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在没有光谱探测器的商用共聚焦显微镜上进行光谱相位成像

Elisa Longo¹, Angelita Costantino^2,3,4, Alessio Andreoni⁵

¹Department of Physics and Astronomy "Ettore Majorana", University of Catania, Via S. Sofia, 64-95123, Catania, Italy.

Scientific reports

|August 26, 2025

概括

这项研究引入了对焦显微镜的新光谱成像方法,提高了活细胞成像的速度和光子效率. 四通道光谱相位分析允许详细的光谱分离和光物种的识别.

科学领域:

生物物理
显微镜
光谱学

背景情况:

光谱成像对于分析光探测器和识别物种至关重要.
在共聚焦显微镜中,传统的兰巴扫描模式存在时间分辨率差和光子低效,限制了活样本分析.
需要改进的光谱成像技术,与标准的共焦显微镜相兼容.

研究的目的:

开发和验证使用4通道同时采集和光谱相位分析的新型光谱成像方法.
在时间分辨率和光子效率方面证明该方法的性能比兰巴扫描模式更好.
展示该方法在活生物样本中分析环境敏感探针,光谱分离和多色成像的应用.

主要方法:

在商用共聚焦激光扫描显微镜上实施4通道同步图像采集系统.
将光谱相位分析应用于获得的四通道数据以进行光谱表征.
使用环境敏感的染料 (ACDAN,尼罗河红色) 和光蛋白 (mEmerald,EYFP) 进行方法验证.
在活细胞和有机体中进行光谱分离和多色成像.

主要成果:

与兰巴扫描模式相比,四通道光谱相机方法提供了更高的光子效率和时间分辨率.
该技术可以在亚细胞区之间辨别小到5nm的光谱发射变化.
成功将尼罗河红色信号分解成两个组成部分,并使活体器官中实现三色成像.
证明了mEmerald和EYFP等光蛋白的重叠辐射光谱的有效分离.

结论:

四通道光谱相位分析是标准共聚焦显微镜的可行和高效的光谱成像技术.
这种方法通过使高分辨率光谱分析和多色成像显著推进生物系统的研究.
该技术为光谱分离和识别具有重叠光谱的光物种提供了强大的工具.

关键词:

在 ACDAN 聚焦显微镜对环境敏感的探测器光蛋白尼罗河红色频谱相子

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