Clarifying Tissue Clearing
Open accessAbstract
Biological specimens are intrinsically three dimensional; however, because of the obscuring effects of light scatter, imaging deep into a tissue volume is problematic. Although efforts to eliminate the scatter by "clearing" the tissue have been ongoing for over a century, there have been a large number of recent innovations. This Review introduces the physical basis for light scatter in tissue, describes the mechanisms underlying various clearing techniques, and discusses several of the major advances in light microscopy for imaging cleared tissue.
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Several tissue clearing techniques have been developed to reduce light scattering in fixed biological tissues by selectively removing lipids in a non-destructive way.
“Recently, several tissue clearing techniques have been developed to reduce light scattering in fixed biological tissues by selectively removing lipids in a non-destructive way3.”
From A crystal-clear zebrafish for in vivo imaging (2016) · cites this paper as [3]
The Modern Clearing Techniques and Aqueous based clearing sections say that clearing reduces light scattering and that several approaches remove lipids. But the aqueous-based section also describes an approach that does not remove lipids, while the Solvent based clearing and Hydrogel embedding sections report shrinkage and risks to tissue proteins. The paper therefore does not support describing tissue-clearing techniques generally as selectively removing lipids in a non-destructive way. (LLM, conf=0.88)
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Cites
Serial Block-Face Scanning Electron Microscopy to Reconstruct Three-Dimensional Tissue Nanostructure
Denk, W., & Horstmann, H. (2004) · PLoS Biology · doi:10.1371/journal.pbio.0020329 · Cited by 1,644 · see citing works
Transparent Adult Zebrafish as a Tool for In Vivo Transplantation Analysis
White, R. M., Sessa, A., Burke, C., Bowman, T., LeBlanc, J., Ceol, C., Bourque, C., Dovey, M., Goessling, W., Burns, C. E., et al. (2008) · Cell Stem Cell · doi:10.1016/j.stem.2007.11.002 · Cited by 1,408 · see citing works
Spectral Imaging and Linear Unmixing in Light Microscopy
Zimmermann, T. (2005) · Advances in Biochemical Engineering/Biotechnology · doi:10.1007/b102216 · Cited by 302 · see citing works
Flagged first, then by citations
Research works printed but not linked: 58
- Alnuami AA Zeedi B Qadri SM Ashraf SS 2008 Oxyradical-induced GFP damage and loss of fluorescence International journal of biological macromolecules 43 182 186 18561996
- Aoyagi Y Kawakami R Osanai H Hibi T Nemoto T 2015 A rapid optical clearing protocol using 2,2′-thiodiethanol for microscopic observation of fixed mouse brain PloS one 10 e0116280 25633541 PMC4310605
- Becker K Jahrling N Saghafi S Weiler R Dodt HU 2012 Chemical clearing and dehydration of GFP expressing mouse brains PloS one 7 e33916 22479475 PMC3316521
- Booth MJ 2007 Adaptive optics in microscopy Philosophical transactions Series A, Mathematical, physical, and engineering sciences 365 2829 2843 17855218
- Bouchard MB Voleti V Mendes CS Lacefield C Grueber WB Mann RS Bruno RM Hillman EM 2015 Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms Nature photonics 9 113 119 25663846 PMC4317333
- Boyde A 1985 Stereoscopic images in confocal (tandem scanning) microscopy Science 230 1270 1272 4071051
- Bunka DH Stockley PG 2006 Aptamers come of age - at last Nature reviews Microbiology 4 588 596 16845429
- Cai D Cohen KB Luo T Lichtman JW Sanes JR 2013 Improved tools for the Brainbow toolbox Nature methods 10 540 547 23866336
- Chalfie M Tu Y Euskirchen G Ward WW Prasher DC 1994 Green fluorescent protein as a marker for gene expression Science 263 802 805 8303295
- Chen BC Legant WR Wang K Shao L Milkie DE Davidson MW Janetopoulos C Wu XS Hammer JA 3rd Liu Z 2014 Lattice light-sheet microscopy imaging molecules to embryos at high spatiotemporal resolution Science 346 1257998 25342811 PMC4336192
- Chen F Tillberg PW Boyden ES 2015 Optical imaging. Expansion microscopy Science 347 543 548 25592419 PMC4312537
- Chiang AS Lin WY Liu HP Pszczolkowski MA Fu TF Chiu SL Holbrook GL 2002 Insect NMDA receptors mediate juvenile hormone biosynthesis Proceedings of the National Academy of Sciences of the United States of America 99 37 42 11773617 PMC117510
- Chung K Wallace J Kim SY Kalyanasundaram S Andalman AS Davidson TJ Mirzabekov JJ Zalocusky KA Mattis J Denisin AK 2013 Structural and molecular interrogation of intact biological systems Nature 497 332 337 23575631 PMC4092167
