Nature Protocols最新文献_第9页

Multiplexed single-molecule characterization at the library scale. 在文库规模上的多路单分子表征。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-04 DOI: 10.1038/s41596-025-01198-w

M Panfilov, G Mao, J Guo, J Aguirre Rivera, A Sabantsev, S Deindl

{"title":"Multiplexed single-molecule characterization at the library scale.","authors":"M Panfilov, G Mao, J Guo, J Aguirre Rivera, A Sabantsev, S Deindl","doi":"10.1038/s41596-025-01198-w","DOIUrl":"https://doi.org/10.1038/s41596-025-01198-w","url":null,"abstract":"Single-molecule techniques are exceptionally well suited for analyzing the complex dynamic behavior of macromolecules involved in fundamental biological processes. Nevertheless, time and cost usually restrict current single-molecule methods to examining a limited number of different samples. At the same time, a broad sequence or chemical space often needs to be investigated to gain a thorough understanding of complex biological phenomena. To address this urgent need, we have developed multiplexed single-molecule characterization at the library scale (MUSCLE), a method that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of complex dynamics on millions of individual molecules spanning thousands of distinct sequences or barcoded entities. In this protocol, we outline the implementation of MUSCLE and present examples from our recent research, such as the sequence-dependent dynamics of Cas9-induced target DNA unwinding and rewinding. This example demonstrates that MUSCLE can be applied to study protein-nucleic acid interactions, going beyond nucleic-acid-only model systems. We detail the sample and library design, high-throughput single-molecule data acquisition, next-generation sequencing, spatial registration of single-molecule fluorescence and sequencing data and downstream data analysis. The ligation-based surface immobilization approach of MUSCLE ensures high clustering efficiency (>40%), increasing throughput and simplifying registration. In addition, MUSCLE includes a 3D-printed flow cell adapter that enables liquid exchange during single-molecule fluorescence microscopy. The complete procedure typically spans 3-4 days and yields a dataset that comprehensively characterizes the dynamic behavior of a library of constructs.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144226011","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Highly stable planar asymmetric suspended membranes for investigating protein dynamics and membrane fusion. 用于研究蛋白质动力学和膜融合的高度稳定的平面不对称悬浮膜。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-03 DOI: 10.1038/s41596-025-01192-2

Manindra Bera, Ramalingam Venkat Kalyana Sundaram, Jeff Coleman, Atrouli Chatterjee, Sikha Thoduvayil, Frederic Pincet, Sathish Ramakrishnan

{"title":"Highly stable planar asymmetric suspended membranes for investigating protein dynamics and membrane fusion.","authors":"Manindra Bera, Ramalingam Venkat Kalyana Sundaram, Jeff Coleman, Atrouli Chatterjee, Sikha Thoduvayil, Frederic Pincet, Sathish Ramakrishnan","doi":"10.1038/s41596-025-01192-2","DOIUrl":"https://doi.org/10.1038/s41596-025-01192-2","url":null,"abstract":"Membrane fusion is central to cellular signaling and trafficking, requiring a detailed understanding of protein-lipid interactions. Studying these dynamic events in live cells presents challenges due to their complexity and heterogeneity. To address this, we developed a reductionist in vitro membrane model system that enables the controlled investigation of individual molecular components. This approach begins with a minimal membrane environment, with the opportunity for the stepwise addition of specific components to incrementally increase complexity achieving a level of experimental precision often unattainable in cellular studies. We developed suspended lipid membranes, a platform that uses pore-spanning lipid bilayers formed on microfabricated silicon chips with micrometer-sized holes. These membranes closely mimic native cellular architecture by maintaining aqueous compartments on both sides, providing a solvent-free, near-native environment with exceptional lateral diffusion properties. Their high stability makes them ideal for time-lapse imaging and dynamic process analysis using total internal reflection fluorescence and confocal microscopy. Here we present a detailed protocol for generating pore-spanning, planar suspended lipid membranes from native and synthetic reconstituted lipids using our silicon chip platform. Using SNARE proteins and molecular chaperones, we demonstrate the system's ability to capture ultrafast membrane fusion events. Additionally, we demonstrate single-molecule protein counting, protein dynamics analysis and single-vesicle fusion assays using fluorescently labeled proteins and vesicles. The ability to preserve native lipid asymmetry, biological composition and lateral diffusion makes this method a powerful tool for dissecting membrane fusion mechanisms and other membrane biological processes with unparalleled precision.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144216392","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Biomimetic membrane in a microfluidic chip for the electrical and optical monitoring of biological reactions. 用于生物反应电学和光学监测的微流控芯片中的仿生膜。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-03 DOI: 10.1038/s41596-025-01171-7

