It seems that using the FocusClear(r) reagent is paramount to obtain the best transparency. However, this mounting medium is expensive, and seems to be back-ordered for a while due to large demand. Also, the formulation is proprietary, which makes further optimization harder.

While trying different mounting reagents for the ETC treated samples, I realized that glycerol does not give great results, even after very long incubations. ScaleA2 (4M Urea) works great on ETC treated brains, full transparency is achieved in just hours, but I'm afraid that it could disrupt the molecular integrity of the sample.

Could we try to design a home-made mounting reagent that would give similar results?

It seems that the best way to achieve index matching is to use a solution with ions presents at a high molarity. FocusClear(r) seems to use diatrizoic acid and meglumine to achieve that. Does anyone had any luck in making a good substitute?


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    I wanted also to stress another important point about imaging and the need to find eventually a alternative mounting mediums for Focusclear.

    Down the road, confocal microscopy might not be convenient enough to image large samples in routine applications : they are slow, and small field of view means a lot of tiles to cover the whole tissue. Light sheet microscopy (Single plane illumination), if compatible with Clarity, could be a great way to image whole brains with short acquisition times.

    However, the current SPIM microscope from LaVision biotech use an imaging chamber that you need to fill completely with the mounting medium. I think the volume is about 30mL (not sure exactly, but it is in this ballpark). At about 200$ for 5mL, it would be too expensive to use this kind of microscope for Clarity routinely. I don't know if microscopes from other manufacturers operate differently.
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    I am very interested in this topic. Although I have not quite obtained a sample worthy of mounting (end of the week perhaps?) I had planned to use the glycerol solution. Now I am a smidge worried when you say that the glycerol does not give great results.

    Do you have any data as to the degree which the urea treatment degrades signal quality?
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    I tried to use ScaleA2 to make a P30 thalamus transparent after an ETC clearing. The mouse was injected with a cholera toxin beta Alexa 488 conjugated tracer in the eye, which reliably labels the LGN in the thalamus. After the whole treatment, I couldn't see the fluorescence. However, I'm not sure if the fluo was already gone after the ETC, or after ScaleA2, so I don't know for sure that ScaleA2 is too blame. I am trying an immunostaining on ETC treated cortices this week, and plan to fix the antibodies with PFA. I'll see if the staining is still there with different mounting mediums.

    I my experience with 80% glycerol on a whole brain after ETC, you only have a good clearing up to 1mm in the tissue, and the deep structures are still cloudy. Also the whole brain is very swollen in glycerol.
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    karlkoehkarlkoeh Posts: 8
    I have also had issues with glycerol. The ventricles of the brain as well as the central canal of the spinal cord become cloudy after prolonged incubation (>3 hr) in glycerol. Apparently everywhere there is no tissue.

    I've been thinking about a FocusClear alternative. The FocusClear patent provides an ingredients list including: DMSO, diatrizoic acid, EDTA, glucamine, NADPH, dodium diatrizoate and Tween-20. Perhaps we could come up with a logical home-brew from this list. I realize this is easier said than done, but the patent does contain 2 claims suggesting that DMSO, diatrizoic acid and sodium diatrizoate are the key ingredients. Dissolving varying concentrations of diatrizoic acid and diatrizoate in DMSO should be a decent starting point.
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    I have been trying mixing those elements already. I'm not sure about the exact proportion, but I found out that the DMSO should be pure. If you add H2O (for instance 80% DMSO, 20%H2O), the transparency decreases. I started with an equimolar amount of n-methyl-glucosamine and diatrizoic acid at about 20% w/v, with 0.1% tween20. It is not all bad, but could be better.

    I'm wondering if Ann-Shyn Chiang would be willing to disclose the formula to the research community?
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    There is a new method published today in Nature Neuroscience about optical clearing of large samples
    "SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction"

    A single solution is used to clear sample in 3 days and it is a simple a mixture of Fructose and Thioglycerol. It may be a good mounting medium for CLARITY processed samples too.

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    Thanks for pointing that out. I'll try that on my samples.

