方法文章

TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis

DOI：

10.3791/64953

⸱

2022年12月16日

 ,  ,  ,  ,  , 
1School of Neuroscience, Virginia Polytechnic Institute and State University, 2CMU-Pitt Joint Computational Biology, School of Medicine, University of Pittsburgh, 3Eastern Virginia Medical School, 4Department of Physics, University of Miami

本文内容

摘要

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TrackMate Analysis of Calcium Imaging (TACI) is an open-source ImageJ plugin for 3D calcium imaging analysis that examines motion on the z-axis and identifies the maximum value of each z-stack to represent a cell's intensity at the corresponding time point. It can separate neurons overlapping in the lateral (x/y) direction but on different z-planes.

摘要

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Research in neuroscience has evolved to use complex imaging and computational tools to extract comprehensive information from data sets. Calcium imaging is a widely used technique that requires sophisticated software to obtain reliable results, but many laboratories struggle to adopt computational methods when updating protocols to meet modern standards. Difficulties arise due to a lack of programming knowledge and paywalls for software. In addition, cells of interest display movements in all directions during calcium imaging. Many approaches have been developed to correct the motion in the lateral (x/y) direction.

This paper describes a workflow using a new ImageJ plugin, TrackMate Analysis of Calcium Imaging (TACI), to examine motion on the z-axis in 3D calcium imaging. This software identifies the maximum fluorescence value from all the z-positions a neuron appears in and uses it to represent the neuron's intensity at the corresponding t-position. Therefore, this tool can separate neurons overlapping in the lateral (x/y) direction but appearing on distinct z-planes. As an ImageJ plugin, TACI is a user-friendly, open-source computational tool for 3D calcium imaging analysis. We validated this workflow using fly larval thermosensitive neurons that displayed movements in all directions during temperature fluctuation and a 3D calcium imaging dataset acquired from the fly brain.

引言

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The level of intracellular calcium is a precise marker of neuronal excitability. Calcium imaging measures the changes in intracellular calcium to understand neuronal activity1. Studies in neuroscience have increasingly used this method due to the development of techniques for measuring intracellular calcium concentration, including genetically encoded calcium indicators (GECIs), such as GCaMP2,3, which can be noninvasively expressed in specific sets of neurons through genetic approaches. The lower costs of lasers and microscope components have also increased the use of calcium imaging

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方案

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1. Calcium imaging

  1. Fly larvae preparation
    NOTE: Flies and larvae are maintained at 25 °C under a 12 h:12 h light:dark cycle.
    1. Anesthetize the flies with CO2. Sort 20-45 males and 20-45 females into each fly vial, and give them at least 24 h to 48 h to recover from the CO2 exposure.
      NOTE: Fly exposure to CO2 should last for the shortest amount of time possible.
    2. To synchronize the larvae age, tap over the flies into new vials containing yeast granules, and allow them 4-8 h to lay eggs. Remove the flies by flipping them into new vials.
    3. Collect the larvae at 72 h usin....

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结果

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Workflow of 3D calcium imaging analysis
In this study, we developed a new ImageJ plugin, TACI, and described a workflow to track z-drift and analyze 3D calcium imaging that pinpoints the responses of individual cells appearing in multiple z-positions (Figure 1). This tool has four functions: RENAME, ORGANIZE, EXTRACT, and MERGE. First, if the image names are not compatible with the ORGANIZE

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讨论

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This study developed a new ImageJ plugin, TACI, and described a workflow analyzing 3D calcium imaging. Many currently available tools focus on correcting the x/y motion, although motion on the z-axis also needs to be explicitly diagnosed or corrected6. During image acquisition in a live organism, movement on the z-axis is unavoidable even when the organism is immobilized, and some stimuli, such as temperature change, often cause significant z-drift. Increasing the height of the z-stacks will allow.......

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披露

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The authors have no conflicts of interest to disclose.

