[{"date_created":"2024-09-10T12:32:38Z","year":"2024","quality_controlled":"1","corr_author":"1","citation":{"short":"D. Kleindienst, T. Costanzo, R. Shigemoto, in:, J.H.R. Lübke, A. Rollenhagen (Eds.), New Aspects in Analyzing the Synaptic Organization of the Brain, 1st ed., Springer Nature, New York, 2024, pp. 123–137.","ieee":"D. Kleindienst, T. Costanzo, and R. Shigemoto, “Automated Imaging and Analysis of Synapses in Freeze-Fracture Replica Samples with Deep Learning,” in <i>New Aspects in Analyzing the Synaptic Organization of the Brain</i>, 1st ed., J. H. R. Lübke and A. Rollenhagen, Eds. New York: Springer Nature, 2024, pp. 123–137.","mla":"Kleindienst, David, et al. “Automated Imaging and Analysis of Synapses in Freeze-Fracture Replica Samples with Deep Learning.” <i>New Aspects in Analyzing the Synaptic Organization of the Brain</i>, edited by Joachim H.R.  Lübke and Astrid Rollenhagen, 1st ed., Springer Nature, 2024, pp. 123–37, doi:<a href=\"https://doi.org/10.1007/978-1-0716-4019-7_8\">10.1007/978-1-0716-4019-7_8</a>.","ista":"Kleindienst D, Costanzo T, Shigemoto R. 2024.Automated Imaging and Analysis of Synapses in Freeze-Fracture Replica Samples with Deep Learning. In: New Aspects in Analyzing the Synaptic Organization of the Brain. Neuromethods, , 123–137.","ama":"Kleindienst D, Costanzo T, Shigemoto R. Automated Imaging and Analysis of Synapses in Freeze-Fracture Replica Samples with Deep Learning. In: Lübke JHR, Rollenhagen A, eds. <i>New Aspects in Analyzing the Synaptic Organization of the Brain</i>. 1st ed. New York: Springer Nature; 2024:123-137. doi:<a href=\"https://doi.org/10.1007/978-1-0716-4019-7_8\">10.1007/978-1-0716-4019-7_8</a>","chicago":"Kleindienst, David, Tommaso Costanzo, and Ryuichi Shigemoto. “Automated Imaging and Analysis of Synapses in Freeze-Fracture Replica Samples with Deep Learning.” In <i>New Aspects in Analyzing the Synaptic Organization of the Brain</i>, edited by Joachim H.R.  Lübke and Astrid Rollenhagen, 1st ed., 123–37. New York: Springer Nature, 2024. <a href=\"https://doi.org/10.1007/978-1-0716-4019-7_8\">https://doi.org/10.1007/978-1-0716-4019-7_8</a>.","apa":"Kleindienst, D., Costanzo, T., &#38; Shigemoto, R. (2024). Automated Imaging and Analysis of Synapses in Freeze-Fracture Replica Samples with Deep Learning. In J. H. R. Lübke &#38; A. Rollenhagen (Eds.), <i>New Aspects in Analyzing the Synaptic Organization of the Brain</i> (1st ed., pp. 123–137). New York: Springer Nature. <a href=\"https://doi.org/10.1007/978-1-0716-4019-7_8\">https://doi.org/10.1007/978-1-0716-4019-7_8</a>"},"ec_funded":1,"title":"Automated Imaging and Analysis of Synapses in Freeze-Fracture Replica Samples with Deep Learning","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","edition":"1","publication_status":"published","abstract":[{"text":"Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) is an electron microscope (EM) sample preparation technique which allows for high-resolution visualization of membrane proteins with high sensitivity. However, image acquisition of specific replica profiles such as synapses in a large field of EM view needs a valid experience and a long time for manual searching. Here, we describe how to utilize deep learning for automatizing image acquisition of specific profiles of interest in replica samples. This protocol facilitates the labor-intensive collection of EM images, in particular for rare profiles. We provide instructions for using SerialEM image acquisition software in conjunction with object detection by our newly developed deep learning software DarEM, to automatically acquire tilt series of all synapses in a selected region. We then show how to perform a mostly automated analysis of gold particle