- Clancy B Cauller LJ 1998 Reduction of background autofluorescence in brain sections following immersion in sodium borohydride Journal of neuroscience methods 83 97 102 9765122
- Conchello JA Lichtman JW 2005 Optical sectioning microscopy Nature methods 2 920 931 16299477
- Costantini I Ghobril JP Di Giovanna AP Mascaro AL Silvestri L Mullenbroich MC Onofri L Conti V Vanzi F Sacconi L 2015 A versatile clearing agent for multi-modal brain imaging Scientific reports 5 9808 25950610 PMC4423470
- Denk W Strickler JH Webb WW 1990 Two-photon laser scanning fluorescence microscopy Science 248 73 76 2321027
- Dodt HU Leischner U Schierloh A Jahrling N Mauch CP Deininger K Deussing JM Eder M Zieglgansberger W Becker K 2007 Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain Nature methods 4 331 336 17384643
- Duong H Han M 2013 A multispectral LED array for the reduction of background autofluorescence in brain tissue Journal of neuroscience methods 220 46 54 23994358 PMC3856220
- Erturk A Becker K Jahrling N Mauch CP Hojer CD Egen JG Hellal F Bradke F Sheng M Dodt HU 2012a Three-dimensional imaging of solvent-cleared organs using 3DISCO Nature protocols 7 1983 1995 23060243
- Erturk A Mauch CP Hellal F Forstner F Keck T Becker K Jahrling N Steffens H Richter M Hubener M 2012b Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury Nature medicine 18 166 171 22198277
- Hama H Kurokawa H Kawano H Ando R Shimogori T Noda H Fukami K Sakaue-Sawano A Miyawaki A 2011 Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain Nature neuroscience 14 1481 1488 21878933
- Hecht E 2001 Optics 4 Addison-Wesley
- Hell S Reiner G Cremer C Stelzer EHK 1993 Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index Journal of microscopy 169 391 405
- Holekamp TF Turaga D Holy TE 2008 Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy Neuron 57 661 672 18341987
- Holliger P Hudson PJ 2005 Engineered antibody fragments and the rise of single domains Nature biotechnology 23 1126 1136 16151406
- Hong G Diao S Chang J Antaris AL Chen C Zhang B Zhao S Atochin DN Huang PL Andreasson KI 2014 Through-skull fluorescence imaging of the brain in a new near-infrared window Nat Photon 8 723 730 PMC5026222 27642366
- Hou B Zhang D Zhao S Wei M Yang Z Wang S Wang J Zhang X Liu B Fan L 2015 Scalable and DiI-compatible optical clearance of the mammalian brain Frontiers in neuroanatomy 9 19 25759641 PMC4338786
- Hua L Zhou R Thirumalai D Berne BJ 2008 Urea denaturation by stronger dispersion interactions with proteins than water implies a 2-stage unfolding Proceedings of the National Academy of Sciences of the United States of America 105 16928 16933 18957546 PMC2579355
- Ichimura K Miyazaki N Sadayama S Murata K Koike M Nakamura K Ohta K Sakai T 2015 Three-dimensional architecture of podocytes revealed by block-face scanning electron microscopy Scientific reports 5 8993 25759085 PMC4355681
- Ke MT Fujimoto S Imai T 2013 SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction Nature neuroscience 16 1154 1161 23792946
- Kuwajima T Sitko AA Bhansali P Jurgens C Guido W Mason C 2013 ClearT: a detergent-and solvent-free clearing method for neuronal and non-neuronal tissue Development 140 1364 1368 23444362 PMC3912244
- Lichtman JW Conchello JA 2005 Fluorescence microscopy Nature methods 2 910 919 16299476
- Marques MJ Conchello JA Lichtman JW 2000 From plaque to pretzel: fold formation and acetylcholine receptor loss at the developing neuromuscular junction The Journal of neuroscience: the official journal of the Society for Neuroscience 20 3663 3675 10804208 PMC6772702
- Martell JD Deerinck TJ Sancak Y Poulos TL Mootha VK Sosinsky GE Ellisman MH Ting AY 2012 Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy Nature biotechnology 30 1143 1148 PMC3699407 23086203
- Mertz J 2011 Optical sectioning microscopy with planar or structured illumination Nature methods 8 811 819 21959136
- Oh SW Harris JA Ng L Winslow B Cain N Mihalas S Wang Q Lau C Kuan L Henry AM 2014 A mesoscale connectome of the mouse brain Nature 508 207 214 24695228 PMC5102064
- Paez-Segala MG Sun MG Shtengel G Viswanathan S Baird MA Macklin JJ Patel R Allen JR Howe ES Piszczek G 2015 Fixation-resistant photoactivatable fluorescent proteins for CLEM Nature methods 12 215 218 214 p following 218 25581799 PMC4344411
- Planchon TA Gao L Milkie DE Davidson MW Galbraith JA Galbraith CG Betzig E 2011 Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination Nature methods 8 417 423 21378978 PMC3626440
- Renier N Wu Z Simon DJ Yang J Ariel P Tessier-Lavigne M 2014 iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging Cell 159 896 910 25417164
