自发荧光寿命流式细胞仪与时间相关单光子计数。

IF 2.5 4区 生物学 Q3 BIOCHEMICAL RESEARCH METHODS
Kayvan Samimi, Ojaswi Pasachhe, Emmanuel Contreras Guzman, Jeremiah Riendeau, Amani A. Gillette, Dan L. Pham, Kasia J. Wiech, Darcie L. Moore, Melissa C. Skala
{"title":"自发荧光寿命流式细胞仪与时间相关单光子计数。","authors":"Kayvan Samimi,&nbsp;Ojaswi Pasachhe,&nbsp;Emmanuel Contreras Guzman,&nbsp;Jeremiah Riendeau,&nbsp;Amani A. Gillette,&nbsp;Dan L. Pham,&nbsp;Kasia J. Wiech,&nbsp;Darcie L. Moore,&nbsp;Melissa C. Skala","doi":"10.1002/cyto.a.24883","DOIUrl":null,"url":null,"abstract":"<p>Autofluorescence lifetime imaging microscopy (FLIM) is sensitive to metabolic changes in single cells based on changes in the protein-binding activities of the metabolic co-enzymes NAD(P)H. However, FLIM typically relies on time-correlated single-photon counting (TCSPC) detection electronics on laser-scanning microscopes, which are expensive, low-throughput, and require substantial post-processing time for cell segmentation and analysis. Here, we present a fluorescence lifetime-sensitive flow cytometer that offers the same TCSPC temporal resolution in a flow geometry, with low-cost single-photon excitation sources, a throughput of tens of cells per second, and real-time single-cell analysis. The system uses a 375 nm picosecond-pulsed diode laser operating at 50 MHz, alkali photomultiplier tubes, an FPGA-based time tagger, and can provide real-time phasor-based classification (i.e., gating) of flowing cells. A CMOS camera produces simultaneous brightfield images using far-red illumination. A second PMT provides two-color analysis. Cells are injected into the microfluidic channel using a syringe pump at 2–5 mm/s with nearly 5 ms integration time per cell, resulting in a light dose of 2.65 J/cm<sup>2</sup> that is well below damage thresholds (25 J/cm<sup>2</sup> at 375 nm). Our results show that cells remain viable after measurement, and the system is sensitive to autofluorescence lifetime changes in Jurkat T cells with metabolic perturbation (sodium cyanide), quiescent versus activated (CD3/CD28/CD2) primary human T cells, and quiescent versus activated primary adult mouse neural stem cells, consistent with prior studies using multiphoton FLIM. This TCSPC-based autofluorescence lifetime flow cytometer provides a valuable label-free method for real-time analysis of single-cell function and metabolism with higher throughput than laser-scanning microscopy systems.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":null,"pages":null},"PeriodicalIF":2.5000,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24883","citationCount":"0","resultStr":"{\"title\":\"Autofluorescence lifetime flow cytometry with time-correlated single photon counting\",\"authors\":\"Kayvan Samimi,&nbsp;Ojaswi Pasachhe,&nbsp;Emmanuel Contreras Guzman,&nbsp;Jeremiah Riendeau,&nbsp;Amani A. Gillette,&nbsp;Dan L. Pham,&nbsp;Kasia J. Wiech,&nbsp;Darcie L. Moore,&nbsp;Melissa C. Skala\",\"doi\":\"10.1002/cyto.a.24883\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Autofluorescence lifetime imaging microscopy (FLIM) is sensitive to metabolic changes in single cells based on changes in the protein-binding activities of the metabolic co-enzymes NAD(P)H. However, FLIM typically relies on time-correlated single-photon counting (TCSPC) detection electronics on laser-scanning microscopes, which are expensive, low-throughput, and require substantial post-processing time for cell segmentation and analysis. Here, we present a fluorescence lifetime-sensitive flow cytometer that offers the same TCSPC temporal resolution in a flow geometry, with low-cost single-photon excitation sources, a throughput of tens of cells per second, and real-time single-cell analysis. The system uses a 375 nm picosecond-pulsed diode laser operating at 50 MHz, alkali photomultiplier tubes, an FPGA-based time tagger, and can provide real-time phasor-based classification (i.e., gating) of flowing cells. A CMOS camera produces simultaneous brightfield images using far-red illumination. A second PMT provides two-color analysis. Cells are injected into the microfluidic channel using a syringe pump at 2–5 mm/s with nearly 5 ms integration time per cell, resulting in a light dose of 2.65 J/cm<sup>2</sup> that is well below damage thresholds (25 J/cm<sup>2</sup> at 375 nm). Our results show that cells remain viable after measurement, and the system is sensitive to autofluorescence lifetime changes in Jurkat T cells with metabolic perturbation (sodium cyanide), quiescent versus activated (CD3/CD28/CD2) primary human T cells, and quiescent versus activated primary adult mouse neural stem cells, consistent with prior studies using multiphoton FLIM. This TCSPC-based autofluorescence lifetime flow cytometer provides a valuable label-free method for real-time analysis of single-cell function and metabolism with higher throughput than laser-scanning microscopy systems.</p>\",\"PeriodicalId\":11068,\"journal\":{\"name\":\"Cytometry Part A\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.5000,\"publicationDate\":\"2024-06-28\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24883\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cytometry Part A\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cyto.a.24883\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cytometry Part A","FirstCategoryId":"99","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cyto.a.24883","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0