labeling in the acquired images by utilizing Darea software.","lang":"eng"}],"_id":"18052","status":"public","publication":"New Aspects in Analyzing the Synaptic Organization of the Brain","publisher":"Springer Nature","article_processing_charge":"No","type":"book_chapter","page":"123-137","day":"27","doi":"10.1007/978-1-0716-4019-7_8","publication_identifier":{"isbn":["9781071640180"],"eissn":["1940-6045"],"eisbn":["9781071640197"],"issn":["0893-2336"]},"editor":[{"first_name":"Joachim H.R. ","last_name":"Lübke","full_name":"Lübke, Joachim H.R. "},{"first_name":"Astrid","full_name":"Rollenhagen, Astrid","last_name":"Rollenhagen"}],"acknowledgement":"This research was supported by the European Research Council Advanced Grant 694539 to RS and by the Scientific Service Units of IST Austria through resources provided by the Electron Microscopy Facility.","department":[{"_id":"EM-Fac"},{"_id":"RySh"}],"date_published":"2024-08-27T00:00:00Z","alternative_title":["Neuromethods"],"place":"New York","author":[{"first_name":"David","id":"42E121A4-F248-11E8-B48F-1D18A9856A87","last_name":"Kleindienst","full_name":"Kleindienst, David"},{"first_name":"Tommaso","orcid":"0000-0001-9732-3815","id":"D93824F4-D9BA-11E9-BB12-F207E6697425","last_name":"Costanzo","full_name":"Costanzo, Tommaso"},{"orcid":"0000-0001-8761-9444","first_name":"Ryuichi","last_name":"Shigemoto","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","full_name":"Shigemoto, Ryuichi"}],"project":[{"_id":"25CA28EA-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","grant_number":"694539"}],"date_updated":"2025-04-14T07:27:15Z","language":[{"iso":"eng"}],"acknowledged_ssus":[{"_id":"EM-Fac"}],"month":"08","oa_version":"None","scopus_import":"1"},{"department":[{"_id":"MaDe"}],"editor":[{"full_name":"Yamamoto, Daisuke","last_name":"Yamamoto","first_name":"Daisuke"}],"acknowledgement":"We thank de Bono lab members for the helpful comments on the manuscript. The biotin-auxotrophic E. coli strain MG1655bioB:kan was a generous gift from J. Cronan (University of Illinois) and was kindly sent to us by Jessica Feldman and Ariana Sanchez (Stanford University). dg398 pEntryslot2_mNeongreen::3XFLAG::stop and dg397 pEntryslot3_mNeongreen::3XFLAG::stop::unc-54 3’UTR entry vector were kindly sent by Dr. Dominique Glauser (University of Fribourg). This work was supported by an Advanced ERC Grant (269058 ACMO) and a Wellcome Investigator Award (209504/Z/17/Z) to MdB and an ISTplus Fellowship to MA (Marie Sklodowska-Curie agreement No 754411).","publication_identifier":{"eissn":["1940-6045"],"isbn":["9781071623206"],"eisbn":["9781071623213"],"issn":["0893-2336"]},"doi":"10.1007/978-1-0716-2321-3_15","scopus_import":"1","oa_version":"None","month":"06","language":[{"iso":"eng"}],"project":[{"_id":"23870BE8-32DE-11EA-91FC-C7463DDC885E","name":"Molecular mechanisms of neural circuit function","grant_number":"209504/A/17/Z"},{"_id":"260C2330-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","name":"ISTplus - Postdoctoral Fellowships","grant_number":"754411"}],"date_updated":"2025-04-14T07:43:58Z","place":"New York","author":[{"full_name":"Artan, Murat","last_name":"Artan","id":"C407B586-6052-11E9-B3AE-7006E6697425","orcid":"0000-0001-8945-6992","first_name":"Murat"},{"orcid":"0000-0001-8347-0443","first_name":"Mario","last_name":"de Bono","full_name":"de Bono, Mario","id":"4E3FF80E-F248-11E8-B48F-1D18A9856A87"}],"date_published":"2022-06-04T00:00:00Z","alternative_title":["Neuromethods"],"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","ec_funded":1,"title":"Proteomic Analysis of C. Elegans Neurons Using TurboID-Based Proximity Labeling","citation":{"chicago":"Artan, Murat, and Mario de Bono. “Proteomic Analysis of C. Elegans Neurons