For anyone who has gotten to imaging yet, have you noticed a lot of autofluorescence in the green and red wavelength ranges? I am working with heart tissue, which I know is notorious for autofluorescence, but sounds like this may be true for other tissues as well. To try to get around this, I am testing blue and far-red dyes, but I would like to know other thoughts or suggestions.

Also, according to the SeeDB article posted about by Bertrand_Vernay on the FocusClear discussion (, this autofluorescence issue could be due to the Maillard reaction, which means it could potentially be prevented... however I don't think the chemicals used to do this would be practical to add to the ETC buffer during the clearing process (2-ME or 1-thiogylcerol). The Maillard reaction is reversible though.


  • Thanks for starting a topic on this issue. I'm also experiencing very high auto-fluorescence that prevents me from imaging anything, either endogenous fluorescence or fluorescent antibodies.

    I'm making a carry over from the ResearchGate thread, but I wanted to point out that the browning of the tissue during electrophoresis could be due to the Maillard reaction, promoted by the high pH and temperature in the chamber.

    I'm wondering if adding a reducing agent in the clearing solution could help with this? In the recent seeDB paper, they add 0.5% of 1-thioglycerol. This looks doable, even in the high volume of clearing solution we use here.
  • aysegulaysegul Posts: 33
    Thanks all of you for valuable discussions. I cant reach the article at that time. Could you please send me the that seeDB paper?

    It seems that the paper suggests that the Maillard reaction that produces the browning of the tissue is due to the fact that they use a fructose solution, and fructose is a major reagent in the Maillard reaction.

    It makes sense to me that the Maillard reaction may be contributing to the browning and autoflourescence in our ETC tissue, but I would imagine that we would see it to a lesser degree than those soaking their brains in fructose.

    I should say that my preliminary images have had very high autoflourescence - which completely prevented me from seeing my native GFP.
  • AReadyAReady Posts: 7
    Glad I'm not the only one seeing autofluorescence in my tissue. While I can definitely discern eGFP after ETC clearing and some index matching, the extra signal is preventing me from getting much data without quite a bit of segmentation.
    I'm honestly wondering if I have a step wrong somewhere at this point, especially since the original paper shows fairly clean eYFP signal - considering where the autofluorescence is showing up (for me at least) it seems like their results would also have it.
  • That's a good point, joelrosiene. Does the neural tissue get brown through the process, or does it just primarily yellow? I have been clearing heart tissue, and it is definitely getting brown.

    My other thought, for the heart at least, was that this could be lipofuscin (collections of oxidized protein). I think this would be a harder problem to deal with than the Maillard.
  • joelrosienejoelrosiene Posts: 63
    edited June 2013

    Here are two pictures which show one of my brains before and after clearing - I didn't remove enough of the surrounding gel on this run, which caused the cleared tissue to be a bit sloppy, but we definitely see some good clearing. We also see yellowing, which is frustrating, but not too bad - I have not attempted index matching.

    When I got this amount of clearing, I decided to run it for one more day to see what would happen.

    This was the result.
    Don't run your brains too long folks - they melt.

    (edit) We also see some of the black tarnish that is building up on the cathode making its way to the sample and causing those black spots. My new chamber has an extra mesh in place to prevent this. I should have some pictures of the new results in a few days.
  • AReadyAReady Posts: 7
    How long did you (initially) run that for? My last brain was clearing for 80 hours at 30V and it still doesn't look that clear.
  • FabioFabio Posts: 61
    I can also add that I see already autofluorescence after hydrogel embedding.
    I have a strong expression of eGFP in my brain. I can see it already in the brain
    under a microscope without any clearing or hydrogel, but after embedding I started
    to see some autofluorescence.
  • @AReady I cleared for 50 hours at 94V (400mAmps) to get that amount of clearing, but there are a few things to note. My chamber is nonstandard- 7cm between electrodes, and the high voltage run produced so much heat that it started deforming my PVC chamber cap. I DIY'd up an aluminum foil heatsink that seemed to help a bit.

    Obviously 74 hours is far too long - melted brains are simply useless.

    Fabio brings up an interesting point. Would the crosslinking step change the amount of autoflourescence we see?
  • I cleared part of a brain for 5 days at 20V (~800mAmps) at 37C and wound up with autofluorescence of my GFP signal and a yellowing of the tissue. I can use a dissecting scope to see my native signal before and I will look at my next sample after the embedding process (didn't look last time).
  • Checked today, and I have minimal autofluorescence in the blue channel and pretty much nothing in the far-red range. My labmates and I are going to be testing a 633 alexa dye, to see if we can't get rid of the autofluorescence, we can at least work around it.
  • @KatherineHolzem would you mind sharing the exact parameters that you used for clearing your latest tissue please? It's really exciting that you got minimal autofluorescence! Was that with the heart tissue you were working with? I'm very interested because I'll be working with clearing the heart pretty soon now. Thanks so much.
  • FabioFabio Posts: 61
    edited June 2013
    @joelrosiene @nicolasrenier
    Have you tried to check for autofluorescence after hydrogel embedding?
    Im really interested in this.
  • edited June 2013
    Yesterday we compared a mouse heart that had been cleared and placed in focusclear to one that had been perfused with hydrogel monomer but not yet crosslinked - both seemed to exhibit significant auto-fluorescence in the green and red channels, but minimal auto-fluorescence in the blue, and basically none in the far-red (see @KatherineHolzem's comment above). I remember reading somewhere on this forum that someone had seen less auto-fluorescence before the crosslinking, but based on what we saw yesterday, this does not seem to be the case (at least not for mouse heart tissue).
  • FabioFabio Posts: 61
    thanks for the comment
  • @Andy_Donaldson... just want to be clear that we do have a lot of autofluorescence in the red and green channels (mentioned in the first post). Blue and far-red seem to be okay. We have tried both mouse and human heart tissue, and both seem to have similar autofluorescent properties.

