Name: automation of the micronucleus assay using imaging flow cytometry and artificial intelligence
Uploaded: 2023-01-27T13:00:00
Description: Watch this Scientific Journal Video about Automation of the Micronucleus Assay Using Imaging Flow Cytometry and Artificial Intelligence at JoVE.com

1. 使用成像流式细胞术采集数据
注意：请参考Rodrigues等人16 进行以下修改，注意使用IFC的采集区域可能需要修改以获得最佳图像捕获：
<ol><li>对于非Cyt-B方法，按照制造商的说明（参见 材料表）在培养前和恢复期后立即对每种培养物进行细胞计数。</li>
	<li>如果在单相机成像流式细胞仪上运行样品，请将明场（BF）置?...

<ol>
	<li>Fenech, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HUMN+project+initiative+and+review+of+validation,+quality+control+and+prospects+for+further+development+of+automated+micronucleus+assays+using+image+cytometry+systems.">HUMN project initiative and review of validation, quality control and prospects for further development of automated micronucleus assays using image cytometry systems.</a> International Journal of Hygiene and Environmental Health. 216 (5), 541-552 (2013).</li><li>OECD. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Test+No.+487:+In+Vitro+Mammalian+Cell+Micronucleus+Test.+Section+4.">Test No. 487: In Vitro Mammalian Cell Micronucleus Test. Section 4.</a> OECD Guidelines for the Testing of Chemicals. , (2016).</li><li>Fenech, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+micronucleus+technique.">The in vitro micronucleus technique.</a> Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 455 (1), 81-95 (2000).</li><li>Bonassi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+increased+micronucleus+frequency+in+peripheral+blood+lymphocytes+predicts+the+risk+of+cancer+in+humans.">An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans.</a> Carcinogenesis. 28 (3), 625-631 (2007).</li><li>Fenech, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokinesis-block+micronucleus+cytome+assay.">Cytokinesis-block micronucleus cytome assay.</a> Nature Protocols. 2 (5), 1084-1104 (2007).</li><li>Fenech, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commentary+on+the+SFTG+international+collaborative+study+on+the+in+vitro+micronucleus+test:+To+Cyt-B+or+not+to+Cyt-B.">Commentary on the SFTG international collaborative study on the in vitro micronucleus test: To Cyt-B or not to Cyt-B.</a> Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 607 (1), 9-12 (2006).</li><li>Seager, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations,+evaluation+and+validation+of+a+semi-automated,+fluorescent-based+scoring+protocol+for+micronucleus+testing+in+human+cells.">Recommendations, evaluation and validation of a semi-automated, fluorescent-based scoring protocol for micronucleus testing in human cells.</a> Mutagenesis. 29 (3), 155-164 (2014).</li><li>Rossnerova, A., Spatova, M., Schunck, C., Sram, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+scoring+of+lymphocyte+micronuclei+by+the+MetaSystems+Metafer+image+cytometry+system+and+its+application+in+studies+of+human+mutagen+sensitivity+and+biodosimetry+of+genotoxin+exposure.">Automated scoring of lymphocyte micronuclei by the MetaSystems Metafer image cytometry system and its application in studies of human mutagen sensitivity and biodosimetry of genotoxin exposure.</a> Mutagenesis. 26 (1), 169-175 (2011).</li><li>Bryce, S. M., Bemis, J. C., Avlasevich, S. L., Dertinger, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+micronucleus+assay+scored+by+flow+cytometry+provides+a+comprehensive+evaluation+of+cytogenetic+damage+and+cytotoxicity.">In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity.</a> Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 630 (1), 78-91 (2007).</li><li>Avlasevich, S. L., Bryce, S. M., Cairns, S. E., Dertinger, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+micronucleus+scoring+by+flow+cytometry:+Differential+staining+of+micronuclei+versus+apoptotic+and+necrotic+chromatin+enhances+assay+reliability.">In vitro micronucleus scoring by flow cytometry: Differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability.</a> Environmental and Molecular Mutagenesis. 47 (1), 56-66 (2006).</li><li>Basiji, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+Amnis+imaging+flow+cytometry.">Principles of Amnis imaging flow cytometry.</a> Methods in Molecular Biology. 1389, 13-21 (2016).</li><li>Rodrigues, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automation+of+the+in+vitro+micronucleus+assay+using+the+Imagestream&#174;+imaging+flow+cytometer.">Automation of the in vitro micronucleus assay using the Imagestream&#174; imaging flow cytometer.</a> Cytometry Part A. 93 (7), 706-726 (2018).</li><li>Verma, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+FlowSight&#174;+imaging+flow+cytometry+as+a+platform+to+assess+chemically+induced+micronuclei+using+human+lymphoblastoid+cells+in+vitro.">Investigating FlowSight&#174; imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro.</a> Mutagenesis. 33 (4), 283-289 (2018).</li><li>Wills, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inter-laboratory+automation+of+the+in+vitro+micronucleus+assay+using+imaging+flow+cytometry+and+deep+learning.">Inter-laboratory automation of the in vitro micronucleus assay using imaging flow cytometry and deep learning.</a> Archives of Toxicology. 95 (9), 3101-3115 (2021).</li><li>Rodrigues, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+micronucleus+assay+using+imaging+flow+cytometry+and+deep+learning.">The in vitro micronucleus assay using imaging flow cytometry and deep learning.</a> Npj Systems Biology and Applications. 7 (1), 20 (2021).</li><li>Rodrigues, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+method+to+perform+the+in+vitro+micronucleus+assay+using+multispectral+imaging+flow+cytometry.">An automated method to perform the in vitro micronucleus assay using multispectral imaging flow cytometry.</a> Journal of Visualized Experiments. (147), e59324 (2019).</li><li>Lovell, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+negative+historical+control+group+data+from+the+in+vitro+micronucleus+assay+using+TK6+cells.">Analysis of negative historical control group data from the in vitro micronucleus assay using TK6 cells.</a> Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 825, 40-50 (2018).</li><li>Berg, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ilastik:+interactive+machine+learning+for+(bio)image+analysis.">ilastik: interactive machine learning for (bio)image analysis.</a> Nature Methods. 16 (12), 1226-1232 (2019).</li><li>Hennig, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open-source+solution+for+advanced+imaging+flow+cytometry+data+analysis+using+machine+learning.">An open-source solution for advanced imaging flow cytometry data analysis using machine learning.</a> Methods. 112, 201-210 (2017).</li></ol>