Using TurboID-Based Proximity Labeling.” In <i>Behavioral Neurogenetics</i>, edited by Daisuke Yamamoto, 181:277–94. NM. New York: Springer Nature, 2022. <a href=\"https://doi.org/10.1007/978-1-0716-2321-3_15\">https://doi.org/10.1007/978-1-0716-2321-3_15</a>.","apa":"Artan, M., &#38; de Bono, M. (2022). Proteomic Analysis of C. Elegans Neurons Using TurboID-Based Proximity Labeling. In D. Yamamoto (Ed.), <i>Behavioral Neurogenetics</i> (Vol. 181, pp. 277–294). New York: Springer Nature. <a href=\"https://doi.org/10.1007/978-1-0716-2321-3_15\">https://doi.org/10.1007/978-1-0716-2321-3_15</a>","ista":"Artan M, de Bono M. 2022.Proteomic Analysis of C. Elegans Neurons Using TurboID-Based Proximity Labeling. In: Behavioral Neurogenetics. Neuromethods, vol. 181, 277–294.","ama":"Artan M, de Bono M. Proteomic Analysis of C. Elegans Neurons Using TurboID-Based Proximity Labeling. In: Yamamoto D, ed. <i>Behavioral Neurogenetics</i>. Vol 181. NM. New York: Springer Nature; 2022:277-294. doi:<a href=\"https://doi.org/10.1007/978-1-0716-2321-3_15\">10.1007/978-1-0716-2321-3_15</a>","mla":"Artan, Murat, and Mario de Bono. “Proteomic Analysis of C. Elegans Neurons Using TurboID-Based Proximity Labeling.” <i>Behavioral Neurogenetics</i>, edited by Daisuke Yamamoto, vol. 181, Springer Nature, 2022, pp. 277–94, doi:<a href=\"https://doi.org/10.1007/978-1-0716-2321-3_15\">10.1007/978-1-0716-2321-3_15</a>.","short":"M. Artan, M. de Bono, in:, D. Yamamoto (Ed.), Behavioral Neurogenetics, Springer Nature, New York, 2022, pp. 277–294.","ieee":"M. Artan and M. de Bono, “Proteomic Analysis of C. Elegans Neurons Using TurboID-Based Proximity Labeling,” in <i>Behavioral Neurogenetics</i>, vol. 181, D. Yamamoto, Ed. New York: Springer Nature, 2022, pp. 277–294."},"quality_controlled":"1","corr_author":"1","date_created":"2022-06-20T08:10:34Z","year":"2022","volume":181,"day":"04","page":"277-294","type":"book_chapter","series_title":"NM","article_processing_charge":"No","publication":"Behavioral Neurogenetics","publisher":"Springer Nature","intvolume":"       181","_id":"11456","abstract":[{"text":"The proteomes of specialized structures, and the interactomes of proteins of interest, provide entry points to elucidate the functions of molecular machines. Here, we review a proximity-labeling strategy that uses the improved E. coli biotin ligase TurboID to characterize C. elegans protein complexes. Although the focus is on C. elegans neurons, the method is applicable regardless of cell type. We describe detailed extraction procedures that solubilize the bulk of C. elegans proteins and highlight the importance of tagging endogenous genes, to ensure physiological expression levels. We review issues associated with non-specific background noise and the importance of appropriate controls. As proof of principle, we review our analysis of the interactome of a presynaptic active zone protein, ELKS-1. Our aim is to provide a detailed protocol for TurboID-based proximity labeling in C. elegans and to highlight its potential and its limitations to characterize protein complexes and subcellular compartments in this animal.","lang":"eng"}],"status":"public","publication_status":"published"},{"doi":"10.1007/978-1-4939-3064-7_17","date_created":"2025-07-10T13:56:06Z","year":"2016","corr_author":"1","quality_controlled":"1","publication_identifier":{"issn":["0893-2336"],"eisbn":["9781493930647"],"isbn":["9781493930630"],"eissn":["1940-6045"]},"citation":{"ama":"Harada H, Shigemoto R. High-Resolution Localization of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL). In: <i>Receptor and Ion Channel Detection in the Brain</i>. Neuromethods. Springer Nature; 2016:233-245. doi:<a