    We have cleared our heart tissue at 30-35 V and keeping the water bath at 32 degrees. Our tissues have taken about 2 weeks for clearing.
  • @KatherineHolzem great, thanks for the information!
  • KeithSiewKeithSiew Posts: 24
    I made a comment on this thread about autofluorescence solutions:

    But to be more succinct there is an excellent guide here:
  • aysegulaysegul Posts: 33
    edited July 2013
    Thanks @KeithSiew your useful guide. It will helps us on.
  • check out my new paper on the reduction of autofluorescence using LED illumination. This should help those who are having issues with lipofuscins and other autofluorescent substances.
  • TomoTomo Posts: 7
    Did anyone end up with a good imaging of endogenous fluorescent proteins such as EGFP, tdTomato or whatever with low autofluorescence? This issue brought up a long time ago in the forum, but it seems no one really got over it. Or is it just biased because only people suffering from this issue keep posting?

    Thanks @haisond, have you photo-irradiated the tissue with any endogenous fluorescent proteins?
  • TomoTomo Posts: 7
    In my hand, I've cleared many organs such as brain, lung, kidney, adipose tissue, intestine and skin from either mTomato or R26-Confetti (YFP/GFP/CFP/RFP after cre recombination) mouse, and what we've found so far under our confocal was just autofluorescence. PFA-fixed thick cryo sections from the same mouse had a strong signal though.

    Here is a quick summary of the posts (thanks everyone!), please correct or update if I'm wrong.

    @joelrosiene has reported the same issue with Thy-1 GFP mouse.
    @Fabio has reported that strong GFP expression was uncovered after clearing.
    @kadipietro found the same autofluorescence issue.
    @KatherineHolzem found minimal autofluorescence in far-red, but still a lot on the other channel where fluorescent protein usually emits.
    @KeithSiew brought up techniques to reduce noise but I'm not sure it's directly related to the fluorescent protein issue.
  • just to add to this discussion i have cleared quite a few thy-1 gfp brains and could never get good pics due to autofluorescence. ive decided to no longer clear brains with an inherent fluorescent signal and instead attempt to stain with antibodies and see if i could find a secondary channel to give me better results
  • TomoTomo Posts: 7
    Thanks @rspence

    Despite the beautiful images in the original paper, no one has ever succeeded to show good pics?? Any ideas?
  • FabioFabio Posts: 61
    I have to say that it works for my brain
  • TomoTomo Posts: 7
    @Fabio, it's really GREAT to know it works for you.
    Earlier you mentioned you also see some autofluorescence after embedding, but do you mean you still have an enough signal over the noise? Could you tell us what kind of mice with EGFP do you test if you don't mind?
  • FabioFabio Posts: 61
    edited September 2013
    You will see autofluorescence under a stereomicroscope, but once I go to the confocal I see my signal. My mice have the GFP coupled to histone H2B. So you have a strong nuclear signal.
  • TomoTomo Posts: 7
    Thank you for the helpful info! We also have H2B-GFP reporter mice in my hand to try. Sounds easier to troubleshoot because of its nuclear localization. Other than H2B-GFP, is there anyone else who could see good signal with less autofluorescent?

    I don't see YFP/GFP/CFP/RFP or TdTomato even under our confocal. One of their expression should be nuclear restricted, two of them should be membrane, others cytoplasmic. I believe that their signal is much weaker than H2B-GFP though. (I could see strong signal of all in thick sections without ETC).
  • rajuraju Posts: 12
    Hi folks, a few general observations for minimizing Autofluorescence concerns:

    1. Make sure you check in the Confocal scan mode to assess the level of auto-fluorescence in your samples. We would expect to see very blurred signal under the eyepiece of a Confocal or a wide-field microscope. In fact, that's a good sign implying that your sample is very god cleared, and the light from out of focus plane is contaminating your focal plane.

    2. Optimize the staining protocol (eg, buffers used: PBS or Boric Acid etc, and wash very well). If using transgenic, use as high expressing transgenic line as available - good for signal-to-noise as well as for photo-bleaching. We have tested Thy1-eYFP, Thy1-GFP, Cre-driven reporter, as well as viral injected brains. All look good.

    3. Make sure your sample looks normal cleared (not much yellowish, not burned or damaged). If it does not, go down in voltage applied.

    4. DO NOT leave your sample in focus clear for longer than needed. The white precipitation from focus clear produce heavy auto-fluorescence and also occlude the imaging depth.

    Hope these help.
  • TomoTomo Posts: 7
    Thank you, @raju, for these suggestions! It's again GREAT to know the clearing on these mice work for you.
    I don't see any strong or blurred epi-fluorescence in my sample, and the loss of signal seems to happen during clearing.
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