摘要

自发荧光寿命成像显微镜(FLIM)根据代谢辅酶 NAD(P)H 蛋白结合活性的变化,对单细胞的代谢变化非常敏感。然而,荧光寿命显微镜通常依赖于激光扫描显微镜上的时间相关单光子计数(TCSPC)检测电子装置,这种装置价格昂贵、吞吐量低,而且需要大量的后处理时间来进行细胞分割和分析。在这里,我们介绍一种荧光寿命敏感流式细胞仪,它能在流式几何结构中提供相同的 TCSPC 时间分辨率,具有低成本的单光子激发光源、每秒数十个细胞的通量以及实时单细胞分析功能。该系统使用 375 nm 皮秒脉冲二极管激光器,工作频率为 50 MHz,配有碱性光电倍增管和基于 FPGA 的时间标记,可对流动细胞进行实时相量分类(即选门)。CMOS 摄像机使用远红照明同时生成明视野图像。第二个 PMT 提供双色分析。使用注射泵以 2-5 毫米/秒的速度将细胞注入微流控通道,每个细胞的整合时间接近 5 毫秒,因此光剂量为 2.65 焦耳/平方厘米,远低于损伤阈值(375 纳米波长下为 25 焦耳/平方厘米)。我们的研究结果表明,细胞在测量后仍能存活,而且该系统对代谢扰动(氰化钠)的 Jurkat T 细胞、静止与活化(CD3/CD28/CD2)的原代人类 T 细胞以及静止与活化的原代成年小鼠神经干细胞的自发荧光寿命变化很敏感,这与之前使用多光子 FLIM 的研究结果一致。这种基于 TCSPC 的自发荧光寿命流式细胞仪为实时分析单细胞功能和代谢提供了一种宝贵的无标记方法,其通量高于激光扫描显微镜系统。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Autofluorescence lifetime flow cytometry with time-correlated single photon counting

Autofluorescence lifetime flow cytometry with time-correlated single photon counting

Autofluorescence lifetime imaging microscopy (FLIM) is sensitive to metabolic changes in single cells based on changes in the protein-binding activities of the metabolic co-enzymes NAD(P)H. However, FLIM typically relies on time-correlated single-photon counting (TCSPC) detection electronics on laser-scanning microscopes, which are expensive, low-throughput, and require substantial post-processing time for cell segmentation and analysis. Here, we present a fluorescence lifetime-sensitive flow cytometer that offers the same TCSPC temporal resolution in a flow geometry, with low-cost single-photon excitation sources, a throughput of tens of cells per second, and real-time single-cell analysis. The system uses a 375 nm picosecond-pulsed diode laser operating at 50 MHz, alkali photomultiplier tubes, an FPGA-based time tagger, and can provide real-time phasor-based classification (i.e., gating) of flowing cells. A CMOS camera produces simultaneous brightfield images using far-red illumination. A second PMT provides two-color analysis. Cells are injected into the microfluidic channel using a syringe pump at 2–5 mm/s with nearly 5 ms integration time per cell, resulting in a light dose of 2.65 J/cm2 that is well below damage thresholds (25 J/cm2 at 375 nm). Our results show that cells remain viable after measurement, and the system is sensitive to autofluorescence lifetime changes in Jurkat T cells with metabolic perturbation (sodium cyanide), quiescent versus activated (CD3/CD28/CD2) primary human T cells, and quiescent versus activated primary adult mouse neural stem cells, consistent with prior studies using multiphoton FLIM. This TCSPC-based autofluorescence lifetime flow cytometer provides a valuable label-free method for real-time analysis of single-cell function and metabolism with higher throughput than laser-scanning microscopy systems.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
Cytometry Part A
Cytometry Part A 生物-生化研究方法
CiteScore
8.10
自引率
13.50%
发文量
183
审稿时长
4-8 weeks
期刊介绍: Cytometry Part A, the journal of quantitative single-cell analysis, features original research reports and reviews of innovative scientific studies employing quantitative single-cell measurement, separation, manipulation, and modeling techniques, as well as original articles on mechanisms of molecular and cellular functions obtained by cytometry techniques. The journal welcomes submissions from multiple research fields that fully embrace the study of the cytome: Biomedical Instrumentation Engineering Biophotonics Bioinformatics Cell Biology Computational Biology Data Science Immunology Parasitology Microbiology Neuroscience Cancer Stem Cells Tissue Regeneration.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信