Delphine Mion, Louis Bunel, Sathish Ramakrishnan, Paul Heo, Frédéric Pincet

{"title":"Biomimetic membrane in a microfluidic chip for the electrical and optical monitoring of biological reactions.","authors":"Delphine Mion, Louis Bunel, Sathish Ramakrishnan, Paul Heo, Frédéric Pincet","doi":"10.1038/s41596-025-01171-7","DOIUrl":"https://doi.org/10.1038/s41596-025-01171-7","url":null,"abstract":"Biological membranes separate distinct inner and outer compartments through the organization of fluid lipids into two-dimensional bilayers. The specific lipid composition varies across different membrane types. Model membranes play a crucial role in replicating certain features of biological membranes. They provide invaluable insights to decipher reactions at biological membranes in physicochemical cues. In this Protocol, we present a comprehensive procedure for creating a biomimetic membrane that encompasses key characteristics of biological membranes. Each leaflet of this horizontal and large (~10,000 µm2) membrane is obtained from a separate set of liposomes, allowing control of the lipid distribution between the two bilayer leaflets. Suspended in a vertical conduit separating two controllable horizontal microfluidic channels, this membrane can be used for the reconstitution of chemical or molecular reactions in close proximity to the membrane on the desired leaflet. The microfluidic chip containing the two channels separated by the vertical conduit is made of poly(dimethylsiloxane) and is fabricated from resin molds. Initially, oil is trapped in the conduit. Liposome solutions are pushed in each channel and spread on the trapped oil-buffer interface, forming a separate leaflet facing each channel. As oil is absorbed by poly(dimethylsiloxane), the two leaflets assemble and form a bilayer. We outline four applications of this biomimetic membrane microfluidic setup, incorporating optical microscopy and/or electrical readouts (patch-clamp amplifiers): single-particle and global diffusion, membrane fusion and channel formation. The entire protocol, covering chip fabrication, membrane formation and various measurements, can be completed within 2-3 d.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144216391","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generation and characterization of vestibular inner ear organoids from human pluripotent stem cells. 人多能干细胞制备前庭内耳类器官及其特性研究。

IF 16 1区生物学

Nature Protocols Pub Date : 2025-06-02 DOI: 10.1038/s41596-025-01191-3

Wouter H van der Valk, Carl Nist-Lund, Jingyuan Zhang, Camila Perea, Jiahe Jin, Kelly Y Gim, Matthew R Steinhart, Jiyoon Lee, Karl R Koehler