致谢

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A Zeiss LSM 880 in the Fralin Imaging Center was used to collect the calcium imaging data. We acknowledge Dr. Michelle L Olsen and Yuhang Pan for their assistance with the IMARIS software. We acknowledge Dr. Lenwood S. Heath for constructive comments on the manuscript and Steven Giavasis for comments on the GitHub README file. This work was supported by NIH R21MH122987 (https://www.nimh.nih.gov/index.shtml) and NIH R01GM140130 (https://www.nigms.nih.gov/) to L.N. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

....

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材料

本文使用的材料清单
姓名公司目录编号评论
Blunt Fill NeedelBD303129
氯化钙二水合物 Fisher Scientific 10035-04-8苍蝇食物成分
二氧化碳AirgasUN1013尺寸 200 高压钢瓶
CO2 起泡器套件Genesee59-180
共聚焦显微镜 LSM880蔡司4109002107876000倒置的 Axio Observer Z1，配备 5 个激光器、2 个标准 PMT 检测器、32 通道 GaAsP 检测器、1 个 Airyscan 检测器和 Definite 检测器Focus.2 的。
DAQami 软件测量计算
葡萄糖Genesee62-113果蝇食品成分
果蝇琼脂Genesee66-111苍蝇食品成分
乙醇Decon Labs， Inc.64-17-5飞行食物成分
飞行线：Ir21a-Gal4Dr. Paul Garrity 实验室善意的礼物
飞行线：Ir21a-Gal80Dr. Lina Ni 实验室
飞行线：Ir68a-Gal4Dr. Aravinthan DT Samuel 实验室善意的礼物
飞行线：Ir93a-Gal4Dr. Paul Garrity 实验室善意的礼物
Fly line： UAS-GCaMP6Bloomington 果蝇库存中心42750
FlypadGenesee59-114
通用锻造黄铜调节器GentecG152
Gibco PBS pH 7.4 （1x）Thermo Fisher Scientific10010-031
绿色果蝇管Genesee59-124
传热化合物MG Chemicals860-60G
散热器Digi-Key ElectronicsATS2193-ND调整大小至 12.9 x 5.5 厘米
照明器AmScopeLED-6W
非活性干酵母Genesee62-108果蝇食品成分
培养箱PervicalDR-41VL亮：暗周期：12h：12h;温度：25 &°C;C;湿度：40-50% RH。
Thermo Scientific126965000苍蝇食品成分
微型盖板玻璃VWR 48382-12622 x 40 mm
显微镜载玻片Fisher Scientific 12-544-225 x 75 x 1.0 mm
指甲油Kleancolor
窄型果蝇样品瓶Genesee32-113RL
物镜 蔡司420852-9871-000LD LCI Plan-Apochromat 25x/0.8 Imm Corr DIC M27
Peltier 冷却模块TE TechnologyTE-127-1.0-0.830 x 30 mm
插头Genesee49-102
电源电路专家CSI1802X10 伏直流 2.0 安培线性工作台电源
Princeton Artist Brush NepturePrinceton Artist Brush Co.4750 系列，尺寸 2
石酸钠钾四水合Thermo Scientific033241-36果蝇食品成分
阶段插页 Wienecke 和 Sinske432339-9030-000
立体显微镜OlympusSZ61任何立体显微镜都可以工作
T-FittingGenesee59-123
热电偶数据采集设备测量计算USB-2001-TC单通道
热电偶微探针物理传感器IT-24P 
黄色玉米面Genesee62-101苍蝇食品成分
Z 轴压电载物台Wienecke 和 Sinske432339-9000-000
酒物

参考文献

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  1. Grienberger, C., Konnerth, A. Imaging calcium in neurons. Neuron. 73 (5), 862-885 (2012).
  2. Nakai, J., Ohkura, M., Imoto, K. A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein. Nature Biotechnology. 19 (2), 137-141 (2001).
  3. Zhang, Y.....

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