I want to make spheroids more clear for confocal imaging. What are the possible ways to clear them? Maybe somebody tried several different commercial solutions or home-made ones and could suggest me some options? Thanks!
Some think of a dilute aqueous solution of NaOH & others think of xylene. However, the US patent (the attached in this link)offers an interesting solution:
http://www.google.ps/patents/US6472216
Having not worked in cell culture for a while, am I correct to understand a spheroid is large cluster of cells? How big are your spheroids? If they are less than 200 um you could incubate them overnight at room temp in 33% TDE (2,2′-Thiodiethanol in PBS) and then once more overnight in 66% TDE before imaging in the solution. It will match the refractive index of tissue and the solution to around 1.45.
The nontoxic embedding medium, 2,2′-thiodiethanol (TDE), which, by being miscible with water at any ratio, allows fine adjustment of the average refractive index of the sample ranging from that of water (1.33) to that of immersion oil (1.52). I mention TDE in the review about the method CLARITY which is used to clear thick (>1 mm) tissue samples.
Article Advances and perspectives in tissue clearing using CLARITY
I would suggest you to try with uDISCO protocol that gives very high transparency.
Here the protocol: https://www.nature.com/nmeth/journal/v13/n10/full/nmeth.3964.html
as far as I remember we had a workshop participant who tried with spheroids.
Dear Vilma Petrikaite,
I think it is hard to say what the best really is. There are very different approaches available. Some are very simple others are more facilitated. However, all of them have different drawbacks and you need to consider what the best solution for your lab/case is.
How big are your spheroids?
What microscope would you like use?
What resolution do you need?
Do you want to add some antibody staining or perform in situ hybridization?
How much effort want/can you to invest?
These are some question you should answer before you can pick the right approach.
One of the best in terms of possibilities and quality is: CLARITY.
uDisco or scale are much faster. CUBIC is somehow in the middle.
All of them should allow you to image at least to a depth of 400 µm.
I used these methods so far for tissue samples however I am sure all of them can be adopted to spheroids.
Best regards
Usually I image 200-400 um spheroids, so all your mentioned solutions should work. Thank you very much!
Vilma Petrikaite You may want to give focusclear a shot: https://bit.ly/2lVOqdL we've seen good results in biologicals and biomaterials.
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