UltraMicroscope II Clearing - LaVision BioTec GmbH

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UltraMicroscope II - Clearing

Clearing with organic solvents and aqueous buffers
Imaging large samples even into the depth of the tissue needs certain procedures to reduce the opacity. The tissue has to be virtually transparent. Some samples like Zebra Fish are mostly transparent by nature but the majority of samples are opaque. This counteracts all attempts to image the sample in total.
Nowadays, two main principles of creating translucent samples have been established. In the case of organic solvent clearing, the principle of operation is matching the different refractive indices. On the other hand, the sample may be cleared by using aqueous buffers which have a certain depolymerizing effect on structures like lipid chains.

Organic Solvent Clearing Protocols
When performing organic solvent clearing, the water has to be removed in the first step by incubating the sample in increasing concentrations of ethanol or another dehydrating solution. After this step, the refractive index of water (1.33) is virtually no longer present. Within a second step, the remaining refractive indices are matched by ether incubation as in case of the 3DISCO (7, 8) or iDISCO (10,11) clearing. The organic solvent clearing leads to very transparent samples and is perfectly suited for dense tissue like tumors, adult tissue or highly myelinated brain. The majority of immunohistochemical staining is well conserved. To preserve the fluorescence of proteins like GFP, the pH has to be adjusted as in case of the ECi protocol (12). The UltraMicroscope II can be used for all current organic solvent clearing procedures including BABB, 3DISCO and iDISCO. Visikol Inc. has developed a patented tissue clearing technique called Visikol HISTO that is reversible and non-destructive to tissue morphology such that tissues can be imaged in three-dimensions followed by traditional (e.g. H&E, IHC) histological processing.

Water-Based Clearing Protocols
The most common operating principle of water-based clearing protocols is by depolymerization. By dividing large structures like lipid chains into small micelles of different sizes, the opacity is remarkably reduced. As depolymerizing reagent aqueous buffers can contain urea as it is used for CUBIC (3, 4, 5) clearing. A SDS buffer and an advanced electrophoresis protocol are used for CLARITY (1) clearing. The clearing protocols differ in complexity and in the degree of translucency which can be achieved. By depolymerization, the entire structure of a sample can be debilitated while the fluorescence of proteins like GFP is well preserved. Both organic solvent-based clearing and water-based clearing methods are powerful tools for successful sample preparation. The variety of clearing protocols shows that clearing procedures have to be optimized for the sample of interest. The UltraMicroscope II is capable to handle all current clearing solutions.

top: mouse embryo E12 tag1 ChAT ; courtesy of Chloé Dominici and Alain Chédotal, Institut de la Vision, Paris, France