href=\"https://doi.org/10.1007/978-1-4939-3064-7_17\">10.1007/978-1-4939-3064-7_17</a>","ista":"Harada H, Shigemoto R. 2016.High-Resolution Localization of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL). In: Receptor and Ion Channel Detection in the Brain. , 233–245.","apa":"Harada, H., &#38; Shigemoto, R. (2016). High-Resolution Localization of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL). In <i>Receptor and Ion Channel Detection in the Brain</i> (pp. 233–245). Springer Nature. <a href=\"https://doi.org/10.1007/978-1-4939-3064-7_17\">https://doi.org/10.1007/978-1-4939-3064-7_17</a>","chicago":"Harada, Harumi, and Ryuichi Shigemoto. “High-Resolution Localization of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL).” In <i>Receptor and Ion Channel Detection in the Brain</i>, 233–45. Neuromethods. Springer Nature, 2016. <a href=\"https://doi.org/10.1007/978-1-4939-3064-7_17\">https://doi.org/10.1007/978-1-4939-3064-7_17</a>.","ieee":"H. Harada and R. Shigemoto, “High-Resolution Localization of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL),” in <i>Receptor and Ion Channel Detection in the Brain</i>, Springer Nature, 2016, pp. 233–245.","short":"H. Harada, R. Shigemoto, in:, Receptor and Ion Channel Detection in the Brain, Springer Nature, 2016, pp. 233–245.","mla":"Harada, Harumi, and Ryuichi Shigemoto. “High-Resolution Localization of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL).” <i>Receptor and Ion Channel Detection in the Brain</i>, Springer Nature, 2016, pp. 233–45, doi:<a href=\"https://doi.org/10.1007/978-1-4939-3064-7_17\">10.1007/978-1-4939-3064-7_17</a>."},"acknowledgement":"We thank Mitsuru Ikeda for preparing replica images used in Fig. 2.","title":"High-Resolution Localization of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL)","department":[{"_id":"RySh"}],"user_id":"ba8df636-2132-11f1-aed0-ed93e2281fdd","date_published":"2016-02-02T00:00:00Z","publication_status":"published","author":[{"last_name":"Harada","full_name":"Harada, Harumi","id":"2E55CDF2-F248-11E8-B48F-1D18A9856A87","first_name":"Harumi","orcid":"0000-0001-7429-7896"},{"orcid":"0000-0001-8761-9444","first_name":"Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","last_name":"Shigemoto","full_name":"Shigemoto, Ryuichi"}],"_id":"19990","abstract":[{"lang":"eng","text":"Visualizing molecular localization at high resolution contributes to understanding of their functions and roles in physiological and pathological conditions. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) is a powerful electron microscopy method to study high-resolution two-dimensional distribution of transmembrane proteins and their tightly associated proteins on platinum-carbon replica. During treatment with SDS, unfixed proteins and intracellular organelle are dissolved and integral membrane proteins captured and stabilized by carbon and platinum deposition are denatured, retaining most of their antigenicity, and exposed on exoplasmic and protoplasmic surfaces of lipid monolayers. The exposure of these antigens on the surface of replica facilitates the accessibility of antibodies and therefore provides higher labeling efficiency than those obtained with other immunoelectron microscopy techniques. In this chapter, we describe the protocols of SDS-FRL adapted for mammalian brain samples and an additional procedure for fluorescence-guided electron microscopy for replica immunolabeling."}],"status":"public","date_updated":"2026-04-07T08:32:03Z","language":[{"iso":"eng"}],"publication":"Receptor and Ion Channel Detection in the Brain","publisher":"Springer Nature","OA_type":"closed access","article_processing_charge":"No","month":"02","series_title":"Neuromethods","type":"book_chapter","oa_version":"None","page":"233-245","day":"02"}]