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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...
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超分辨率光显微镜的可交换的HaloTag连接物

Julian Kompa1, Jorick Bruins1, Marius Glogger2

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概括

我们开发了可逆的HaloTag配体 (xHTLs) 用于先进的生物成像. 这些可交换的配体减少光白,并使新的超分辨率显微镜技术如PAINT和MINFLUX成为可能.

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科学领域:

  • 细胞和分子成像
  • 生物技术
  • 超分辨率显微镜

背景情况:

  • 在活细胞中,HaloTag蛋白质标记对于生物成像至关重要.
  • 共价探针不可逆地结合HaloTag,限制了应用,并导致光变白.

研究的目的:

  • 为HaloTag (xHTLs) 引入可交换的配体,使其能够进行可逆的短暂结合.
  • 为了克服不可逆转的共价HaloTag标签的局限性,用于先进的成像.

主要方法:

  • 用于光标记HaloTag (xHTLs) 的可交换配体的开发.
  • 用于多色成像的xHTL与罗达胺染料的合.
  • 在刺激辐射耗尽 (STED) 显微镜,纳米尺度拓图像积分 (PAINT) 和MINFLUX显微镜中应用xHTL.
  • 用于双色超分辨率成像的xHTL和HaloTag突变对的设计.

主要成果:

  • 在STED显微镜中,xHTLs对HaloTag表现出可逆的结合,减少光漂白.
  • 在超分辨率的PAINT和MINFLUX成像中,原性xHTLs有助于暂时结合.
  • 使用xHTL和HaloTag突变体成功展示了双色 PAINT和STED显微镜.

结论:

  • 可交换联体 (xHTL) 为HaloTag标记提供了对共价探针的多功能替代品.
  • 在多个显微镜平台上扩展成像可能性,包括超分辨率技术.
  • 这种方法通过使短暂的结合和减少光漂白来增强生物成像.