We are commonly asked by researchers how to minimize autofluorescence in tissues for fluorescent imaging as a high background can ruin your ability to acquire data from your tissues. Here we discuss what causes autofluorescence and approaches to reduce it in your tissues.

Autofluorescence has several causes:

1. Fixation Induced Autofluorescence: Cross-linking of tyrosine and tryptophan residues in proteins with formaldehyde generates increased autofluorescence due to formation of fluorescent formaldehyde adducts. Glutaraldehyde exacerbates this issue even further. The best way to avoid autofluorescence due to fixation is to fix tissues for the minimum amount of time required for the size and type of tissue. Generally, tissues should be fixed in freshly prepared 4% PFA (paraformaldehyde) or 10% NBF (neutral buffered formalin) overnight at 4°C and then at room temperature for 1-2 hours. Larger tissues may require longer incubation times, but in general, immersion fixed tissues should be less than 6 mm thick. After fixation, tissues should be transferred to PBS containing 0.05% sodium azide or similar preservative, and stored at 4°C.  While fixation induced autofluorescence can occur to some degree in most tissues, it can be avoided by using non-crosslinking fixatives. Non-crosslinking fixatives will also increase the ability of antibodies and stains to penetrate the tissues. However, the use of non-crosslinking fixatives may lead to loss of some epitopes if the protein of interest is not bound to the structure of the tissues.

2. Heat and Dehydration induced Autofluorescence: Treatment of tissues at elevated temperatures can increase autofluorescence in fixed tissues. To minimize autofluorescence, incubate tissues at room temperature for dehydration, staining, and clearing steps. Dehydration of tissues is a necessary step in clearing tissues with Visikol HISTO. Alcohol dehydration increases background autofluorescence by shifting equilibrium of formalin-adducts in fixed tissues with the effect of increasing the content intensity of autofluorescence. This effect is much greater in the red (530-600 nm) region than in the green or blue.

3. Endogenous Pigments: Heme Autofluorescence: The heme group is the primary pigment in blood cells. This porphyrin ring structure exhibits broad autofluorescence in tissues and can complicate analysis. The best way to control heme autofluorescence is simply to perfuse tissues with PBS prior to fixation at the time of sacrifice. This procedure will remove blood cells from tissues and eliminate this problem at the source. If it is not possible to perfuse the tissue (i.e. archived specimens), there are techniques that can be applied to bleach the tissue and reduce autofluorescence of heme. The procedure involves incubating tissues in 5% H2O2 in Methanol/DMSO (1 part 30% H2O2, 4 parts methanol, 1 part 100% DMSO) at 4°C overnight prior to staining and clearing steps.

4. Endogenous pigments: Lipofuscin, Collagen, and Others: Lipofuscin is a lipophilic pigment that accumulates through normal aging processes in animal tissue and is significantly autofluorescent. Lipofuscin often appears as small yellow granules in fluorescent imaging. Collagen is a structural protein which occurs in high concentrations in many tissues (e.g. liver, muscle) and is strongly autofluorescent in the blue region (350-450 nm), and lesser so in the green region (475-550 nm).  There are also several other biomolecules that are innate to animal tissue that can exhibit autofluorescence. These include nicotinamides (NADP), retinols and carotenoids, bile acids and downstream products like bilirubin.

Methods to manage autofluorescence

1. Perfuse tissues with PBS to remove blood and other soluble pigments prior to fixation.

2. Incubate tissues at room temperature or 4°C for antigen retrieval, labeling and clearing steps. Incubation times should be increased by 1.5-2x at 4°C due to slow diffusion.

3. Bleach tissues with 5% H2O2 during antigen retrieval step (not compatible with fluorescent protein). The procedure involves incubating tissues in 5% H2O2 in Methanol/DMSO (1 part 30% H2O2, 4 parts methanol, 1 part 100% DMSO) at 4°C overnight prior to staining and clearing steps.

4. Autofluorescence can be subtracted during image processing to reveal signals otherwise difficult to detect. Autofluorescence is highest in the red channel (TRITC, 530-600 nm), so select green, or far red for important labels, and use blue for nuclear staining. During imaging, capture your signal channel, and a channel of autofluorescence, and subtract the autofluorescent image stack from the signal channels to decouple autofluorescence from signal.

Author: Dr. Tom Villani