{"doi":"10.3791/62271","title":"Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton","abstract":"The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these \"time-resolved\" studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.","journal":"Journal of Visualized Experiments","year":2021,"id":185141,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":9,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9585,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":741638,"name":"Luis Allegri","orcid":null,"position":1,"is_corresponding":false},{"id":741639,"name":"Venkat Dandey","orcid":null,"position":2,"is_corresponding":false},{"id":253672,"name":"Clinton S. Potter","orcid":"0000-0003-2394-0831","position":3,"is_corresponding":false},{"id":253673,"name":"Bridget Carragher","orcid":"0000-0002-0624-5020","position":4,"is_corresponding":false},{"id":258056,"name":"William C. Budell","orcid":"0000-0002-5538-668X","position":0,"is_corresponding":true}],"reference_count":22,"raw_metadata":null,"created_at":"2026-07-18T23:48:34.992499Z","pmid":"33720116","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}