Imaging Three-Dimensional Brain Organoid Architecture from Meso- to Nanoscale across Development

Organoids are human stem cell-derived three-dimensional cultures offering a new avenue to model human development and disease. Brain organoids allow studying various aspects of human brain development in the finest details in vitro in a tissue-like context. However, spatial relationships of subcellular structures such as synaptic contacts between distant neurons are hardly accessible by conventional light microscopy. This limitation can be overcome by systems that quickly image the entire organoid in three dimensions and in super-resolution. To that end we have developed a setup combining tissue expansion and light sheet fluorescence microscopy for imaging and quantifying diverse spatial parameters during organoid development. This technique enables zooming from a mesoscopic perspective into super-resolution within a single imaging session, thus revealing cellular and subcellular structural details in three spatial dimensions, including unequivocal delineation of mitotic cleavage planes as well as the alignment of pre- and postsynaptic proteins. We expect light sheet fluorescence expansion microscopy (LSFEM) to facilitate qualitative and quantitative assessment of organoids in developmental and disease-related studies. Summary statement The combination of light sheet fluorescence and expansion microscopy enables imaging of mature human brain organoids in toto and down to synaptic resolution

[1]  U. Kubitscheck,et al.  Expansion light sheet fluorescence microscopy of extended biological samples: Applications and perspectives. , 2021, Progress in biophysics and molecular biology.

[2]  C. Clouchoux,et al.  Recent Trends and Perspectives in Cerebral Organoids Imaging and Analysis , 2021, Frontiers in Neuroscience.

[3]  P. Arlotta,et al.  Multiscale 3D phenotyping of human cerebral organoids , 2020, Scientific Reports.

[4]  H. Brismar,et al.  High-Resolution Imaging of Tumor Spheroids and Organoids Enabled by Expansion Microscopy , 2020, Frontiers in Molecular Biosciences.

[5]  Ó. Martínez-Matos,et al.  Ultra-long light sheets via curved beam intercrossing , 2020 .

[6]  Madeline A. Lancaster,et al.  An early cell shape transition drives evolutionary expansion of the human forebrain , 2020, Cell.

[7]  S. Baron-Cohen,et al.  Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids , 2020, bioRxiv.

[8]  Jens F Schweihoff,et al.  Hard-wired lattice light-sheet microscopy for imaging of expanded samples. , 2020, Optics express.

[9]  Bjoern H Menze,et al.  Cellular and Molecular Probing of Intact Human Organs , 2020, Cell.

[10]  Pavel Tomancak,et al.  Tissue clearing and its applications in neuroscience , 2020, Nature Reviews Neuroscience.

[11]  Maximilian Haeussler,et al.  Cell Stress in Cortical Organoids Impairs Molecular Subtype Specification , 2019, Nature.

[12]  Sean K. Simmons,et al.  Individual brain organoids reproducibly form cell diversity of the human cerebral cortex , 2019, Nature.

[13]  J. Visvader,et al.  High-resolution 3D imaging of fixed and cleared organoids , 2019, Nature Protocols.

[14]  G. Ming,et al.  Brain organoids: advances, applications and challenges , 2019, Development.

[15]  Gerald M. Rubin,et al.  Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution , 2019, Science.

[16]  C. Henneberger,et al.  Light-sheet fluorescence expansion microscopy: fast mapping of neural circuits at super resolution , 2019, Neurophotonics.

[17]  Ian T. Fiddes,et al.  Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution , 2018, Cell.

[18]  J. Chang,et al.  Expansion microscopy , 2018, Journal of microscopy.

[19]  Laura Masullo,et al.  Cerebral organoids at the air-liquid interface generate diverse nerve tracts with functional output , 2018, Nature Neuroscience.

[20]  Elly M. Tanaka,et al.  Broad applicability of a streamlined ethyl cinnamate-based clearing procedure , 2018, Development.

[21]  Kyle L Ellefsen,et al.  Dynamic Ca2+ imaging with a simplified lattice light-sheet microscope: A sideways view of subcellular Ca2+ puffs. , 2018, Cell calcium.

[22]  Arnold R. Kriegstein,et al.  The use of brain organoids to investigate neural development and disease , 2017, Nature Reviews Neuroscience.

[23]  F. J. Livesey,et al.  Guided self-organization and cortical plate formation in human brain organoids , 2017, Nature Biotechnology.

[24]  Daniel R. Berger,et al.  Cell diversity and network dynamics in photosensitive human brain organoids , 2017, Nature.

[25]  Alex A. Pollen,et al.  Human iPSC-Derived Cerebral Organoids Model Cellular Features of Lissencephaly and Reveal Prolonged Mitosis of Outer Radial Glia. , 2017, Cell stem cell.

[26]  F. Müller,et al.  An Organoid-Based Model of Cortical Development Identifies Non-Cell-Autonomous Defects in Wnt Signaling Contributing to Miller-Dieker Syndrome. , 2017, Cell reports.

[27]  P. Arlotta,et al.  The promises and challenges of human brain organoids as models of neuropsychiatric disease , 2016, Nature Medicine.

[28]  Kwanghun Chung,et al.  Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues , 2016, Nature Biotechnology.

[29]  Edward S Boyden,et al.  Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies , 2016, Nature Biotechnology.

[30]  Joshua C Vaughan,et al.  Expansion microscopy with conventional antibodies and fluorescent proteins , 2016, Nature Methods.

[31]  Liang Gao,et al.  Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy , 2016, Nature Communications.

[32]  Gaudenz Danuser,et al.  Deconvolution-free Subcellular Imaging with Axially Swept Light Sheet Microscopy , 2015, Biophysical journal.

[33]  D. Geschwind,et al.  Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture , 2015, Nature Methods.

[34]  Wesley R. Legant,et al.  Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution , 2014, Science.

[35]  Lisle W. Blackbourn,et al.  A Simple and Efficient System for Regulating Gene Expression in Human Pluripotent Stem Cells and Derivatives , 2014, Stem cells.

[36]  K. Dholakia,et al.  Light-sheet microscopy using an Airy beam , 2014, Nature Methods.

[37]  Madeline A. Lancaster,et al.  Cerebral organoids model human brain development and microcephaly , 2013, Nature.

[38]  A. Kriegstein,et al.  Mitotic spindle orientation predicts outer radial glial cell generation in human neocortex , 2013, Nature Communications.

[39]  Ulrich Kubitscheck,et al.  Scanned light sheet microscopy with confocal slit detection. , 2012, Optics Express.

[40]  G. Iannello,et al.  Confocal light sheet microscopy: micron-scale neuroanatomy of the entire mouse brain. , 2012, Optics express.

[41]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[42]  Stephan Saalfeld,et al.  Globally optimal stitching of tiled 3D microscopic image acquisitions , 2009, Bioinform..

[43]  A. Wynshaw-Boris,et al.  Neuroepithelial Stem Cell Proliferation Requires LIS1 for Precise Spindle Orientation and Symmetric Division , 2008, Cell.

[44]  A. Schierloh,et al.  Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain , 2007, Nature Methods.

[45]  Rafael C. González,et al.  Digital image processing using MATLAB , 2006 .

[46]  F. Del Bene,et al.  Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy , 2004, Science.