Whole-Brain Profiling of Cells and Circuits in Mammals by Tissue Clearing and Light-Sheet Microscopy
2020; Cell Press; Volume: 106; Issue: 3 Linguagem: Inglês
10.1016/j.neuron.2020.03.004
ISSN1097-4199
AutoresHiroki R. Ueda, Hans‐Ulrich Dodt, Pavel Osten, Michael N. Economo, Jayaram Chandrashekar, Philipp Keller,
Tópico(s)Single-cell and spatial transcriptomics
ResumoTissue clearing and light-sheet microscopy have a 100-year-plus history, yet these fields have been combined only recently to facilitate novel experiments and measurements in neuroscience. Since tissue-clearing methods were first combined with modernized light-sheet microscopy a decade ago, the performance of both technologies has rapidly improved, broadening their applications. Here, we review the state of the art of tissue-clearing methods and light-sheet microscopy and discuss applications of these techniques in profiling cells and circuits in mice. We examine outstanding challenges and future opportunities for expanding these techniques to achieve brain-wide profiling of cells and circuits in primates and humans. Such integration will help provide a systems-level understanding of the physiology and pathology of our central nervous system. Tissue clearing and light-sheet microscopy have a 100-year-plus history, yet these fields have been combined only recently to facilitate novel experiments and measurements in neuroscience. Since tissue-clearing methods were first combined with modernized light-sheet microscopy a decade ago, the performance of both technologies has rapidly improved, broadening their applications. Here, we review the state of the art of tissue-clearing methods and light-sheet microscopy and discuss applications of these techniques in profiling cells and circuits in mice. We examine outstanding challenges and future opportunities for expanding these techniques to achieve brain-wide profiling of cells and circuits in primates and humans. Such integration will help provide a systems-level understanding of the physiology and pathology of our central nervous system. The beginning of the 20th century saw the birth of two technologies: tissue clearing and light-sheet microscopy. The earliest report on tissue clearing of opaque biomedical samples, which appeared in 1911 in Leipzig, was by the German anatomist Walter Spalteholz. He tried to make human hearts transparent to study their vascular system (Spalteholz, 1911Spalteholz W. Über das Durchsichtigmachen von menschlichen und tierischen Präparaten. S. Hirzel, 1911Google Scholar). Using hydrophobic tissue-clearing reagents (Wintergrünöl) such as methyl salicylate and benzyl benzoate on dehydrated specimens, he succeeded in visualizing macroscopic structures in transparent samples for the first time. However, without a technology like light-sheet microscopy, it was not possible to quantify his findings. So even this key advance brought only some qualitative insights into human anatomy. Not far from Leipzig, Austrian chemist Richard A. Zsigmondy and German physicist Henry Siedentopf, working in Jena, developed the first light-sheet microscope, the Ultramicroscope (Siedentopf and Zsigmondy, 1902Siedentopf H. Zsigmondy R. Über Sichtbarmachung und Größenbestimmung ultramikoskopischer Teilchen, mit besonderer Anwendung auf Goldrubingläser.Ann. Phys. 1902; 315: 1-39Crossref Google Scholar). Unlike Spalteholz, Zsigmondy was looking for very small things (Ultramikronen), colloidal particles in solution, which he tried to quantify. In principle, it would have been possible to integrate these two technologies more than 100 years ago, but at that time, it would not have led far. Light sheets traversing cleared specimens might create optical sections if one looks at the specimen at the correct angle, but these images still had to be recorded. Most importantly, a three-dimensional (3D) model of the specimen must be reconstructed digitally. Without concurrent inventions of electronic cameras and computers, even a hypothetical encounter of Spalteholz and Zsigmondy would not have produced the same impact that these methods have recently achieved. Approximately 90 years after these seminal works, the first relevant step toward integration of tissue clearing and light-sheet microscopy was made by Arno Voie and colleagues (Voie et al., 1993Voie A.H. Burns D.H. Spelman F.A. Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens.J. Microsc. 1993; 170: 229-236Crossref PubMed Google Scholar). They designed a modern version of light-sheet microscopy (orthogonal-plane fluorescence optical sectioning microscopy [OPFOS]), based on lasers and digital camera technology. After clearing the bony structure of the inner ear using EDTA and Spalteholz’s hydrophobic tissue-clearing reagents (methyl salicylate and benzyl benzoate), Voie and colleagues performed the first fluorescence optical imaging of a tissue-cleared biological specimen, an excised guinea-pig cochlea labeled with fluorescein (Voie et al., 1993Voie A.H. Burns D.H. Spelman F.A. Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens.J. Microsc. 1993; 170: 229-236Crossref PubMed Google Scholar). They recorded the images with a charge-coupled device (CCD) camera and successfully reconstructed the spiral in the inner ear with rudimentary homemade 3D reconstruction software. The approach unfortunately remained largely unnoticed by the broader scientific community for nearly two decades. Light-sheet microscopy rapidly gained momentum in biological imaging in the early 21st century, with applications in diverse fields including microbial oceanography, developmental biology, and neuroscience (Dodt et al., 2007Dodt H.U. Leischner U. Schierloh A. Jährling N. Mauch C.P. Deininger K. Deussing J.M. Eder M. Zieglgänsberger W. Becker K. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.Nat. 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Optical sectioning deep inside live embryos by selective plane illumination microscopy.Science. 2004; 305: 1007-1009Crossref PubMed Scopus (1437) Google Scholar). They reconstructed largely transparent living biological samples, including Medaka fish embryos. Despite the success of this approach for developmental biology, it remained restricted to naturally transparent samples. The first use of light-sheet imaging on neural tissues artificially rendered transparent was made by Hans-Ulrich Dodt and colleagues (Dodt et al., 2007Dodt H.U. Leischner U. Schierloh A. Jährling N. Mauch C.P. Deininger K. Deussing J.M. Eder M. Zieglgänsberger W. Becker K. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.Nat. Methods. 2007; 4: 331-336Crossref PubMed Scopus (786) Google Scholar). They took advantage of hydrophobic tissue-clearing reagents, benzyl alcohol and benzyl benzoate (BABB), that were originally developed by Andrew Murray and Marc Kirschner around 1989 and first applied to fluorescent- and peroxidase-based whole-mount immunocytochemistry of Xenopus oocytes and embryos (Dent et al., 1989Dent J.A. Polson A.G. Klymkowsky M.W. A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus.Development. 1989; 105: 61-74Crossref PubMed Google Scholar). Combining this clearing method with ultramicroscopy and image processing enabled the visualization of neuronal networks at the resolution of neural dendrites in whole mouse brains (Dodt et al., 2007Dodt H.U. Leischner U. Schierloh A. Jährling N. Mauch C.P. Deininger K. Deussing J.M. Eder M. Zieglgänsberger W. Becker K. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.Nat. 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The RI of a certain material is defined as the ratio of the speed of light in a vacuum to that in the material. These high-RI solutions included X-ray contrast agents (Trazograph), a series of alcohols (polyethylene glycol, glycerol, and propylene glycol), sugars (glucose and dextran), and dimethyl sulfoxide (DMSO) (V.V. Bakutkin et al., 1995, Int. Soc. Optic. Photon., conference; A.N. Bashkatov et al., 1999, Int. Soc. Optic. Photon., conference; V.V. Tuchin et al., 1999, Proc. SPIE, conference; Tuchin et al., 1997Tuchin V.V. Maksimova I.L. Zimnyakov D.A. Kon I.L. Mavlyutov A.H. Mishin A.A. Light propagation in tissues with controlled optical properties.J. Biomed. Opt. 1997; 2: 401-417Crossref PubMed Google Scholar, Tuchin et al., 2002Tuchin V.V. Xu X. Wang R.K. Dynamic optical coherence tomography in studies of optical clearing, sedimentation, and aggregation of immersed blood.Appl. Opt. 2002; 41: 258-271Crossref PubMed Google Scholar, Xu et al., 2003Xu X. Wang R.K. Elder J.B. 