What kind of clearing is best for my project?
There are several protocols for tissue clearing available today and it is not really easy to identify the best protocol for your sample. There are two large groups of clearing procedures: the aqueous buffer based protocols and the organic solvent clearing. Every procedure has its advantages and disadvantages. If you start with aqueous buffer based clearing please consider that samples might swell and get very soft because the stabilizing structures will be depolymerized. Furthermore, it might take quite a while to get the samples cleared. Another disadvantage might be a stronger background and some turbid appearance of the tissue. But this is not the case for all aqueous buffers based clearing protocols.
From what users report we know that CLARITY (1) and CUBIC (2, 3, 4) deliver great results. The CUBIC protocol is not very complex and can be followed in detail from the different publications. A brain hemisphere is ready for imaging within about 11-13 days. Chemicals which are needed for this kind of clearing are not that expensive and the CUBIC buffer R2 is very useful for imaging samples cleared with RapiClear®. Please consider that there are different RapiClear® (SunJin Lab Co.) solutions available with different refractive indices. The CUBIC samples do not swell that much and they get really clear.
The CLARITY protocol also generates perfect samples but it is a bit more complex. It may take a while to establish this protocol in your lab but today there are also systems available like SmartClear® (LifeCanvas Technologies) doing the clearing for you. For the imaging solution of CLARITY samples one can choose between different liquids like FocusClear® (CelExplorer Labs Co.), 80% glycerol, 63% thiodiethanol or RIMS (refractive index matching solution) (5). You may also try the CUBIC R2 buffer. There might be a slight difference regarding the refractive index between the sample and the imaging solution. In that case just let the solution settle for 20 minutes and then start imaging. Further assistance on using CLARITY you will find on this page: http://clarityresourcecenter.org/
There are several further aqueous buffers based clearing protocols but some of them are only suitable for embryonic tissue. Other procedures require a buffer which is very viscous and is not really easy to handle due to the formation of small bubbles inside. Some protocols are very time consuming with incubation times of several weeks. There are also protocols generating tissue which is like gel and expanded to more than twice the original size.
Very often it is difficult to clear adult and dense tissue only with aqueous buffers. In that case you may try the other group of clearing protocols which is based on organic solvents. The first who described this technique was Werner Spalteholz (6). After using clove oil he searched for solvents to clear tissue. Besides the methyl salicylate protocol a mixture of benzyl alcohol and benzyl benzoate showed convincing results. This protocol is still in use today. Users should always be aware of the fact that this clearing solution is very harmful and should only be handled under a fume hood. A less harmful clearing procedure is based on tetrahydrofuran and benzyl ether (7). This protocol is today mostly known as the 3DISCO protocol (8). The advantage of organic solvent clearing is that samples are getting really clear like glass. The clearing itself is fast. Depending on the size of the sample it can be done within a few hours. It has been for a long time the disadvantage of these clearing protocols that GFP and other proteins were not preserved. Today this issue is not relevant anymore. On the one hand the FluoClearBABB (9) preserves GFP very well while the sample is cleared. On the other hand the GFP can be immunohistochemically stained following the iDISCO protocol (10, 11). This outstanding protocol describes how to combine the gold standard immunostaining with sample clearing. The advantage is that one can target several proteins without generating the GFP mouse model for each target. This protocol also enables the usage of chromophores in the far red. GFP is excited with 488 nm but this wavelength is not really suitable for deep imaging because light at this wavelength is absorbed and scattered by the tissue. Immunolabeling with a far red dye is much more suitable for deep imaging. With that kind of imaging you may get much deeper into your sample than with a 488 nm excitation. The protocol has to be optimized for your tissue; and please be aware that this may take a few attempts until you have the perfect sample but there is also great assistance. The iDISCO developers put their knowledge to the internet: http://idisco.info/


1) Can I use DAPI?
DAPI is excited with 405 nm. This is a pretty short wavelength which is scattered and absorbed by the tissue. It is absolutely not suitable for deep imaging. One should try DRAQ5™ or TO-PRO®.

2) My sample contains GFP which clearing should I try?
You may try CUBIC, CLARITY, FluoClearBABB or iDISCO. iDISCO maybe of advantage due to the fact that you can label it with a far red dye for better penetration of exciting wavelength.

3) The tissue I am working with is very dense. What clearing is capable to get it transparent?
Users working with dense samples often use BABB or 3DISCO.
4) The sample I would like to image is colorized (e.g.: liver)? How can I cope with colorization?
You may try the CUBIC-perfusion protocol. This protocol was designed to decolorize sample tissue by perfusion.
5) I would like to label my sample with fluorophores which are suitable?
Every fluorophore with an excitation maximum between 500 nm and 785 nm is suitable. The longer the excitation wavelength the better the penetration of the light. DiI or DiAsp are not compatible with all clearing protocols. The membranes as binding site for these dyes is often altered or affected by the clearing. Users have for example good results with using several Alexa Fluor® dyes (Thermo Fisher Scientific Inc.), ATTO-fluorescent dyes (ATTO-TEC GmbH), VivoTag® (PerkinElmer Inc.), Cy7, or IRDye ®(LI-COR, Inc.).
6) My sample is very soft. How can I mount it for the image acquisition?
You may try some glue. picodent® (dentist glue, Dental-Produktions- und Vertriebs-GmbH) is even stable in organic solvents.
7) The sample I would like to image is very small. How can I mount it?
If you work with organic solvents you may prepare an empty but cleared cube with 1% low melting agarose or Phytagel™ (Sigma-Aldrich Co. LLC.). After clearing the cube you make a small cut on top of it where you insert your sample. One can also directly embed the sample into agarose and do the clearing of the sample within the agarose. In that case please extend the dehydration (100% over night) so that the agarose is completely dry. If there is only a small amount of water remaining in the cube it will start shrinking and it will get milky as soon as it is transferred to the clearing solution. If you work with aqueous buffers you may insert it into a FEP tube. For some protocols embedding in collagen works also.
8) I cleared my sample but it is still not transparent and shows a strong background.
If you have applied an aqueous buffer based protocol you may give a try to an organic solvent protocol. Methanol treatment might reduce the background. Please consider to dehydrate the sample completely. If the used dehydration solution (tetrahydrofuran or ethanol) is stored in a bottle which is opened again and again there will be already a remarkable amount of water in that solution due to the hydroscopic characteristic of the solvent.
9) I cleared my sample using the CLARITY protocol. What kind of solution should I use for imaging?
Some CLARITY imaging solutions a very expensive. To reduce costs you may try 80% glycerol, RIMS, or 63% thiodiethanol.
10)  The iDISCO protocol resulted in a sample with a strong labelling only on the surface. What should I do?
Increase the dilution of the antibody and find further information on: http://idisco.info/

