TY - JOUR
T1 - High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding
AU - Bykov, Yury S.
AU - Cohen, Nir
AU - Gabrielli, Natalia
AU - Manenschijn, Hetty
AU - Welsch, Sonja
AU - Chlanda, Petr
AU - Kukulski, Wanda
AU - Patil, Kiran R.
AU - Schuldiner, Maya
AU - Briggs, John A. G.
N1 - We are grateful to Markus Mund (European Molecular Biology Laboratory [EMBL], Heidelberg, Germany) and Albert Mas (Universitat Rovira i Virgili, Ramón y Cajal, Tarragona, Spain) for yeast strains and to Nadav Shai, Maria Bohnert, and Michal Eisenberg-Bord (Weizman Institute of Science, Rehovot, Israel) for sharing unpublished reagents. We thank Morgane Wartel, Lisa Maier, Einat Zalckvar, Nassos Typas, and Anne-Claude Gavin for advice and discussions. We are grateful to Michael Knop for discussions and comments on the manuscript. We thank Martin Schorb, Yannick Schwab, and Rachel Mellwig for their help and advice on setting up the MultiCLEM method at the EMBL. This study was technically supported by the EMBL Advanced Light Microscopy Facility, EMBL Electron Microscopy Core Facility, Weizmann Institute of Science Electron Microscopy Facility, and Medical Research Council Laboratory of Molecular Biology Light Microscopy Facility. This work was financially supported by grants from the Deutsche Forschungsgemeinschaft (SFB1129 Z2 to J.A.G. Briggs), EMBL (to J.A.G. Briggs), the Medical Research Council (MC_UP_1201/16 to J.A.G. Briggs), and the German Ministry of Education and Research (031A605 to K.R. Patil). The Schuldiner laboratory is supported by the European Research Council CoG 646604 Peroxisystem, the Deutsche Forschungsgemeinschaft (grant SFB1190 and a Deutsch-Israelische Projektkooperation [DIP] collaborative grant). N. Gabrielli was supported by the EMBL interdisciplinary postdoctoral program. M. Schuldiner is an incumbent of the Dr. Gilbert Omenn and Martha Darling Professorial Chair in Molecular Genetics. The authors declare no competing financial interests. Author contributions: Y.S. Bykov, conceptualization, formal analysis, methodology, investigation, writing – original draft; N. Cohen, conceptualization, formal analysis, methodology, investigation, writing – original draft; N. Gabrielli, investigation, resources, writing – review and editing; H. Manenschijn, investigation, methodology, writing – review and editing; S. Welsch, conceptualization, investigation, methodology, writing – review and editing; P. Chlanda, conceptualization, investigation, methodology, writing – review and editing; W. Kukulski, conceptualization, investigation, methodology, writing – review and editing; K.R. Patil, conceptualization, supervision, funding acquisition, writing – review and editing, project administration; M. Schuldiner, conceptualization, supervision, funding acquisition, writing – original draft, project administration; and J.A.G. Briggs, conceptualization, methodology, supervision, funding acquisition, writing – original draft, project administration.
PY - 2019/8/5
Y1 - 2019/8/5
N2 - Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called "MultiCLEM," allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique.
AB - Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called "MultiCLEM," allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique.
UR - http://www.scopus.com/inward/record.url?scp=85071070526&partnerID=8YFLogxK
U2 - https://doi.org/10.1083/jcb.201812081
DO - https://doi.org/10.1083/jcb.201812081
M3 - مقالة
SN - 0021-9525
VL - 218
SP - 2797
EP - 2811
JO - Journal of Cell Biology
JF - Journal of Cell Biology
IS - 8
ER -