This document discusses the history and developments of tissue clearing techniques. It provides a timeline of key developments from 129 AD to present day, including early animal dissections, the publication of anatomy books in 1543, and the development of light sheet microscopy and two-photon microscopy in the 1990s. Recent years have seen new clearing agents and methods introduced that build on prior techniques to further homogenize tissue refractive index and allow deep tissue imaging while preserving fluorescence and structure.

1. Tiago Pinheiro
May 2015
Supervision: András Simon Macmillan Publishers Ltd: Nature 497:
332–37, copyright 2013

2. Key concepts
Refractive index
Refractive index variation
Main obstacle to imaging of intact tissues
Refractive index of material needs to be homogeno
http://www.microscopyu.com/articles/formulas/images/refractiveindexf

3. Key concepts
Major types of tissue clearing
Refractive index of material needs to be homogeno
1
 Dry the sample
e.g. ethanol,
tetrahydrofuran
 Refill with high-
index solvent that
matches the tissue
e.g. BABB, DBE,
glycerol
Fluorophore quenching
Sample shrinkage
3
4
 Replace the water in
the tissue with polar
solvents with high
refractive index
e.g. formamide
2
 Increase the
refractive index of
the tissue by adding
water-soluble
compounds
e.g. glucose, fructose
Sample expansion

4. More transparency
Fluorochrome degradation
Less transparency
Fluorochrome preservation
Usual problem
https://www.leica-microsystems.com/science-lab/clarity/clearing-procedures-for-deep-tissue-imaging

5. Timeline
 First commercial light sheet microscope – Lavision
 BABB clearing - Dodt
 Deep tissue application of two-photon microscopy - Helmchen
 FocusClear – Liu
 First light sheet microscope – Voie
 Two-photon laser scanning fluorescence microscopy - Denk
 Superman x-ray vision research – Pittenger JB.
 Other derivative procedures - clove oil and cedarwood oil
 Method for reducing refractive index variations in tissue - Spalteholz
 Publication of 6 anatomy books - Vesallii
 Dissection of animals – Galen 129
1543
1914
...1914
1983
1990
1993
2003
2005
2007
2010

6.  Dissection of animals – Galen 129

7.  Publication of 6 anatomy books - Vesallii 1543

8.  Method for reducing refractive index variations in tissue - Spalteholz 1914

9.  Other derivative procedures - clove oil and cedarwood oil ...1914

10.  Superman x-ray vision research – Pittenger JB. 1983

11.  Two-photon laser scanning fluorescence microscopy - Denk 1990

12. 1990 First light sheet microscope – Voie 1993
Resolution of 26 um

13.  FocusClear – Liu 2003
Proprietary and expensive 50 ml – 12.000 kr

14.  Deep tissue application of two-photon microscopy - Helmchen 2005

15.  BABB clearing - Dodt 2007
BABB clearing quenches fluorophores

16. 1990 First commercial light sheet microscope – Lavision 2010
Rapid zstack acquisition in high resolution

17. Timeline
 First commercial light sheet microscope – Lavision
 BABB clearing - Dodt
 Deep tissue application of two-photon microscopy - Helmchen
 FocusClear – Liu
 First light sheet microscope – Voie
 Two-photon laser scanning fluorescence microscopy - Denk
 Superman x-ray vision research – Pittenger JB.
 Other derivative procedures - clove oil and cedarwood oil
 Method for reducing refractive index variations in tissue - Spalteholz
 Publication of 6 anatomy books - Vesallii
 Dissection of animals – Galen 129
1543
1914
...1914
1983
1990
*
1993
2003
2005
2007
2010

19. Timeline ...1914
*
Developments in technology and tissue clearing techniques converge
 Scale – Hama 2011
 More GFP friendly agents discovered DBE and tetrahydrofuran – Becker
 CUBIC Whole-body – Tainaka
 CUBIC Whole-brain – Susaki
 iDISCO – Renier
 ClearT – Kuwajima
 SeeDB – Ke
 Clarity – Chung
 3DISCO – Ertürk
2012
2012
2013
2013
2013
2014
2014
2014

20.  Scale – Hama 2011
2
 Increase the refractive
index of the tissue by
adding water-soluble
compounds
e.g. glucose, fructose
Sample expansion
3 weeks to 6 months
Protein loss
Non-proprietary

21.  More GFP friendly agents discovered DBE and tetrahydrofuran – Becker 2012
Gives rise to
3DISCO later
on
…

22.  3DISCO – Ertürk 2012
1
 Dry the sample
e.g. ethanol,
tetrahydrofuran
 Refill with high-index
solvent that matches
the tissue
e.g. BABB, DBE,
glycerol
Non-proprietary
1 day half-time of
GFP signal
No tissue expansion
No fluorescent
protein quenching
2-5 days

23.  Clarity – Chung 2013
Non-proprietary
2 weeks
Good antibody labelling
No fluorophore quenching
Keeps tissue morphology
Custom setup, long protocol
4

24.  SeeDB – Ke 2013
3 days
Keeps tissue morphology
No fluorescent protein
quenching
Non-proprietary 1 week
2
 Increase the refractive
index of the tissue by
adding water-soluble
compounds
e.g. glucose, fructose

25.  ClearT – Kuwajima
2013
2013
3
 Replace the water in
the tissue with polar
solvents with high
refractive index
e.g. formamide
1 day - ?
No or mild expansion
Compatible with
lipophilic tracers
Non-proprietary
Not compatible
with fluorescent
proteins

26.  iDISCO – Renier
2014
2014
Same as 3DISCO but for large samples

27.  CUBIC Whole-brain – Susaki 2014
Mix of compounds joining
the best of many
techniquesSimple clearing and
analysis pipeline
Non-proprietary
5
Single-cell resolution,
axons and dendrites

28. 2014 CUBIC Whole-body – Tainaka 2014
Same as whole-brain
CUBIC but applied to
other organs…
5

29. More applications
Viewing neuronal populations and
projections;
Intact tissue imaging of long-range
projections, , local circuit wiring, cellular
relationships, subcellular structures,
protein complexes, nucleic acids, and
neurotransmitters;
Quantitation of distances of neural stem
cells to blood vessels;
…

30. “Both complete structural analysis (i.e. not reconstructed across tissue
sections) and molecular phenotyping are desired to gain full insights into
the relationships and functional mechanisms of biological systems.”
“Techniques that focus on molecular labelling require thin tissue
sectioning which limits structural reconstruction.”
Advantages
• No damage or thin sectioning required to visualize whole intact tissue samples
• Allows marking and visualization of long-range projections and subcellular structures
• Allows multiple rounds of molecular phenotyping
• Applicable to multiple tissue types and sizes
Disadvantages
• Multiple-step process that takes place over several days/weeks
• Immunostaining is time-consuming for thicker tissue samples
• High start-up and consumable material costs
Advantages of
tissue clearing to sectioning
http://wiki.claritytechniques.org/index.php/Main_Page