Challenges of Autofluorescence in Neuroscience

If you have ever imaged biological samples, you have likely encountered autofluorescence. That pesky background coloration you see under the microscope, which can make it difficult to distinguish your actual signal from the noise.¹ When you are trying to look for something as delicate as RNA, you don’t want to be hunting for your signal amongst the background!

Autofluorescence can present an even greater challenge when looking at brain or neural tissues due to the variety of cell types and the accumulation of lipofuscins that tend to stick around in neural cells. Lipofuscin, known as an aging pigment, accumulates in neural tissues and causes that annoying autofluorescence.² Intact tissues, particularly neural tissue, also have lipid bilayers that cause light scattering and autofluorescence—all of which can make it practically impossible to image your sample.

In samples as complex as neural tissue, accuracy counts. Also, you want to be able to see the signal you worked so hard to stain for! So, how can you reduce autofluorescence in your sample? There are a variety of ways to lower autofluorescence—from changing your fixation method to using different reagents (like Stellaris® RNA FISH, an updated take on RNA ISH or in situ hybridization).³ The first step to a better image is reducing autofluorescence.

Wash Your Sample Well to Reduce Autofluorescence

A simple way to help reduce autofluorescence in your sample is to make sure you wash your sample extremely well. You don’t want excess antibody, serum, fixative, or any other particulates to stay behind and increase your background. Avoid using BSA, if you chose to block with serum, as it is notorious for being difficult to wash away and creates noise in your images. Use a different kind of serum (such as goat or human) to block instead, but make sure it won’t react with your secondary antibodies and generate a false signal.⁴ And remember, it never hurts to do one more gentle wash—just to be safe!

Treat Your Samples Gently

While reducing autofluorescence is important, you don’t want to do that at the cost of the integrity of the structures you are trying to image in the first place. Some methods, like boiling your embedded sample in PBS, can be harsh and potentially damage the RNA and structures you are interested in visualizing.⁵ However, these methods can be effective at reducing background fluorescence. If you choose to go this route, it is important to be mindful of what you are looking to image!

Gentler Sample Preparation Methods

Recently developed tissue clearing methods have had major impact on the imaging field, especially neuroscience, because it generates a more optically efficient sample.^6,8 These methods can be generally classified as (1) refractive index (RI) matching, (2) lipid removal either through dehydration or hyperhydration followed by RI matching, or (3) hybridizing the tissue with a gel followed by lipid removal and RI matching.

RI matching. Light scattering is decreased by matching the RI of the medium to that of the tissue.

Lipid Removal. Lipids are a major source of autofluorescence. These can be removed through either dehydrating the tissue using an organic solvent or hyperhydrating the sample using a detergent followed by a denaturing agent.

Gel-Tissue Hybridization. One of the most popular forms of this method is the CLARITY method. The method removes the lipids, but keeps other structural components of your tissue by inserting a hydrogel matrix to maintain the structure after the lipids are removed.⁷ The only major downside to this method is the increase in time needed for sample processing. To process a sample by this method, you can expect for it to take several days. The initial cost barrier is high, however, it can be a small price to pay to get higher quality images and allows for imaging of RNA transcripts in an entire intact animal or organ, such as a brain

Other Sample Preparation Adjustments

With a few simple adjustments to your processing and tissue preparation, you can easily reduce unwanted fluorescence of your sample. For example, you can add TEA buffer with acetic anhydride when processing your sample. An addition of 63 mL of acetic anhydride to a 1X TEA buffer solution can help maintain the integrity of your sample structure. In this case, the acetic anhydride is believed to neutralize the charge in your tissue sample by acetylating free amines in the sample (such as lysine), but the exact science of how this works is not known. This little change also helps to maintain the integrity of the sample, although it is not as effective as gel-tissue hybridization.

Dye Choice and Autofluorescence

Certain channels are known to have higher autofluorescence (for example, the green channel that is used for fluorescein or Alexa 488 dyes). The colors in these channels have shorter wavelengths and, therefore, scatter when passing through tissue. This scattering results in increased autofluorescence. You can avoid this by using longer wavelength dyes, such as the far red Quasar® 670 or Cy5 dyes. The green channel should always be avoided in tissue, and most especially in high background tissues like the brain.

Imaging Your Sample

Once you have your sample prepared, you need a way to visualize your target. While you have many options depending on your target type, including immunohistochemistry, fluorescent methods are becoming more popular. One such fluorescent method is RNA FISH. It can be customized to use several different fluorophores in order to identify and co-localize multiple RNA targets of interest. There are several RNA FISH methods that you can follow. One such method is the Stellaris RNA FISH, which prevents background noise and weak signaling from confounding your sample by binding to a series of oligonucleotides. The binding of the oligonucleotide is more specific as it has to bind to multiple sections of your target, rather than just one segment.  This technology also combines with antibodies to allow for simultaneous visualization of both proteins and RNA in situ.

Keep in mind your particular application when you do prepare any sample for imaging. Not all methods are created equal. Depending on your target or your end goal, you may need more gentle processing methods or a different detection technique. How you prepare your sample and determine the method for your staining can be just as complicated as doing the analysis of the images themselves!

References

1. Microscopy U. Autofluorescence in Microscopy Literature.
2. Schnell SA, Staines WA, Wessendorf MW. (1999) Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem. 47(6):719–30.
3. McNeill MS, Kapheim KM, Brockmann A, McGill TA, Robinson GE. (2016) Brain regions and molecular pathways responding to food reward type and value in honey bees. Genes Brain Behav. 15(3):305–17.
4. The Huett Lab. Immunocytochemistry protocol – version 2.2, 23 Sept 2010.
5. Mundegar RR, Franke E, Schäfer R, Zweyer M, Wernig A. (2008) Reduction of high background staining by heating unfixed mouse skeletal muscle tissue sections allows for detection of thermostable antigens with murine monoclonal antibodies. J Histochem Cytochem. 56(11):969–75.
6. Sylwestrak EL, Rajasethupathy P, Wright MA, Jaffe A, Deisseroth K. (2016) Multiplexed Intact-Tissue Transcriptional Analysis at Cellular Resolution. Cell. 164(4):792–804.
8. Seo J, Choe M, Kim SY. (2016) Clearing and Labeling Techniques for Large-Scale Biological Tissues. Mol Cells. 39(6):439–46.