Causes of Autofluorescence

One of Visikol’s main areas of focus is histology and multiplex imaging. Visikol’s multiplex imaging is driven by patented Histo clearing reagents that work to make images of cells clearer and fit for scientific research. A problem that often occurs while prepping and clearing tissues for imaging is something known as Autofluorescence. Autofluorescence is the fluorescence of the naturally occurring substances in a cell, and these substances usually gain fluorescence through accidental excitation. Autofluorescence can happen for multiple reasons, including issues during fixation, heat and dehydration, and fluorescence of heme. Autofluorescence can complicate the analysis of a section and skew the results of an experiment. However, Autofluorescence can be helpful if it labels something that would otherwise require labeling.

Fixation-Induced Autofluorescence

Since there are several causes of Autofluorescence, there are many things to consider when attempting to mitigate the effects of Autofluorescence. In fixation-induced Autofluorescence, the cause of the fluorescence is the cross-linking of tyrosine and tryptophan residues in proteins that contain formaldehyde. This cross-linking forms a fluorescent formaldehyde adduct that can cause this autofluorescence to appear. The best way to avoid this is to ensure that the tissues have been fixed for the minimum amount of time required for the size and type of tissue. In Visikol’s Tissue Clearing eBook, the best protocol for slide fixations is outlined.

Heat and Dehydration-Induced Autofluorescence

The next cause of Autofluorescence to discuss is heat and dehydration-induced Autofluorescence. Treatment of fixed tissues at too high a temperature can cause background fluorescence. When dehydrating, staining, and clearing your tissues, be sure to do so with tissues incubated at room temperature. The effect of heat-induced Autofluorescence is much more significant in the 530-600 nm (red) region.

Endogenous Pigments

The last type of Autofluorescence to discuss is Autofluorescence by endogenous pigments. Heme Autofluorescence is a type of fluorescence caused by the pigment in blood cells. To control this, you should perfuse tissues with phosphate buffer solution before fixation. This will remove the blood cells from tissues and eliminate problems pre-processing. Other types of endogenous pigment Autofluorescence include fluorescence through lipofuscin, collagen, and other lipophilic pigments. The fluorescent spectra of these pigments vary along the spectrum, and the protocol to get rid of their fluorescence can vary.

Visikol has found that background Autofluorescence can be managed if you follow methods correctly. In Visikol’s Tissue Clearing eBook, there are many tips and strategies for keeping Autofluorescence down, including tables and charts documenting the wavelengths at which common pigments tend to be excited. Visikol offers high-quality clearing reagents and kits that are great for your next clearing or imaging project. If you are interested in working with Visikol, don’t hesitate to get in touch with us.

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