In our previous post, we discussed how naturally occurring fluorescence, or “autofluorescence” is present in many samples. This inherent signal can sometimes interfere with the desired fluorescence emission from applied probes, and is often discarded as “background”. However, sometimes autofluorescence can provide useful information about your sample rather than being something undesirable that is to be avoided.
Biological tissue contains diverse naturally occurring fluorophores. A quick literature search, or simply imaging an unlabeled sample, can reveal autofluorescent structures. Further, taking the time to identify and decode the significance of the autofluorescence features can reward you with new and unexpected insights about your research question(s). These details can be missed unless you take the time to image the unstained sample.
There are many examples of autofluorescence from the literature, but one of our favorite examples is from a local lab here at the University of Calgary. Brain tissue contains many autofluorescent features, and while this can be a nuisance, this autofluorescence can also be used as a marker of degeneration due to stress or age. In brain tissue slices, accumulation and quantification of lipofuscin can be used to assess tissue health. Moreover, many amyloid-containing plaques, indicative of brain disease, have proven to be autofluorescent and can be identified and quantified without the need for added labeling. These methods were developed only by observing the autofluorescence and realizing that there was value in the signal rather than discarding it.
Figure 1 shows a section of 5xFAD mouse brain that has not been stained with any external fluorophores. Note the lipofuscin deposits in yellow, amyloid plaques in blue, and a general tissue background in green, all from autofluorescence. Generation of lipofuscin debris has been correlated to demyelination and aging, and by quantifying the debris it is possible to determine differences between normally aging mice and mice that have undergone a demyelinating event. This observation allows lipofuscin, normally an obstacle to be avoided in imaging, to be used as a label-free metric of myelin damage.
Liver tissue also contains a wealth of autofluorescent emission. Figure 2, left, shows mouse hepatocytes (liver cells) within a tissue section. While the cells themselves are emitting green, the dark nuclei are still clearly identifiable against the background. Figure 2, right, shows autofluorescence from the connective tissue that comprises Glisson’s Capsule, which surrounds the liver. The long strands in the image are likely elastin fibers, which are a component of the capsule wall. The organization of these fibers is known to change in certain disease states, such as cirrhosis of the liver.
Metabolic activity in the liver can also be monitored by observing NAD+/NADH ratios. The autofluorescence of NAD can be used to image metabolic activity in the liver when combined with fluorescence lifetime techniques which are sensitive to NAD+/NADH ratios. This illustrates how advanced fluorescence techniques such as lifetime imaging still work with autofluorescent signals, and shows how careful consideration of the “background” can reveal new information about your sample.
So the next time you see strange spots or structures in your “background”, perhaps consider a second look. You might just find something fascinating and useful!
Fig.1 Autofluorescent “Alheimer’s-like” cortical plaques (blue) and lipofuscin (yellow) in 5xFAD mouse brain slice with 405 nm excitation (Spectral confocal 410-730nm, 25x NA 1.1). No labeling was applied to the sample. (Image courtesy Megan Morgan, Stys Laboratory, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary) .
Fig.2 Autofluorescent liver slice excited by 488 nm (Spinning Disk Confocal, GFP and Cy5 channels 20x NA 0.95). Left, hepatocytes exhibiting characteristic double nuclei and Right, Glisson’s capsule, the connective tissue surrounding the liver (images courtesy Dr. Bjoern Petri, Mouse Phenomics Resource Laboratory, Joan Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary).
What to do with high autofluorescence background in pancreatic tissues – an efficient Sudan black B quenching method for specific immunofluorescence labelling – Erben – 2016 – Histopathology – Wiley Online Library
Autofluorescence characterisation of isolated whole crypts and primary cultured human epithelial cells from normal, hyperplastic, and adenomatous colonic mucosa | Journal of Clinical Pathology (bmj.com)
Connective tissue configuration in the human liver hilar region with special reference to the liver capsule and vascular sheath – Hayashi – 2008 – Journal of Hepato-Biliary-Pancreatic Surgery – Wiley Online Library