    I made some progress on the focusclear, and I just wanted to do one more try with a new chemical before sharing my experience on this.
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    In this paper, the authors advise against using SeeDB as a mounting medium because evaporation of the water in solution lead to distortion/uneven refractive index distribution (imaging section of Methods). If we used a completely sealed chamber, as in the CLARITY imagine protocol, do you think this would still be an issue? I was thinking of index-matching ETC cleared tissue with the SeeDB for a couple days, then mounting in 80-90% thiodiethanol as in the SeeDB paper. I have very little background in refraction/imaging so any thoughts regarding this plan would be appreciated.
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    The authors of the SeeDB paper also stated that they had to get a manufacturer to design them a customized objective in order to image using their 80-90% thiodiethanol, because it has a really high RI. With a properly sealed chamber, evaporation shouldn't be an issue - although perhaps settling and separation of the solution might lead to uneven refractive indexes?

    I am tempted to give this SeeDB a shot - if nothing else it's fairly cheap, although the thioglycerol might add a bit of $$.
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    Question about matching the refractive index of the tissue (with FocusClear, Scale A2, etc.). In the literature, it says,

    "... after immersion in refractive-index-specified solutions matching the CLARITY hybrid (for example, 85% glycerol or FocusClear, both with a refractive index of ~1.45), the intact brain (including heavily myelinated white matter, thalamus and brainstem) becomes uniformly transparent."

    Additionally, this is in the literature:

    "Preparing the sample for imaging: (1) Incubate the sample in FocusClear or 80% glycerol solution for 2 days before imaging; these have refractive indices matching that of clarified tissue."

    When are you all placing your samples in the final clearing solution (FocusClear, Scale A2, etc.) and for how long? @nicolasrenier , you mentioned that "full transparency is achieved in just hours" when using Scale A2, but the literature says to do this for 2 days. In addition, is this step carried out after ETC and immunohistochemistry, or directly after ETC?

    Thanks everyone!
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    @Andy_Donaldson Index matching with whatever solution you want to use should be the very last step of the protocol, just before imaging. How long will depend on the solution you use, and your sample. Luckily, it is easy to tell when the incubation is done, as your sample becomes transparent.

    In my experience, ScaleA2 has a very fast diffusion in the sample (hours), Focusclear is slower (1 to 2 days), and is glycerol even slower (weeks).
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    FabioFabio Posts: 62
    @nicolasrenier I would love to see a picture of one of your brains in glycerol and Focusclear.
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    @nicolasrenier Any luck with that knock-off focusclear? I ordered up some diatrizoic acid and am about to start trying my own solutions out, but I was wondering if you have made any progress.
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    @Fabio & @joelrosiene :

    Here you can see a summary of some tests I did :


    My current focusclear recipe is not all bad, but it doesn't get rid of the yellow color. I should point that I haven't bought the real focusclear, so I don't know how better it would be. Note that the "focusclear" brain in this picture looks better than on the picture : the cloudy part in the center is in fact the meninges stuck at the ventral side.

    The current recipe is an equimolar mix of diatrizoic acid and meglumine diatrizoate in DMSO, at the solubility limit at RT (cf the hypaque(c) solution) with 0.1% Tween20. I read the patent again yesterday, and I'm not sure about adding or not H2O. The patent mentions "an aqueous solution" of apparently neutral pH. So far I don't have any water in my solution, so the pH doesn't make much sense (protons might not exchange easily in DMSO I think). Maybe I'll try to start over with some water in the mix.
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    FabioFabio Posts: 62
    edited July 2013
    Can you tell me your average temperature when running your ETC? do you stay below or at 37°C?
    Have you tried to add some reducing agent to the clearing solution?

    When you meet the Deisseroth team can you ask also about the source of autofluorescence. and how to avoid/minimize it?
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    Runs at 30V / 40°c. I plan to try 20V / 37°c next time...