这里介绍的工作描述了使用深度学习算法来自动对 MN 测定进行评分。最近的几篇出版物表明，直观的交互式工具允许创建深度学习模型来分析图像数据，而无需深入的计算知识18，19。这项工作中描述的协议使用用户界面驱动的软件包，旨在很好地处理非常大的数据文件，并允许轻松创建深度学习模型。讨论了在AI软件包中创建和训练RF和CNN模型的所有?...

微核 （MN） 测定是遗传毒理学的基础，用于评估人类使用的化妆品、药品和化学品开发中的 DNA 损伤1，2，3，4。微核由整个染色体或染色体片段形成，这些染色体或染色体片段在分裂后不会合并到细胞核中，并凝结成与细胞核分离的小圆形体。因此，MN可用作量化遗传毒性检测中DNA损伤的终点1。
定量 MN 的首选方法是通过使用细胞松弛素-B （Cyt-B） 阻断分裂在一次分裂的双核细胞 （BNC） 内。在此版本的测定中，还通过评分单核（MONO）和多核（POLY）细胞来评估细胞毒性。该测定也可以通过在未阻塞的单核细胞中对MN进行评分来进行，该评分更快，更容易评分，并使用暴露前和暴露后的细胞计数来评估细胞毒性以评估增殖5，6。
历来通过手动显微镜进行检测的物理评分，因为这允许目视确认所有关键事件。然而，手动显微镜具有挑战性且主观1。因此，已经开发了自动化技术，包括显微镜载玻片扫描和流式细胞术，每种技术都有自己的优点和局限性。虽然玻片扫描方法允许可视化关键事件，但必须以最佳细胞密度创建载玻片，这可能难以实现。此外，这种技术通常缺乏细胞质可视化，这可能会影响单核细胞和POLY细胞7，8的评分。虽然流式细胞术提供高通量数据采集，但必须裂解细胞，因此不允许使用Cyt-B形式的测定。此外，作为一种非成像技术，传统的流式细胞术不能提供关键事件的视觉验证9，10。
因此，已经研究了成像流式细胞术（IFC）来执行MN测定。ImageStreamX Mk II 将传统流式细胞术的速度和统计稳健性与显微镜的高分辨率成像功能结合在一个系统中11.研究表明，通过使用IFC，可以使用基于特征的12，13或人工智能（AI）技术14，15捕获并自动评分所有关键事件的高分辨率图像。通过使用IFC进行MN测定，与显微镜相比，可以在更短的时间内自动对更多的细胞进行评分。
这项工作偏离了前面描述的图像分析工作流程16 ，并讨论了使用Amnis AI软件（以下简称“AI软件”）开发和训练随机森林（RF）和/或卷积神经网络（CNN）模型所需的所有步骤。描述了所有必要的步骤，包括使用人工智能辅助标记工具填充真实数据，解释模型训练结果，以及应用模型对其他数据进行分类，允许计算遗传毒性和细胞毒性15。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleanser - Coulter Clenz&#160;</td>
			<td>Beckman Coulter</td>
			<td>8546931</td>
			<td>Fill container with 200 mL of Cleanser.&#160; https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=8546931#2/10//0/25/ 
			1/0/asc/2/8546931///0/1//0/</td>
		</tr>
		<tr>
			<td>Colchicine</td>
			<td>MilliporeSigma</td>
			<td>64-86-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning bottle-top vacuum filter&#160;</td>
			<td>MilliporeSigma</td>
			<td>CLS430769</td>
			<td>0.22 &#181;m filter, 500 mL bottle</td>
		</tr>
		<tr>
			<td>Cytochalasin B</td>
			<td>MilliporeSigma</td>
			<td>14930-96-2</td>
			<td>5 mg bottle</td>
		</tr>
		<tr>
			<td>Debubbler - 70% Isopropanol</td>
			<td>MilliporeSigma</td>
			<td>1.3704</td>
			<td>Fill container with 200 mL of Debubbler.&#160; http://www.emdmillipore.com/US/en/product/2-Propanol-70%25-%28V%2FV%29-0.1-%C2%B5m-filtred,MDA_CHEM-137040?ReferrerURL=https%3A%2F%2Fwww.google.com%2F</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>MilliporeSigma</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline 1X</td>
			<td>EMD Millipore</td>
			<td>BSS-1006-B</td>
			<td>PBS Ca++MG++ Free&#160;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde, 10%, methanol free, Ultra Pure</td>
			<td>Polysciences, Inc.</td>
			<td>04018</td>
			<td>This is what is used for the 4% and 1% Formalin. CAUTION: Formalin/Formaldehyde toxic by inhalation and if swallowed.&#160; Irritating to the eyes, respiratory systems and skin.&#160; May cause sensitization by inhalation or skin contact. Risk of serious damage to eyes.&#160; Potential cancer hazard.&#160; http://www.polysciences.com/default/catalog-products/life-sciences/histology-microscopy/fixatives/formaldehydes/formaldehyde-10-methanol-free-pure/</td>
		</tr>
		<tr>
			<td>Guava Muse Cell Analyzer</td>
			<td>Luminex</td>
