Autofluorescence

Micrograph of paper autofluorescing under ultraviolet illumination.

Autofluorescence is the natural emission of light by biological structures such as mitochondria and lysosomes when they have absorbed light, and is used to distinguish the light 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 vizualisation 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]

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

Autofluorescence in banana skin under different light conditions.
Molecule
 Excitation (nm)
Fluorescence (nm)
 Organisms
 Reference
NAD(P)H 340 450 All [8]
Chlorophyll 465, 665 673, 726 Plants
Collagen 270-370 305-450 Animals [8]
Retinol 500 Animals & bacteria [9]
Riboflavin 550 All [9]
Cholecalciferol 380-460 Animals [9]
Folic acid 450 All [9]
Pyridoxine 400 All [9]
Tyrosine 270 305 All [2]
Dityrosine 325 400 Animals [2]
Excimer-like aggregate 270 360 Animals collagen[2]
Glycation adduct 370 450 Animals [2]
Indolamine Animals
Lipofuscin 410-470 500-695 Eukaryotes [10]
Polyphenol Plants
Tryptophan 280 300-350 All
Flavin 380-490 520-560 All
Melanin 340–400 360–560 Animals [11]

References

  1. 1 2 3 Monici M. (2005). "Cell and tissue autofluorescence research and diagnostic applications". Biotechnol Annu. Rev. 11: 227–56. doi:10.1016/S1387-2656(05)11007-2. PMID 16216779.
  2. 1 2 3 4 5 Julian M. Menter (2006). "Temperature dependence of collagen fluorescence". Photochem. Photobiol. Sci. 5 (4): 403–410. doi:10.1039/b516429j. PMID 16583021.
  3. ↑ Chia, Thomas; Michael Levene (17 November 2009). "Detection of counterfeit U.S. paper money using intrinsic fluorescence lifetime". Optics Express 17 (24): 22054–22061. doi:10.1364/OE.17.022054. Retrieved January 31, 2012.
  4. ↑ Fritzsche M, Mandenius CF (September 2010). "Fluorescent cell-based sensing approaches for toxicity testing". Anal Bioanal Chem 398 (1): 181–91. doi:10.1007/s00216-010-3651-6. PMID 20354845.
  5. ↑ Gerrits EG, Smit AJ, Bilo HJ (March 2009). "AGEs, autofluorescence and renal function". Nephrol. Dial. Transplant. 24 (3): 710–3. doi:10.1093/ndt/gfn634. PMID 19033250. Retrieved 2011-04-23.
  6. ↑ James R. Mansfield, Kirk W. Gossage, Clifford C. Hoyt, and Richard M. Levenson "Autofluorescence removal, multiplexing, and automated analysis methods for in-vivo fluorescence imaging" J. Biomed. Opt., Vol. 10, 041207 (2005)
  7. ↑ Rainer Kaufmann, Patrick Müller, Michael Hausmann, Christoph Cremer: Imaging label-free intracellular structures by localisation microscopy. Micron (2010), doi:10.1016/j.micron.2010.03.06
  8. 1 2 Georgakoudi I, Jacobson BC, Müller MG, Sheets EE, Badizadegan K, Carr-Locke DL, Crum CP, Boone CW, Dasari RR, Van Dam J, Feld MS (2002-02-01). "NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes". Cancer Res. 62 (3): 682–687. PMID 11830520.
  9. 1 2 3 4 5 Zipfel WR, Williams RM, Christie R, Nikitin AY, Hyman BT, Webb WW (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. doi:10.1073/pnas.0832308100. PMC 165832. PMID 12756303.
  10. ↑ Schönenbrücher, Holger; 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.
  11. ↑ James M. Gallas and Melvin Eisner (May 1987). "Fluorescence of Melanin-Dependence upon Excitation Wavelength and Concentration". Photochem. And Photobiol. 45 (5): 595–600. doi:10.1111/j.1751-1097.1987.tb07385.x.
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