{"id":1187,"date":"2016-09-12T09:41:07","date_gmt":"2016-09-12T09:41:07","guid":{"rendered":"http:\/\/blogs.cardiff.ac.uk\/bioimaging\/?p=1187"},"modified":"2022-04-13T16:27:04","modified_gmt":"2022-04-13T16:27:04","slug":"in-focus-cutting-through-the-fog-reducing-background-autofluorescence-in-microscopy","status":"publish","type":"post","link":"https:\/\/blogs.cardiff.ac.uk\/bioimaging\/in-focus-cutting-through-the-fog-reducing-background-autofluorescence-in-microscopy\/","title":{"rendered":"IN FOCUS: Cutting Through the Fog: Reducing Background Autofluorescence in Microscopy."},"content":{"rendered":"
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<\/figure>\n<\/figure>\n
\"Autofluorescent<\/a><\/figure>\n

Above: Autofluorescence from mixed connective tissues imaged by confocal microscopy (left). The autofluorescent emissions can be spectrally-resolved through wavelength scanning (right). Excitation at 488nm.<\/em><\/p>\n

Whilst autofluorescence from endogenous fluorophores can reveal much about the biochemical composition of a sample, it can also hamper the microscopic detection of targeted fluorochromes if they emit light at the same wavelengths as endogenous fluors. Indeed, without proper controls, complex background autofluorescence can lead to misinterpretation of image data and generation of false positive results.<\/p>\n

Autofluorescence derives from multiple sources within the sample – the main culprits are  NADH and NADPH, lipofuscins, flavins, elastin and collagen (and lignin and chlorophyll in plants). The excitation and emission ranges of the worst offenders have been shown below. It follows that tissues with high collagen and elastin contents, e.g. skin, tendon and cartilage, autofluoresce very brightly; as do tissues that are rich in metabolic breakdown products such as lipofuscin, e.g. liver, spleen etc.<\/p>\n

\"Autofluorescent<\/a><\/figure>\n

Adding to the problem is the effect of chemical fixatives (e.g. formalin, glutaraldehyde etc) and solvents used to preserve tissue architecture for microscopy: the cross-linkages generated by these chemicals increase autofluorescence, which can be worsened further by long-term storage of the fixed processed tissues.<\/p>\n

So, dear reader, here’s some simple advice on steps that you can take to address this common problem:<\/p>\n

1. Include an unlabelled control to evaluate the level of autofluorescence within your sample.<\/strong><\/p>\n