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A different cocktail—FocusClear, which contains another X-ray contrast agent (diatrizoate acid) and a detergent (Tween 20)—was used by Chiang and colleagues for whole-brain imaging of cockroach using a confocal microscope (Chiang et al., 2001Chiang A.S. Liu Y.C. Chiu S.L. Hu S.H. Huang C.Y. Hsieh C.H. Three-dimensional mapping of brain neuropils in the cockroach, Diploptera punctata.J. Comp. Neurol. 2001; 440: 1-11Crossref PubMed Scopus (0) Google Scholar). In 2011, Atsushi Miyawaki and colleagues developed the hydrophilic tissue-clearing method Scale, which hyperhydrates and delipidates mouse brains with urea-based reagents and a detergent, respectively, resulting in semi-transparent mouse brains (Hama et al., 2011Hama H. Kurokawa H. Kawano H. Ando R. Shimogori T. Noda H. Fukami K. Sakaue-Sawano A. Miyawaki A. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.Nat. 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CUBIC protocols for whole-body clearing also seem to permit light-sheet imaging of heart, lung, liver, kidney, pancreas, and other organs (Susaki et al., 2014Susaki E.A. Tainaka K. Perrin D. Kishino F. Tawara T. Watanabe T.M. Yokoyama C. Onoe H. Eguchi M. Yamaguchi S. et al.Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.Cell. 2014; 157: 726-739Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar, Susaki et al., 2015Susaki E.A. Tainaka K. Perrin D. Yukinaga H. Kuno A. Ueda H.R. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.Nat. Protoc. 2015; 10: 1709-1727Crossref PubMed Scopus (251) Google Scholar, Tainaka et al., 2014Tainaka K. Kubota S.I. Suyama T.Q. Susaki E.A. Perrin D. Ukai-Tadenuma M. Ukai H. Ueda H.R. 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In parallel with the development of hydrophobic and hydrophilic tissue-clearing methods, a hydrogel-based tissue-clearing method called clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining/in situ-hybridization-compatible tissue hydrogel (CLARITY) was also developed in 2013 by Karl Deisseroth and Kwanghun Chung. In this process, lipids are removed by perfusion of sodium dodecyl sulfate (SDS), a strong detergent, and tissue is subsequently transformed into a clear acrylamide gel retaining biological elements (Chung et al., 2013Chung K. Wallace J. Kim S.Y. Kalyanasundaram S. Andalman A.S. Davidson T.J. Mirzabekov J.J. Zalocusky K.A. Mattis J. Denisin A.K. et al.Structural and molecular interrogation of intact biological systems.Nature. 2013; 497: 332-337Crossref PubMed Scopus (1114) Google Scholar). CLARITY employs an electrophoresis step to accelerate tissue clearing and is therefore more complex than many other hydrophobic and hydrophilic tissue-clearing methods that rely on passive diffusion. Following SDS perfusion, proteins and nucleic acids are retained, presumably because of their stabilization by the exogenous acrylamide gel. Using CLARITY, it may be possible to visualize endogenous fluorescent proteins and to label epitopes with fluorescent antibodies (Chung et al., 2013Chung K. Wallace J. Kim S.Y. Kalyanasundaram S. Andalman A.S. Davidson T.J. Mirzabekov J.J. Zalocusky K.A. Mattis J. Denisin A.K. et al.Structural and molecular interrogation of intact biological systems.Nature. 2013; 497: 332-337Crossref PubMed Scopus (1114) Google Scholar). Although reliable fluorescence preservation and immunolabeling remain challenging, intact CLARITY-processed brains have been successfully imaged at high resolution (numerical aperture [NA] = 1.0) with light-sheet microscopy (Tomer et al., 2014Tomer R. Ye L. Hsueh B. Deisseroth K. Advanced CLARITY for rapid and high-resolution imaging of intact tissues.Nat. Protoc. 2014; 9: 1682-1697Crossref PubMed Scopus (478) Google Scholar). Raju Tomer and colleagues developed the passive CLARITY technique (PACT) by decreasing gel density to improve tissue permeability and probe penetration (Tomer e
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