{"title":"Generation and characterization of vestibular inner ear organoids from human pluripotent stem cells.","authors":"Wouter H van der Valk, Carl Nist-Lund, Jingyuan Zhang, Camila Perea, Jiahe Jin, Kelly Y Gim, Matthew R Steinhart, Jiyoon Lee, Karl R Koehler","doi":"10.1038/s41596-025-01191-3","DOIUrl":"10.1038/s41596-025-01191-3","url":null,"abstract":"The inner ear has a pivotal role in auditory and vestibular perception. Despite the vast number of individuals worldwide affected by hearing loss and balance disorders, therapeutic options have been largely limited to technological aids. The recent advent of gene therapies for genetic hearing loss in human patients underscores the urgency of developing scalable platforms to investigate a broader spectrum of inner ear disorders. Although animal models are powerful for assessing auditory and vestibular dysfunction, in vitro human inner ear models have shown promise in disease modeling and as platforms for studying developmental biology. Several studies have demonstrated that stem cells can be guided to differentiate into otic progenitor cells by mimicking environmental cues present during normal fetal inner ear development. Here we present a step-by-step approach to creating inner ear organoids (IEOs), which is an extension of our previous method for skin organoid generation, with which it shares foundational methodology and reagents. We used these organoids to elucidate the subtle signaling cues that govern their developmental trajectories. Generating sensory hair cells takes about 40 d, and cultures can be maintained for up to 150 d to allow further development. Moreover, we outline methods for assessing late-stage organoids, including whole-mount imaging of cleared IEOs, vibratome sectioning of live and fixed IEOs and other endpoint analyses, to study inner ear biology. IEOs are ideal for investigating human inner ear development, studying the mechanisms of inner ear disorders and developing therapeutic strategies. This protocol requires proficiency in basic stem cell culture techniques.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12447924/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144208930","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Author Correction: Transcriptomic profiling of individual bacteria by MATQ-seq. 作者更正：通过MATQ-seq对单个细菌进行转录组学分析。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-30 DOI: 10.1038/s41596-025-01213-0

Christina Homberger, Fabian Imdahl, Regan J Hayward, Lars Barquist, Antoine-Emmanuel Saliba, Jörg Vogel

引用次数: 0

Fibrous polyisocyanide hydrogels for 3D cell culture applications. 纤维多异氰化物水凝胶用于3D细胞培养应用。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-30 DOI: 10.1038/s41596-025-01159-3

Hongbo Yuan, Kaizheng Liu, Melissa J J van Velthoven, Jyoti Kumari, Yuying Bao, Susana Rocha, Paul H J Kouwer

{"title":"Fibrous polyisocyanide hydrogels for 3D cell culture applications.","authors":"Hongbo Yuan, Kaizheng Liu, Melissa J J van Velthoven, Jyoti Kumari, Yuying Bao, Susana Rocha, Paul H J Kouwer","doi":"10.1038/s41596-025-01159-3","DOIUrl":"https://doi.org/10.1038/s41596-025-01159-3","url":null,"abstract":"Three-dimensional (3D) cell culture models based on hydrogels are rapidly evolving into a prominent tool for tissue engineering, mechanobiology, disease modeling and drug screening. While a vast variety of synthetic gels have emerged in recent years, they fail to penetrate the market substantially for two major reasons: they poorly mimic the extracellular matrix or they are difficult to use in gel formation and cell extraction. Mimicking the complexity of nature is challenging: the extracellular matrix plays a crucial role in cell development and function, which goes well beyond simple mechanical support. Recently, we introduced polyisocyanide (PIC) hydrogels for 3D cell culture applications. The fibrous architecture and associated (non)linear mechanical behavior closely mimic the physical properties of biogels such as collagen and fibrin. As fully synthetic materials, PIC gels benefit from high tailorability and reproducibility. Moreover, the thermoresponsive properties of PIC gels make them easy to handle in the lab; the gels form instantly at 37 °C and cells are easily extracted after cooling to 5 °C. The potential of PIC gels has been demonstrated in a quickly expanding library of papers discussing different cell lines, primary cells and organoids, as well as in vivo experiments. This manuscript provides protocols on how to handle PIC gels in the chemistry and cell biology laboratories. Material preparation requires 72 h. Cell encapsulation takes 1 h and the time for downstream analysis depends on the (commercial) methods used. The protocols described are suitable for researchers with expertise in cell culture and molecular biology.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144187418","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A scalable protocol for the radiosynthesis of clinical grade lutetium-177-labeled theranostic agents. 放射合成临床级镥-177标记治疗剂的可扩展方案。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-27 DOI: 10.1038/s41596-025-01176-2

William W Hunt, Mathew Long, Usama Kamil, Sunil Kellapatha, Wayne Noonan, Peter D Roselt, Brittany Emmerson, Michael S Hofman, Mohammad B Haskali