			<td>0500-3115</td>
			<td>A standard configuration Guava Muse Cell Analyzer was used.</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher</td>
			<td>H3570</td>
			<td>10 mg/mL solution</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>MilliporeSigma</td>
			<td>69-65-8</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids 100X</td>
			<td>HyClone</td>
			<td>SH30238.01</td>
			<td></td>
		</tr>
		<tr>
			<td>MIFC - ImageStreamX Mark II</td>
			<td>Luminex, a DiaSorin company</td>
			<td>100220</td>
			<td>A 2 camera ImageStreamX Mark II eqiped with the 405 nm, 488 nm, and 642 nm lasers was used.</td>
		</tr>
		<tr>
			<td>MIFC analysis software - IDEAS</td>
			<td>Luminex, a DiaSorin company</td>
			<td>100220</td>
			<td>&#34;Image analysis sofware&#34; 
			The companion software to the MIFC (ImageStreamX MKII)</td>
		</tr>
		<tr>
			<td>MIFC software - INSPIRE</td>
			<td>Luminex, a DiaSorin company</td>
			<td>100220</td>
			<td>&#34;Image acquisition software&#34; 
			This is the software that runs the MIFC (ImageStreamX MKII)</td>
		</tr>
		<tr>
			<td>Amnis AI software</td>
			<td>Luminex, a DiaSorin company</td>
			<td>100221</td>
			<td>&#34;AI software&#34; 
			This is the software that permits the creation of artificial intelligence models to analyze data</td>
		</tr>
		<tr>
			<td>Mitomycin C</td>
			<td>MilliporeSigma</td>
			<td>50-07-7</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA Mixture 100x</td>
			<td>Lonza BioWhittaker</td>
			<td>13-114E</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicllin/Streptomycin/Glutamine solution 100X</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>MilliporeSigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Rinse - Ultrapure water or deionized water</td>
			<td>NA</td>
			<td>NA</td>
			<td>Use any ultrapure water or deionized water.&#160; Fill container with 900 mL of Rinse.</td>
		</tr>
		<tr>
			<td>RNase</td>
			<td>MilliporeSigma</td>
			<td>9001-99-4</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium 1x</td>
			<td>HyClone</td>
			<td>SH30027.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheath - PBS</td>
			<td>MilliporeSigma</td>
			<td>BSS-1006-B</td>
			<td>This is the same as Dulbecco&#39;s Phosphate Buffered Saline 1x&#160; Ca++MG++ free.&#160; Fill container with 900 mL of Sheath.</td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>HyClone</td>
			<td>SH30529.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizer - 0.4%&#8211;0.7% Hypochlorite</td>
			<td>VWR</td>
			<td>JT9416-1</td>
			<td>This is assentually 10% Clorox bleach that can be made by deluting Clorox bleach with water.&#160; Fill container with 200 mL of Sterilzer.</td>
		</tr>
		<tr>
			<td>T25 flask</td>
			<td>Falcon</td>
			<td>353109</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Falcon</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>TK6 cells</td>
			<td>MilliporeSigma</td>
			<td>95111735</td>
			<td></td>
		</tr>
	</tbody>
</table>