    I haven't tried the reducing agents in the clearing solution. I tried BME and 1-thioglycerol after the clearing, and it didn't worked. I am trying SeeDB as a mounting solution on clarity samples, I'll let you know how it goes.
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    Tomorrow I will start playing with the DMSO/diatrizoic acid, as well as the mounting solution suggested by the SeeDB paper, which was dithioethanol. Will report back.
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    I tried SeeDB on "Clarity" samples last week, and it didn't worked (after only 3d of incubation). I'm leaving the sample for a while to see if it improves. I would be interested to see if anyone else tried seeDB at the end of the clarity protocol...
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    Just reporting here that I have had some success using a saturated solution of diatrizoic acid in DMSO, with NaOH at a 50% Molar ratio to the diatrizoic acid, and a 0.1% Tween20. The only issue I have had with this solution so far is the production of bubbles within the tissue. Also, mounting samples is Incredibly difficult.
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    Back in March my PI came up with a recipe for what we're calling "FauxcusClear." We tested it against the real stuff using 2-photon laser microscopy and saw no difference. Ours is a qualified success: we've had other problems that are most likely due to previous steps (autofluorescence, loss of true fluorescence) so I can't say whether or not our version works for sure. But if you want to try it, below is our recipe. Please help me make it better if you see anything that we're doing wrong:

    We make a 70 mM stock of NADPH in 0.01N NaOH and add 1.425 ul in 100 ml final volume DMSO. We also add 0.27 mM EDTA (from a 500 mM pH8 stock I had for other uses), 1% Tween 20, and 12% w/v each of diatrizoic acid and sodium diatrizoate. Then we sonicate the solution until everything is dissolved. I've been storing the solution in 20 ml aliquots in scintillation vials (one brain/vial) in the dark at room temperature. We've already shared this recipe with @nicolasrenier and @nparker327. Maybe you guys can chime in?

    To answer Laura, I just followed your recipe, and had very good results (except autofluorescence). The only change I made was to replace the sodium diatrizoate with meglumine diatrizoate, and had slightly better results. Here is my recipe :

    - 19mL DMSO
    - 1.65g diatrizoic acid
    - 2.17g Meglumine diatrizoate
    - 11µL EDTA 0.5M
    - 200µL Tween20

    I will try to add NaOH as you do, to see if it gets better. I'm not sure what happens with the protonation of OH- in DMSO... Also, adding NaOH would add H20 to the mix, which I found can decrease transparency. But let me know what you found out.
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    LauraLynchLauraLynch Posts: 31
    I used NaOH in order to dissolve the NADPH. When I tried to add it straight to the DMSO, it wouldn't dissolve. When I tried to adjust the pH back to neutral, I got a solution that looked like Mountain Dew and an ugly yellow precipitate. Amusing, but useless. Making a stock NADPH solution in NaOH solved the problem. NADPH is autofluorescent, though, so I tried a batch of FauxcusClear without it. Our sample autofluoresced just as much, so whatever our autofluorescence problem is, NADPH didn't add to it.
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    JalalJalal Posts: 11
    You guys are amazing. Can't wait to try it! Will report back
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    achungachung Posts: 7
    This could be an ignorant question, but fluoromount-g has a pretty close refraction index of 1.4 (focusclear = 1.43). Anyone tried it?
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    Fluoromount G doesn't work. RI is important, but it seems that very viscous medium (glycerol, seedb, fluoromount) don't work well with large samples
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    rchurtrchurt Posts: 6
    Possibly a dumb question: Why can't we just put the clarified brain in a dish full of more hydrogel monomer solution, polymerize it, and just image that block of gel? That should have the same refractive index as the brain, right?
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    The hydrogel itself will probably have a similar refractive index as water. The refractive index of the tissue is determined by all the biomolecules attached inside the embedded tissue.
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    DavidDavid Posts: 2
    If the mounting medium is water soluble, it possible to counteract it's lack of penetration in the brain tissue namely it's viscosity by simply :
    (1) heating up the medium, max temperature around 45 Celcius (glycerol-PBS, fluoromount-G, etc)
    (2) and/or making successive dilution of the mounting medium and water (for exemple 20, 40, 80, 90 ... 100%)
    I did try it with glycerol and it seem to work fine. In previous test I also did encounter the opacifying phenomenon. But if the glycerol-PBS is warm up prior to embeding and combine with succesive dilution it work just fine. Note: instead of using 85% glycerol, I use 90%.
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    jjsun90jjsun90 Posts: 1

    What was the catalogue number for the NADPH used in your FauxcusClear recipe? We're going to try clearing brains with FocusClear as well as a number of other recipes on this forum to compare how each turns out. Thanks!
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    arvidarvid Posts: 9
    @David, cool concept, For how long time did you keep the tissue in the respective concentrations of glycerol? For tissues of what size? Can you post pictures with the results?
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