{"title":"A scalable protocol for the radiosynthesis of clinical grade lutetium-177-labeled theranostic agents.","authors":"William W Hunt, Mathew Long, Usama Kamil, Sunil Kellapatha, Wayne Noonan, Peter D Roselt, Brittany Emmerson, Michael S Hofman, Mohammad B Haskali","doi":"10.1038/s41596-025-01176-2","DOIUrl":"https://doi.org/10.1038/s41596-025-01176-2","url":null,"abstract":"Theranostics utilizes tandem targeted diagnostic and therapeutic agents that are molecularly analogous. In a theranostic approach, the diagnostic agent is a tracer typically radiolabeled with a positron emission tomography radionuclide such as fluorine-18 or gallium-68. Utilizing the selectivity of the tracer, the therapeutic agent is subsequently radiolabeled with an ablative radionuclide such as the β- emitting lanthanide lutetium-177 (177Lu). 177Lu is typically incorporated into theranostics using the chelators 2,2',2'',2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) and 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAGA) that are used to prepare the 177Lu-radiopharmaceutical [177Lu]Lu-DOTA-TATE, [177Lu]Lu-PSMA-617 and [177Lu]Lu-PSMA-I&T. Here we describe the scalable and validated production for these 177Lu-radiopharmaceuticals and further include the necessary quality control protocols. The procedures can be generalized and support both carrier added and noncarrier added 177Lu sources for use in a clinical setting. With robust procedures that accommodate 177Lu activity levels from 5 to 100 GBq, the procedures ensure stability for up to 8 h postproduction and achieve an average activity yield of 98%. As proven in over 1,000 patient cycles, this methodology is adaptable to both centralized production facilities and regional centers, enabling versatile application across small and large-scale production settings.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144159740","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Hybridization chain reaction-based DNA nanoframeworks for biosensing and therapeutic applications. 基于杂交链反应的DNA纳米框架的生物传感和治疗应用。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-26 DOI: 10.1038/s41596-025-01183-3

Zhaoyue Lv, Peiran Li, Mingxing Liu, Chi Yao, Dayong Yang

{"title":"Hybridization chain reaction-based DNA nanoframeworks for biosensing and therapeutic applications.","authors":"Zhaoyue Lv, Peiran Li, Mingxing Liu, Chi Yao, Dayong Yang","doi":"10.1038/s41596-025-01183-3","DOIUrl":"https://doi.org/10.1038/s41596-025-01183-3","url":null,"abstract":"Artificial DNA nanostructures, with their sequence programmability, precise molecular recognition and tunable stimuli responsiveness, bridge material chemistry and biomedicine. Here we detail the design and construction of hybridization chain reaction (HCR)-based DNA nanoframeworks, a class of DNA nanostructures with programmable sequences and customizable functions. HCR is an efficient, enzyme-free amplification strategy that isothermally produces nicked double-stranded DNA with periodically repeated modules via the assembly of two DNA hairpins, triggered by a DNA initiator. In contrast to other available assembly methods for the synthesis of DNA nanostructures, such as tile-mediated assembly, DNA origami and rolling circle amplification, the HCR method offers improved stability and efficiency under mild conditions, without reliance on enzymatic activity. The procedure uses radical polymerization to integrate DNA initiator into nanoframeworks, with overhangs complementary to functional sequences - termed linkers -which are amplified and incorporated through HCR. The linkers enable the incorporation of functional nucleic acid sequences. The HCR-based DNA nanoframeworks facilitate the loading capability of the delivered molecules, showing notable therapeutic efficacy and biosensing sensitivity. Preparation time for HCR-based DNA nanoframeworks ranges from 30 h to 45 h, depending on the payload.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144151147","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Facile synthesis of mesoporous TiO₂ architectures with tunable configurations and nanometer precision. 具有可调结构和纳米精度的介孔TiO2结构的简单合成。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-23 DOI: 10.1038/s41596-025-01175-3

Jingyu Zhang, Jialong Li, Xu Wen, Lu Liu, Qiulong Wei, Pengfei Zhang, Jun-Ye Zhang, Dongyuan Zhao, Kun Lan

