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tissue clearing methods for plants and animals

     When you want to image thick tissue samples with fluorescent proteins, the depth of imaging is limited by tissue transparency. To simplify, the level of transparency is the determining factor of how deep you can image. The more transparent the sample, the deeper you can image.

     The older methods used for rendering tissue transparent are mostly based dehydrating sample in a step by step manner and mounting them in high refractive index organic solvents. Other methods use chloral hydrate and Gum Arabica. Although these methods render tissue transparent, these are not compatible with fluorescent proteins.

     The tissue clearing methods that are listed below are compatible with fluorescent proteins. With these methods, you can image fluorescent samples 100s of microns deep that are otherwise difficult to image even 20-50 microns deep.

Plant tissue clearing methods

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An Optical Clearing Technique for Plant Tissues Allowing Deep Imaging and Compatible with Fluorescence Microscopy

Journal: Plant Physiology

Authors: Warner CA et al. (2014)

Material used:

Arabidopsis thaliana: Leaf

Medicago truncatula: Leaf

Pisum sativum: Leaf and Root nodules

Nicotiana benthamiana: Leaf 

Zea mays: Leaf

Chemicals used:

6M Urea; Other reagents in the clearing solution are different for each tissue type. 

Time required for clearing:

48 hours to 3 weeks.

Dyes and/or Fluorescent proteins used

Citrine (yellow range)

Compatible with immunolabelling: Alexa 568

Calcofluor white (cell wall)

Refractive index of mounting medium:

NA

How deep tissue imaged:

Fresh samples 100 µm; Cleared tissue up to 350 µm.

Microscope used: 

Zeiss LSM 510 NLO multiphoton confocal microscope.  Zeiss LSM 710 confocal microscope.40X LD water immersion and 25X LD multi immersion (30% glycerol)

Conclusion:

I guess first of its kind in the plant clearing method. Now there are other methods that are faster in clearing (hours to days).

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A Versatile Optical Clearing Protocol for Deep Tissue Imaging of Fluorescent Proteins in Arabidopsis thaliana

Journal: PLoS One

Authors: Musielak TJ et al. (2016)

Material used:

Arabidopsis thaliana: Leaf, Root, Cotyledons, Isolated ovules, Siliques, Anthers and Shoot.

Chemicals used:

20-95% Thiodiethanol, Glycerol for mounting, 4%PFA for fixation. 

Time required for clearing:

1 hr to O/N fixation time + 1 - 3 hrs incubation in TDE. 

Dyes and Fluorescent proteins used

EGFP

Citrine (yellow range)

YFP

tdTomato

mCherry

Refractive index of mounting medium:

TDE (mounting medium) refractive index is adjustable up to 1.515 depending on the dilution.

How deep tissue imaged:

>240 microns with clearing method.

Microscope used: 

AxioImager Z.1         - 20X, 0.8NA

Leica TCS SP8            - 40X, 1.1NA W Corr

Olympus FV1200       - 40X

Zeiss LSM 780 NLO (2 photon) - 25x/0,8 Imm Korr and 40x/1,1 W Korr.

Conclusion:

This method clears tissue quicker than the Urea based method. The authors also showed the higher photostability of tdTomato in TDE. Despite the advantages mentioned above, tissue shrinkage may be a drawback in certain situations.

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ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging

Journal: Development

Authors: Kurihara D et al. (2015)

Material used:

Physcomitrella patens (Moss)

Arabidopsis thaliana: Leaf, Root, seedlings, pollinated  Pistil.

Chemicals used:

4%PFA for fixation; 

ClearSee:

Xylitol powder 10% (W/V),  sodium deoxycholate 15% (W/V), urea 25% (W/V) in water.

Time required for clearing:

Fixation time + 4 days to 4 weeks or more. 

Dyes and Fluorescent proteins used

mTFP1

sGFP

mGFP5

Venus 

mCitrine

YFP

mClover

tdTomato

mApple

mRFP1

Calcofluor White (cell wall stain)

Hoechst 33342

Refractive index of ClearSee:

1.41; For ClearSee V2 - 1.395.

How deep tissue imaged:

100-150 microns with clearing method + confocal laser scanning microscope.

400-670 microns with 2 photon microscope.

Microscope used: 

Olympus bx53: 10X

Nikon A1R MP (multiphoton): 25× WI, NA = 1.10, WD = 2.00 mm

Zeiss LSM780-DUO-NLO:  20× NA = 0.8

Storage of the cleared samples:

Cleared samples can be stored in the ClearSee reagent  5 (tested up to) months at room temperature.

Conclusion:

The most significant advantage of this method is the compatibility with fluorescent proteins (shown with ten different FPs) and also with cell wall staining (Calcofluor White) and nuclear stains(Hoechst 33342). The only drawback I can think of is the duration of tissue clearing (4 weeks in some cases). However, due to the advantages mentioned above waiting for 4 weeks may not be such a disadvantage.

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Animal tissue clearing methods

lab mice

RTF: a rapid and versatile tissue optical clearing method

Journal: Scientific Reports

Author: Yu T et al., 2018

Material used:

Adult mice brain sections

Neonatal whole brain

Whole embryo

Chemicals used:

Sample fixation: 4% Paraformaldehyde.

RTF-R1: 30% Triethanolamine + 40% Formamide + 30% Water 

RTF-R2: 60% Triethanolamine + 25% Formamide + 15% Water

RTF-R1: 70% Triethanolamine + 15% Formamide + 15% Water 

The time required for clearing:

hours to a day.

Dyes or Fluorescent proteins used

Yellow Fluorescent Protein (YFP)

Enhanced Green Fluorescent Protein (EGFP)

dsRed

Dil - membrane labelling dye

Alexa 594

Compatibility with Immunostaining: 

Yes; Anti-GFP antibody with Alexa 594.

Refractive index of mounting medium:

1.46;

pH of the mounting medium:

10

How deep tissue imaged:

 

Microscope used: 

Zeiss LSM710 and light sheet microscope (La VisisonBioTec) 

Conclusion:

This method was reported to be quicker and better compared to ClearT2. When compared with other methods like FRUIT and ScaleSQ.

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