Autofluorescence

General comments on autofluorescence

The following review paper has a useful summary of autofluorescent molecules, including excitation/emission wavelength ranges. (information by: Iain Johnson, Molecular Probes)

Billinton N, Knight AW., "Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence." Analytical Biochemistry (2001) 291:175-197.

Autofluorescence due to Lipofuschin

This is commonly seen in older tissues, particularly brain and in the retinal pigmented epithelium (RPE layer) of the eye. This autofluorescence typically has similar excitation and emission characteristics to fluorescein and will, therefore, interfere with the detection of FITC and GFP fluorescence.

The following information comes from a discussion on the confocal microscopy listserver, an email based discussion list of approximately 1500 confocal and related microscopists.

1. It's also possible to quench lipofuschin autofluorescence with CuSO4 or Sudan Black. Reference: SA Schnell, W Staines and M Wessendorf. "Reduction of lipofuschin-like autofluorescence in fluorescently labeled tissue." J. Histochem Cytochem, 47(6): 719-30, 1999.
   
   



2. Sudan Black B (BDH Laboratory Supplies, Poole, BH15 1TD, England) with success against autofluorescence in human brain tissue. As described in: Romijn et al, J Histochem Cytochem, 47(2): 229-235. It must be dissolved in the dark to 0.3% in 70% EtOH at room temp (RT), stirring, left to stand overnight and then filtered (can be stored at this point up to 2 months) before used on tissue. They recommend inc with SBB for 10 mins at RT between primary and secondary, rinsing rapidly and repeatedly (10x) in Tris buffered saline (pH 7.5). It may be especially useful against lipofuschin granules. It appears to get taken up in a vesicular pattern, reminescent of the absent autofluorescence.

Connective tissue autofluorescence

Elastin is highly autofluorescent particularly when using the typical excitation/emission filter sets for fluorescein or GFP. By staining the tissue with Pontamine Sky Blue and using what is most likely a FRET (Fluorescence Resonance Energy Transfer) method, the emission from this autofluorescence can be moved into a longer wavelength region where it does not interfere with FITC or GFP emission.

Notes from our lab manual: Pontamine Sky Blue (BDH 34138 2A). Reference Cowen et al, Histochemistry, 82: 205 - 8. Used to reduce autofluorescence on FITC labelled sections only (fluoresces in the TRITC range). Use at 0.5% in PBS for 10 - 15 minutes with a 5 minute wash for sections or a 30 minute wash for thick wholemounts.

From Confocal Listserver (provided by Ian Clements, Molecular Probes):

The following article cites the use of a dye called Pontamine Sky Blue (also known as C.I. Direct Blue 1) as a selective quencher of autofluorescence in mesenteric vessels and carotid arteries. The dye is available through BDH. Reference: "Pontamine Sky Blue: A counterstain for background autofluorescence in fluorescence and immunofluorescence histochemistry", T. Cowen, A.J. Haven and G. Burnstock, Histochemistry, 82, 205-208, 1985.

• Alfa Aesar (cat A14242) called Pontamine Sky Blue 6BX
• TCI (cat B0782) Direct Blue 1
• Pfaltz and Bauer, Inc (cat C07550) Chicago Blue 6b, C.I. No 24410
• Acros (cat 19147) Chicago Sky Blue 6B