]
Indolamine			Z
Lipofuscin	410–470	500–695	Z	F	P	[10]
Lignin (a polyphenol)	335–488	455–535			P	[11]
Tryptophan	280	300–350	Z	F	P
Flavin	380–490	520–560	Z	F	P
Melanin	340–400	360–560	Z	F	P	[12]

Substances luminous in animal tissue are, by taxonomic inclusion, also luminous in human tissue.

See also

• Autoluminescence
• Phosphorescence
• Fluorescence in the life sciences

References

1. Monici, M. (2005). "Cell and tissue autofluorescence research and diagnostic applications". Biotechnology Annual Review. 11: 227–256. doi:10.1016/S1387-2656(05)11007-2. ISBN   9780444519528. PMID   16216779.
2. Menter, Julian M. (2006). "Temperature dependence of collagen fluorescence". Photochemical & Photobiological Sciences. 5 (4): 403–410. doi:10.1039/b516429j. PMID   16583021. S2CID   34205474.
3. Chia, Thomas; Levene, Michael (17 November 2009). "Detection of counterfeit U.S. paper money using intrinsic fluorescence lifetime". Optics Express. 17 (24): 22054–22061. Bibcode:2009OExpr..1722054C. doi: 10.1364/OE.17.022054 . PMID   19997451.
4. Fritzsche, M.; Mandenius, C.F. (September 2010). "Fluorescent cell-based sensing approaches for toxicity testing". Anal Bioanal Chem. 398 (1): 181–191. doi:10.1007/s00216-010-3651-6. PMID   20354845. S2CID   22712460.
5. Gerrits, E.G.; Smit, A.J.; Bilo, H.J. (March 2009). "AGEs, autofluorescence and renal function". Nephrol. Dial. Transplant. 24 (3): 710–713. doi: 10.1093/ndt/gfn634 . PMID   19033250.
6. Mansfield, James R.; Gossage, Kirk W.; Hoyt, Clifford C.; Levenson, Richard M. (2005). "Autofluorescence removal, multiplexing, and automated analysis methods for in-vivo fluorescence imaging". Journal of Biomedical Optics. 10 (4): 041207. Bibcode:2005JBO....10d1207M. doi: 10.1117/1.2032458 . PMID   16178631. S2CID   35269802.
7. Kaufmann, R.; Müller, P.; Hausmann, M.; Cremer, C. (2010). "Imaging label-free intracellular structures by localisation microscopy". Micron. 42 (4): 348–352. doi:10.1016/j.micron.2010.03.006. PMID   20538472.
8. Georgakoudi, I.; Jacobson, B.C.; Müller, M.G.; Sheets, E.E.; Badizadegan K.; Carr-Locke, D.L.; et al. (2002-02-01). "NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes". Cancer Research. 62 (3): 682–687. PMID   11830520.
9. Zipfel, W.R.; Williams, R.M.; Christie, R.; Nikitin, A.Y.; Hyman, B.T.; Webb, W.W. (2003-06-10). "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation". Proceedings of the National Academy of Sciences of the United States of America. 100 (12): 7075–7080. Bibcode:2003PNAS..100.7075Z. doi: 10.1073/pnas.0832308100 . PMC   165832 . PMID   12756303.
10. Schönenbrücher, Holger; Adhikary, Ramkrishna; Mukherjee, Prasun; Casey, Thomas; Rasmussen, Mark; Maistrovich, Frank; et al. (2008). "Fluorescence-based method, exploiting lipofuscin, for real-time detection of central nervous system tissues on bovine carcasses". Journal of Agricultural and Food Chemistry. 56 (15): 6220–6226. doi:10.1021/jf0734368. PMID   18620407.
11. Donaldson, Lloyd; Williams, Nari (February 2018). "Imaging and spectroscopy of natural fluorophores in pine needles". Plants. 7 (1): 10. doi: 10.3390/plants7010010 . PMC   5874599 . PMID   29393922.
12. Gallas, James M. & Eisner, Melvin (May 1987). "Fluorescence of melanin-dependence upon excitation wavelength and concentration". Photochemistry and Photobiology. 45 (5): 595–600. doi:10.1111/j.1751-1097.1987.tb07385.x. S2CID   95703924.