11) Is there an easy to use kit for clearing?
You may try Visikol Histo which is a non-destructive tissue interrogation technique that can be conducted on tissues prior to traditional histological evaluation.

1) DOI: 10.1523/ENEURO.0022-15.2015
Optimization of CLARITY for Clearing Whole-Brain and Other Intact Organs
Jonathan R. Epp, Yosuke Niibori, Hwa-Lin (Liz) Hsiang, Valentina Mercaldo, Karl Deisseroth, Sheena A. Josselyn, Paul W. Frankland
2) Cell. 2014 Nov 6;159(4):911-24
Whole-body imaging with single-cell resolution by tissue decolorization.
Tainaka K, Kubota SI, Suyama TQ, Susaki EA, Perrin D, Ukai-Tadenuma M, Ukai H, Ueda HR.
3) Cell. 2014 Apr 24
Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis
Susaki EA, Tainaka K, Perrin, Kishino F, Tawara T, Watanabe TM, Yokoyama C, Onoe H, Eguchi M, Yamaguchi S, Abe T, Kiyonari H, Shimizu Y, Miyawaki A, Yokota H, Ueda HR.
4) Nat Protoc. 2015 Nov;10(11):1709-27. doi: 10.1038/nprot.2015.085. Epub 2015 Oct 8.
Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.
Susaki EA, Tainaka K, Perrin D, Yukinaga H, Kuno A, Ueda HR.
5) Cell. 2014 158 (4). pp. 945-958
Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing
Yang B, Treweek JB, Kulkarni RP, Deverman BE, Chen CK, Lubeck E, Shah S, Cai L, Gradinaru V.
6) Über das Durchsichtigmachen von menschlichen und tierischen Präparaten
Spalteholz, Werner
Leipzig, 1911

7) PLoS One. 2012;7(3):e33916. doi: 10.1371/journal.pone.0033916. Epub 2012 Mar 30.
Chemical clearing and dehydration of GFP expressing mouse brains.
Becker K, Jährling N, Saghafi S, Weiler R, Dodt HU.
8) Nature protocols. 2012 Oct
Three-dimensional imaging of solvent-cleared organs using 3DISCO
Ali Ertürk, Klaus Becker, Nina Jährling, Christoph P Mauch, Caroline D Hojer, Jackson G Egen, Farida Hellal, Frank Bradke, Morgan Sheng & Hans-Ulrich Dodt
9) PLoS One. 2015; 10(5): e0124650. PMCID: PMC4439039
Fluorescent-Protein Stabilization and High-Resolution Imaging of Cleared, Intact Mouse Brains
Schwarz MK, Scherbarth A, Sprengel R, Engelhardt J, Theer P, and G Giese

10) Cell. 2014 Nov 6;159(4):896-910
iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging.
Renier N, Wu Z, Simon DJ, Yang J, Ariel P, Tessier-Lavigne M.
11) Cell Rep. 2014 Nov 20;9(4):1191-201.
A Simple Method for 3D Analysis of Immunolabeled Axonal Tracts in a Transparent Nervous System.
Belle M, Godefroy D, Dominici C, Heitz-Marchaland C, Zelina P, Hellal F,  Bradke F, Chédotal A

12) J Am Soc Nephrol. 2016 Aug 3. pii: ASN.2016020232. [Epub ahead of print] PubMed PMID: 27487796.
Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy.
Klingberg A, Hasenberg A, Ludwig-Portugall I, Medyukhina A, Männ L, Brenzel A, Engel DR, Figge MT, Kurts C, Gunzer M.

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