automation of the micronucleus assay using imaging flow cytometry and artificial intelligence

微核（MN）测定在全球范围内被监管机构用于评估化学品的遗传毒性。该测定可以通过两种方式进行：在一次分裂、细胞分裂阻断的双核细胞或完全分裂的单核细胞中对 MN 进行评分。从历史上看，光学显微镜一直是对测定进行评分的金标准方法，但它既费力又主观。近年来，流式细胞术已被用于对测定进行评分，但由于无法直观地确认细胞图像的关键方面而受到限制。成像流式细胞术（IFC）结合了高通量图像捕获和自动图像分析，已成功应用于快速获取MN检测中所有关键事件的图像并对其进行评分。最近，已经证明，基于卷积神经网络的人工智能（AI）方法可用于对IFC获取的MN测定数据进行评分。本文介绍了使用 AI 软件创建深度学习模型以对所有关键事件进行评分并应用此模型自动对其他数据进行评分的所有步骤。AI 深度学习模型的结果与手动显微镜相比效果很好，因此可以通过结合 IFC 和 AI 实现 MN 检测的全自动评分。

图 1 显示了使用 AI 软件创建 MN 检测模型的工作流程。用户将所需的 .daf 文件加载到 AI 软件中，然后使用 AI 辅助集群（图 2）和预测（图 3）标记算法将对象分配给真实模型类。一旦所有真实模型类都填充了足够的对象，就可以使用 RF 或 CNN 算法训练模型。训练后，可以使用包括类分布直方图、准确性统计和交互式混淆矩阵在内?...

Watch this Scientific Journal Video about Automation of the Micronucleus Assay Using Imaging Flow Cytometry and Artificial Intelligence at JoVE.com

Automation of the Micronucleus Assay Using Imaging Flow Cytometry and Artificial Intelligence

微核 （MN） 测定是量化 DNA 损伤的成熟测试。然而，使用常规技术（如手动显微镜或基于特征的图像分析）对测定进行评分既费力又具有挑战性。本文描述了开发人工智能模型以使用成像流式细胞术数据对 MN 测定进行评分的方法。

使用成像流式细胞术和人工智能实现微核检测自动化

The micronucleus (MN) assay is used worldwide by regulatory bodies to evaluate chemicals for genetic toxicity. The assay can be performed in two ways: by ...

