使用光谱聚焦显微镜进行高复合成像,以最大限度地减少非特异性组织光.
Saven Denha1, Vitoria M Olyntho1, Jake Colautti1
1McMaster Immunology Research Centre, Department of Medicine, Faculty of Health Sciences, McMaster University; Schroeder Allergy and Immunology Research Institute, Faculty of Health Sciences, McMaster University.
Journal of visualized experiments : JoVE
|November 17, 2025
概括
频谱代漂白扩展多重性 (IBEX) 通过减少背景噪声和自光来增强高度多重化的成像. 这种优化的技术允许在具有挑战性的组织中进行详细的空间蛋白质组分析.
科学领域:
- 生物医学成像技术 生物医学成像技术
- 蛋白质组学是指蛋白质组学.
- 免疫组织化学 免疫组织化学
背景情况:
- 多复合成像对于研究细胞功能和本地组织环境中的相互作用至关重要.
- 区分真标记表达与背景噪声对于在光显微镜中准确解释数据至关重要.
- 传统的多重成像技术可能受到组织自光和非特异性染色的限制.
研究的目的:
- 适应Iterative Bleaching Extends Multiplexity (IBEX) 协议,以改善信号与背景的比率和减少自发光.
- 开发一个强大的工作流程,在复杂组织中进行高维,空间分辨的蛋白质组分析.
- 在具有挑战性的组织模型中证明光谱IBEX的疗效,例如人类鼻.
主要方法:
- 整合光谱对焦检测与计算不混合.
- 结合肝素阻断来最大限度地减少基于电荷的目标外结合.
- 适用于光谱成像的循环免疫标记和光体失活 (IBEX) 协议.
主要成果:
- 信号与背景比的显著改善和抑制组织自光.
- 与传统方法相比,最大限度地减少了光谱透射和整体采集时间.
- 成功对人类鼻息肉组织中的26个标记物进行成像,捕获结构,免疫和细胞状态信息.
结论:
- 光谱IBEX为多重成像提供了一个强大的和广泛适用的战略.
- 最优化的方法特别适用于具有高自光和非特异性染色的组织.
- 这种方法产生了高维,空间解析的蛋白质组数据,这对于理解复杂的组织架构和细胞所必不可少.
相关概念视频
Confocal Fluorescence Microscopy
19.9K
Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
19.9K
Super-resolution Fluorescence Microscopy
12.1K
Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
12.1K