{"title":"Facile synthesis of mesoporous TiO2 architectures with tunable configurations and nanometer precision.","authors":"Jingyu Zhang, Jialong Li, Xu Wen, Lu Liu, Qiulong Wei, Pengfei Zhang, Jun-Ye Zhang, Dongyuan Zhao, Kun Lan","doi":"10.1038/s41596-025-01175-3","DOIUrl":"https://doi.org/10.1038/s41596-025-01175-3","url":null,"abstract":"Titanium dioxide (TiO2) can be used in various applications such as catalysis, sensing, energy storage and conversion due to its semiconducting and crystalline properties. Ordered mesoporous TiO2 materials with high porosity values may further enable improvements in mass diffusion and surface access. However, despite the syntheses of TiO2-based bulks and polymorphs in the past, it remains challenging to control mesoscopic TiO2 morphology and dimension, pore shape and size, crystallographic phase and orientation. Here we describe a facile and robust solution-processed methodology for the preparation of a series of mesoporous TiO2 structures with highly tailored architectures at the atomic scale, nanoscale and mesoscale. The process relies on the preformation of flexible micelle hydrogels and the stepwise assembly, under specific conditions to synthesize diverse mesoporous TiO2 configurations with tunable parameters, such as bouquet-like spheres, chapped spheres, monolayered nanosheets, sandwich, vertical films and so on. The synthetic conditions and procedures are provided in detail to ensure the reproducibility of the experiments. The preparation of micelle hydrogels takes ~21 h, and the subsequent synthesis time to obtain versatile mesoporous TiO2 is usually ~50 h. Our protocol is suitable for researchers in nanomaterials, porous and inorganic materials and other related disciplines.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144132686","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Integrating optical neuroscience tools into touchscreen operant systems 将光学神经科学工具集成到触摸屏操作系统中。

IF 16 1区生物学

Nature Protocols Pub Date : 2025-05-23 DOI: 10.1038/s41596-025-01143-x

Patrick T. Piantadosi, Oren Princz-Lebel, Miguel Skirzewski, Julie R. Dumont, Daniel Palmer, Sara Memar, Lisa M. Saksida, Vania F. Prado, Marco A. M. Prado, Tim J. Bussey, Andrew Holmes

{"title":"Integrating optical neuroscience tools into touchscreen operant systems","authors":"Patrick T. Piantadosi, Oren Princz-Lebel, Miguel Skirzewski, Julie R. Dumont, Daniel Palmer, Sara Memar, Lisa M. Saksida, Vania F. Prado, Marco A. M. Prado, Tim J. Bussey, Andrew Holmes","doi":"10.1038/s41596-025-01143-x","DOIUrl":"10.1038/s41596-025-01143-x","url":null,"abstract":"Unlocking the neural regulation of complex behavior is a foundational goal of brain science. Touchscreen-based assessments of behavior have been used extensively in the pursuit of this goal, with traditional pharmacological and neurochemical approaches being employed to provide key insights into underlying neural systems. So far, optically based approaches to measure and manipulate neural function, which have begun to revolutionize our understanding of relatively simple behaviors, have been less widely adopted for more complex cognitive functions of the type assessed with touchscreen-based behavioral tasks. Here we provide guidance and procedural descriptions to enable researchers to integrate optically based manipulation and measurement techniques into their touchscreen experimental systems. We focus primarily on three techniques, optogenetic manipulation, fiber photometry and microendoscopic imaging, describing experimental design adjustments that we have found to be critical to the successful integration of these approaches with extant touchscreen behavior pipelines. These include factors related to surgical procedures and timing, alterations to touchscreen operant environments and approaches to synchronizing light delivery and task design. A detailed protocol is included for each of the three techniques, covering their use from implementation through data analysis. The procedures in this protocol can be conducted in as short a time as a few days or over the course of weeks or months. This protocol extension enables researchers to integrate optically based manipulation and measurement techniques into their touchscreen experimental systems, focusing on optogenetic manipulation, fiber photometry and microendoscopic imaging.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 9","pages":"2418-2452"},"PeriodicalIF":16.0,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144132688","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0