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用于光遗传学、光激活和多色成像的新型红色荧光钙指示剂。

New red-fluorescent calcium indicators for optogenetics, photoactivation and multi-color imaging.

作者信息

Oheim Martin, van 't Hoff Marcel, Feltz Anne, Zamaleeva Alsu, Mallet Jean-Maurice, Collot Mayeul

机构信息

CNRS, UMR 8154, Paris F-75006, France; INSERM, U603, Paris F-75006, France; University Paris Descartes, PRES Sorbonne Paris Cité, Laboratory of Neurophysiology and New Microscopies, 45 rue des Saints Pères, Paris F-75006, France.

CNRS, UMR 8154, Paris F-75006, France; INSERM, U603, Paris F-75006, France; University Paris Descartes, PRES Sorbonne Paris Cité, Laboratory of Neurophysiology and New Microscopies, 45 rue des Saints Pères, Paris F-75006, France; University of Florence, LENS - European Laboratory for Non-linear Spectroscopy, Via Nello Carrara 1, I-50019 Sesto Fiorentino, Italy.

出版信息

Biochim Biophys Acta. 2014 Oct;1843(10):2284-306. doi: 10.1016/j.bbamcr.2014.03.010. Epub 2014 Mar 27.

Abstract

Most chemical and, with only a few exceptions, all genetically encoded fluorimetric calcium (Ca(2+)) indicators (GECIs) emit green fluorescence. Many of these probes are compatible with red-emitting cell- or organelle markers. But the bulk of available fluorescent-protein constructs and transgenic animals incorporate green or yellow fluorescent protein (GFP and YFP respectively). This is, in part, not only heritage from the tendency to aggregate of early-generation red-emitting FPs, and due to their complicated photochemistry, but also resulting from the compatibility of green-fluorescent probes with standard instrumentation readily available in most laboratories and core imaging facilities. Photochemical constraints like limited water solubility and low quantum yield have contributed to the relative paucity of red-emitting Ca(2+) probes compared to their green counterparts, too. The increasing use of GFP and GFP-based functional reporters, together with recent developments in optogenetics, photostimulation and super-resolution microscopies, has intensified the quest for red-emitting Ca(2+) probes. In response to this demand more red-emitting chemical and FP-based Ca(2+)-sensitive indicators have been developed since 2009 than in the thirty years before. In this topical review, we survey the physicochemical properties of these red-emitting Ca(2+) probes and discuss their utility for biological Ca(2+) imaging. Using the spectral separability index Xijk (Oheim M., 2010. Methods in Molecular Biology 591: 3-16) we evaluate their performance for multi-color excitation/emission experiments, involving the identification of morphological landmarks with GFP/YFP and detecting Ca(2+)-dependent fluorescence in the red spectral band. We also establish a catalog of criteria for evaluating Ca(2+) indicators that ideally should be made available for each probe. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

摘要

大多数化学荧光钙(Ca(2+))指示剂,以及除少数例外的所有基因编码荧光钙指示剂(GECIs)均发出绿色荧光。其中许多探针与发出红色荧光的细胞或细胞器标记物兼容。但是,现有的荧光蛋白构建体和转基因动物大多采用绿色或黄色荧光蛋白(分别为GFP和YFP)。这部分不仅源于早期发出红色荧光的荧光蛋白的聚集倾向及其复杂的光化学性质,还由于绿色荧光探针与大多数实验室和核心成像设施中 readily available的标准仪器的兼容性。诸如有限的水溶性和低量子产率等光化学限制也导致发出红色荧光的Ca(2+)探针相对于绿色探针相对较少。GFP和基于GFP的功能报告基因的使用日益增加,以及光遗传学、光刺激和超分辨率显微镜的最新发展,加剧了对发出红色荧光的Ca(2+)探针的需求。自2009年以来,响应这一需求而开发的发出红色荧光的化学和基于荧光蛋白的Ca(+)敏感指示剂比之前三十年还要多。在本专题综述中,我们调查了这些发出红色荧光的Ca(2+)探针的物理化学性质,并讨论了它们在生物Ca(2+)成像中的效用。使用光谱可分离性指数Xijk(Oheim M.,2010.《分子生物学方法》591:3 - 16),我们评估了它们在多色激发/发射实验中的性能,包括用GFP/YFP识别形态学标志以及在红色光谱带中检测Ca(2+)依赖性荧光。我们还建立了一个评估Ca(2+)指示剂的标准目录,理想情况下应为每个探针提供该目录。本文是名为:健康与疾病中的钙信号传导的特刊的一部分。客座编辑:Geert Bultynck、Jacques Haiech、Claus W. Heizmann、Joachim Krebs和Marc Moreau。

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