Mosaic analysis with double markers (MADM) mouse models closely mimic the clonal origin of human cancers by generating sporadic, GFP-labeled cancer-initiating cells. Traditional clonal analysis pipelines are labor intensive, hindering throughput and disrupting the 3D architecture. Here, we present a protocol that integrates tissue clearing and light-sheet imaging to analyze pre-malignant clones in whole-mount MADM-labeled tissues. We describe steps for generating mosaic-labeled cancer mouse models, tissue harvesting, fixation, and clearing. We then detail procedures for light-sheet imaging and clonal size analysis. For complete details on the use and execution of this protocol, please refer to Zeng et al..
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http://dx.doi.org/10.1016/j.xpro.2024.103092 | DOI Listing |
STAR Protoc
January 2025
Gladstone Institutes, San Francisco, CA, USA; Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, CA, USA; Department of Pediatrics, Cardiovascular Research Institute, Institute for Human Genetics, and Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:
As light sheet fluorescence microscopy (LSFM) becomes widely available, reconstruction of time-lapse imaging will further our understanding of complex biological processes at cellular resolution. Here, we present a comprehensive workflow for in toto capture, processing, and analysis of multi-view LSFM experiments using the ex vivo mouse embryo as a model system of development. Our protocol describes imaging on a commercial LSFM instrument followed by computational analysis in discrete segments, using open-source software.
View Article and Find Full Text PDFAlzheimers Dement
December 2024
Massachusetts Alzheimer's Disease Research Center, Charlestown, MA, USA.
Background: In cerebral amyloid angiopathy, amyloid beta accumulates within the walls of blood vessels and contributes to impaired vascular integrity and function. In this work, we observe that tau protein similarly builds up along blood vessels in Alzheimer's disease brain.
Method: We obtained frozen inferior temporal cortex from the Massachusetts Alzheimer's Disease Research Center from n = 7 neuropathological confirmed Alzheimer's disease donors and n = 6 normal aging controls.
Nat Commun
January 2025
Laboratory of High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, USA.
Optical aberrations hinder fluorescence microscopy of thick samples, reducing image signal, contrast, and resolution. Here we introduce a deep learning-based strategy for aberration compensation, improving image quality without slowing image acquisition, applying additional dose, or introducing more optics. Our method (i) introduces synthetic aberrations to images acquired on the shallow side of image stacks, making them resemble those acquired deeper into the volume and (ii) trains neural networks to reverse the effect of these aberrations.
View Article and Find Full Text PDFMethods Mol Biol
January 2025
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Heraklion, Crete, Greece.
Biotechniques
January 2025
Biomedical Engineering, The University of Arizona, Tucson, AZ, USA.
Current dorsal skin flap window chambers with flat glass windows are compatible with optical coherence tomography (OCT) and multiphoton microscopy (MPM) imaging. However, light sheet fluorescence microscopy (LSFM) performs best with a cylindrical or spherical sample located between its two 90° objectives and when all sample materials have the same index of refraction (). A modified window chamber with a domed viewing window made from fluorinated ethylene propylene (FEP), with n similar to water and tissue, was designed.
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