- Reynaud EG Krzic U Greger K Stelzer EH 2008 Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage HFSP journal 2 266 275 19404438 PMC2639947
- Rost FWD 1992 Fluorescence microscopy Cambridge; New York Cambridge University Press
- Spalteholz W 1898 Hand-atlas of human anatomy 2nd Philadelphia; London J.B. Lippincott
- Staudt T Lang MC Medda R Engelhardt J Hell SW 2007 2,2′-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy Microscopy research and technique 70 1 9 17131355
- Steinke H Wolff W 2001 A modified Spalteholz technique with preservation of the histology Annals of anatomy = Anatomischer Anzeiger: official organ of the Anatomische Gesellschaft 183 91 95 11206989
- Susaki EA Tainaka K Perrin D Kishino F Tawara T Watanabe TM Yokoyama C Onoe H Eguchi M Yamaguchi S 2014 Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis Cell 157 726 739 24746791
- Tainaka K Kubota SI Suyama TQ Susaki EA Perrin D Ukai-Tadenuma M Ukai H Ueda HR 2014 Whole-body imaging with single-cell resolution by tissue decolorization Cell 159 911 924 25417165
- Theer P Denk W 2006 On the fundamental imaging-depth limit in two-photon microscopy Journal of the Optical Society of America A, Optics, image science, and vision 23 3139 3149 17106469
- Theer P Hasan MT Denk W 2003 Two-photon imaging to a depth of 1000 microm in living brains by use of a Ti:Al2O3 regenerative amplifier Optics letters 28 1022 1024 12836766
- Toga AW Ambach KL Schluender S 1994 High-resolution anatomy from in situ human brain NeuroImage 1 334 344 9343583
- Toga AW Goldkorn A Ambach K Chao K Quinn BC Yao P 1997 Postmortem cryosectioning as an anatomic reference for human brain mapping Computerized medical imaging and graphics: the official journal of the Computerized Medical Imaging Society 21 131 141 9152579
- Tomer R Ye L Hsueh B Deisseroth K 2014 Advanced CLARITY for rapid and high-resolution imaging of intact tissues Nature protocols 9 1682 1697 24945384 PMC4096681
- Tsai PS Blinder P Migliori BJ Neev J Jin Y Squier JA Kleinfeld D 2009a Plasma-mediated ablation: an optical tool for submicrometer surgery on neuronal and vascular systems Current opinion in biotechnology 20 90 99 19269159 PMC3123732
- Tsai PS Kaufhold JP Blinder P Friedman B Drew PJ Karten HJ Lyden PD Kleinfeld D 2009b Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocalization of nuclei and vessels The Journal of neuroscience: the official journal of the Society for Neuroscience 29 14553 14570 19923289 PMC4972024
- Verveer PJ Swoger J Pampaloni F Greger K Marcello M Stelzer EH 2007 High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy Nature methods 4 311 313 17339847
- Wang K Milkie DE Saxena A Engerer P Misgeld T Bronner ME Mumm J Betzig E 2014 Rapid adaptive optical recovery of optimal resolution over large volumes Nature methods 11 625 628 24727653 PMC4069208
- Wu Y Wawrzusin P Senseney J Fischer RS Christensen R Santella A York AG Winter PW Waterman CM Bao Z 2013 Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy Nature biotechnology 31 1032 1038 PMC4105320 24108093
- Yang B Treweek JB Kulkarni RP Deverman BE Chen CK Lubeck E Shah S Cai L Gradinaru V 2014 Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing Cell 158 945 958 25088144 PMC4153367
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- Authors
- Richardson, D. S., & Lichtman, J. W.
- Affiliations
- Harvard University (US)
- Corresponding author
- Richardson, Douglas S.
- Lichtman, Jeff W.
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2015-07
- Volume
- 162
- Issue
- 2
- Pages
- 246-257
- ISSN
- 0092-8674 (ISSN-L)
- PMC
- PMC4537058
- OpenAlex
- W1603300429
- Cited by
- 1,095
- References
- 3
- Citations by year
- 2026: 65 · 2025: 79 · 2024: 68 · 2023: 78 · 2022: 108 · 2021: 140 · 2020: 151 · 2019: 136 · 2018: 114 · 2017: 77 · 2016: 68 · 2015: 10
- FWCI
- 9.14
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Cell (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Science Foundation
- Johns Hopkins University
- National Institutes of Health
- National Institute of Mental Health (P50MH094271)
- National Institute of Neurological Disorders and Stroke (U01NS090449, R01NS076467)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Optical Imaging and Spectroscopy Techniques
- Keywords
- tissue clearing, tissue scattering, optical microscopy, 3D imaging, deep tissue imaging
- MeSH terms
- Animals, Histological Techniques, Histology, Humans, Light, Microscopy, Microscopy, Confocal, Imaging, Three-Dimensional
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