使用成像流式细胞术进行微核测定克服了传统方法的许多局限性，包括通量低、分数可变性和缺乏事件的视觉确认。该技术的主要优点是可以使用成像流式细胞术获取所有关键事件，并使用人工智能进行分析。该方法有可能用于化学品和其他化合物的大规模筛选，以比目前更高的通量测试毒性。在构建新的AI模型时，主要挑战是获得足够的微核细胞图像，因此使用图像标记算法非常重要。首先，启动人工智能或人工智能软件。在“实验类型”下，单击“训练”旁边的单选按钮以启动用于构建卷积神经网络或 CNN 模型的训练实验，然后单击“下一步”。在“类名”上，单击“添加”。在弹出窗口中，键入单核，然后单击“确定”将单核类添加到类名列表中。重复此过程以添加其他类名称，例如 MN 单核、双核、MN 双核、多核和不规则形态。在“选择文件”下，单击“添加文件”，然后浏览要添加到 AI 软件的所需文件，以构建真实数据。接下来，在“选择基本种群”屏幕上，从种群层次结构中找到“非凋亡种群”。然后右键单击非凋亡群体，选择选择所有匹配群体，然后单击下一步。要分配具有微核的单核细胞的标记真实群体，请单击左侧模型类下的 MN 单核类，然后单击右侧的相应标记真实群体。分配所有适当的真值总体后，单击下一步。在“选择通道”屏幕上，确保为实验选择了适当的通道。在这里，选择明场或BF作为通道一，选择DNA群作为通道七，然后单击下一步。最后，在“确认”屏幕上，单击“创建实验”。单击标记以启动标记工具界面。然后单击缩放工具裁剪图像以便于查看。然后单击滑块以调整图像大小并确定要在图库中显示的图像数量。单击“显示设置”选项并选择最小-最大值，这将为识别所有关键事件提供最佳对比度图像。接下来，单击设置库显示将DNA图像的颜色更改为黄色或白色，这将改善小物体的可视化。单击聚类以运行算法，将具有相似形态的图像分组在一起。聚类完成后，单击各个聚类并开始将图像分配给适当的模型类。将至少 25 个对象分配给每个模型类后，预测算法将变为可用。单击预测。预测算法完成运行后，将预测类中的对象添加到相应的模型类。将至少 100 个对象分配给每个模型类后，单击屏幕顶部的“训练”选项卡，然后单击“训练”按钮。模型训练完成后，单击“查看结果”以评估模型准确性。模型训练完成后，单击菜单按钮以定义新实验。在实验类型下，单击分类旁边的单选按钮以启动分类实验，然后单击下一步。单击要用于分类的模型，然后单击“下一步”。在“选择文件”屏幕上，单击“添加文件”。浏览要按 CNN 模型分类的文件，然后单击“下一步”。接下来，在“选择基本种群”屏幕上，单击其中一个加载文件中非凋亡群体旁边的复选框。右键单击“非凋亡群体”，单击“选择所有匹配群体”以从所有加载的文件中选择此群体，然后单击“下一步”。在“选择通道”屏幕上，验证是否为明场选择了通道一，是否为 DNA 染色选择了通道 7，然后单击下一步。最后，在确认屏幕上，单击创建实验。AI软件从所选数据文件中加载所选模型和所有图像。加载后，单击完成。接下来，单击分类以启动分类屏幕。并使用复选框选择使用随机森林或射频以及 CNN。然后点击分类按钮。这将开始使用 RF 和 CNN 模型对其他数据进行分类并识别属于指定模型类的所有对象的过程。分类完成后，单击查看结果。单击更新 DAF 按钮以显示具有分类结果的更新 DAF，然后单击确定以更新 DAF 文件。要生成报告，请在结果屏幕上单击生成报告。如果需要为每个输入 DAF 提供单独的报告，请选中为每个输入 DAF 创建报告旁边的复选框。否则，只需单击“确定”即可获取报告。AI 辅助聚类算法根据未分类对象和已分配给真实对象模型类的对象的形态，将段内的相似对象分组在一起。包含单核细胞的簇落在对象映射的一侧，而多核细胞位于另一侧。双核细胞簇介于单核细胞簇和多核细胞簇之间。最后，具有不规则形态的簇落在对象映射的不同区域。在识别图像中的细微形态方面，预测算法比聚类算法更可靠。例如，具有 MN 的单核细胞与没有 MN 的单核细胞。可以使用工具评估模型的性能，包括类分布直方图、准确性统计和交互式混淆矩阵。在类分布直方图中，真值和预测总体之间的百分比值越接近，模型就越准确。在准确性统计中，这些指标越接近 100%，模型在识别模型类中的事件时就越准确。最后，交互式混淆矩阵指示模型错误分类事件的位置。对于甘露醇、依托泊苷和丝裂霉素 C，使用细胞松弛素 B 和非细胞松弛素 B 方法，通过显微镜测量微核的百分比（用透明条表示）和 AI 用虚线条表示，用透明条表示，AI 在 3 小时暴露和 24 小时恢复后。创建训练实验时，请确保加载的数据包含来自阳性和阴性对照样本的图像。微核很少见，需要足够的图像来构建准确的AI模型。该程序允许创建AI模型来分析任何研究领域的成像流微核数据，例如辐射生物剂量学。一种快速而强大的基于人工智能的微核识别方法可以扩展到其他应用，例如量化可能预测癌症发展风险的微核。

Research

JoVE Journal

Bioengineering

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

科研

生物工程

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

一个手机上的宽视场荧光显微镜和荧光成像流式细胞仪

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

与FRET流式细胞仪灵敏地检测Proteopathic播种活动

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

使用光声流细胞测定卵巢癌检测

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

高维流式细胞术用于解剖种植体组织的免疫功能分析

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Blood Flow Imaging with Ultrafast Doppler

血流成像与超快多普勒

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...