Novel full-spectral flow cytometry with multiple spectrally-adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement.
Novel full-spectral flow cytometry with multiple spectrally-adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement.
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DOI：
10.1002/cyto.a.22725
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发表时间：
2015-09
期刊：
影响因子：
3.7
通讯作者：
Tomura, Michio
中科院分区：
文献类型：
--
作者：
Futamura, Koji;Sekino, Masashi;Hata, Akihiro;Ikebuchi, Ryoyo;Nakanishi, Yasutaka;Egawa, Gyohei;Kabashima, Kenji;Watanabe, Takeshi;Furuki, Motohiro;Tomura, Michio
关键词：
多色荧光探针流式细胞分析是检测细胞生物学特征的重要方法。在这里，我们提出了一种新的全光谱流式细胞仪（spectral - FCM）。与传统流式细胞仪不同的是，该光谱流式细胞仪采用32通道顺序PMT单元，经棱镜色散后，获取每个细胞全光谱上所有探针发出的荧光，并根据每个荧光探针的光谱形状，采用独特的算法提取每个荧光探针的信号，具有高速、高灵敏、准确、自动和实时的特点。光谱FCM以高光谱分辨率检测光转换蛋白KikGR的发射光谱从绿色到红色的连续变化，并分离光谱相邻的荧光探针，如FITC（发射峰（Em） 519 nm）和EGFP （Em 507 nm）。此外，光谱FCM可以测量和减去每个细胞的自身荧光，提供更高的信噪比和更高的暗样品分辨率，这导致了单细胞状态和功能研究的变革性技术。这些进展使得利用表达KikGR的小鼠进行11色荧光分析来观察多系免疫细胞的运动成为可能。因此，新型的光谱流式细胞术提高了在代谢活性细胞中光谱相邻的各种FPs和多色荧光染料的组合使用，不仅用于免疫系统的研究，还用于其他研究和临床领域的使用。©2015作者。《细胞术》A部分由Wiley期刊公司代表ISAC出版
Flow cytometric analysis with multicolor fluoroprobes is an essential method for detecting biological signatures of cells. Here, we present a new full‐spectral flow cytometer (spectral‐FCM). Unlike conventional flow cytometer, this spectral‐FCM acquires the emitted fluorescence for all probes across the full‐spectrum from each cell with 32 channels sequential PMT unit after dispersion with prism, and extracts the signals of each fluoroprobe based on the spectral shape of each fluoroprobe using unique algorithm in high speed, high sensitive, accurate, automatic and real‐time. The spectral‐FCM detects the continuous changes in emission spectra from green to red of the photoconvertible protein, KikGR with high‐spectral resolution and separates spectrally‐adjacent fluoroprobes, such as FITC (Emission peak (Em) 519 nm) and EGFP (Em 507 nm). Moreover, the spectral‐FCM can measure and subtract autofluorescence of each cell providing increased signal‐to‐noise ratios and improved resolution of dim samples, which leads to a transformative technology for investigation of single cell state and function. These advances make it possible to perform 11‐color fluorescence analysis to visualize movement of multilinage immune cells by using KikGR‐expressing mice. Thus, the novel spectral flow cytometry improves the combinational use of spectrally‐adjacent various FPs and multicolor fluorochromes in metabolically active cell for the investigation of not only the immune system but also other research and clinical fields of use. © 2015 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC
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