Laboratory for Molecular Photonics, Department of Chemistry, University of Miami, Coral Gables, Florida 33146-0431, United States.
Bioconjug Chem. 2020 Apr 15;31(4):1052-1062. doi: 10.1021/acs.bioconjchem.0c00073. Epub 2020 Mar 18.
Optical diffraction fundamentally limits the spatial resolution of conventional fluorescence images to length scales that are, at least, 2 orders of magnitude longer than the dimensions of individual molecules. As a result, the development of innovative probes and imaging schemes to overcome diffraction is very much needed to enable the investigation of the fundamental factors regulating cellular functions at the molecular level. In this context, the chemical synthesis of molecular constructs with photoactivatable fluorescence and the ability to label subcellular components of live cells can have transformative implications. Indeed, the fluorescence of the resulting assemblies can be activated with spatiotemporal control, even in the intracellular environment, to permit the sequential localization of individual emissive labels with precision at the nanometer level and the gradual reconstruction of images with subdiffraction resolution. The implementation of these operating principles for subdiffraction imaging, however, is only possible if demanding photochemical and photophysical requirements to enable photoactivation and localization as well as stringent structural requisites to allow the covalent labeling of intracellular targets in live cells are satisfied. Because of these complications, only a few synthetic photoactivatable fluorophores with appropriate performance for live-cell imaging at the nanoscale have been developed so far. Significant synthetic efforts in conjunction with spectroscopic analyses are still very much needed to advance this promising research area further and turn photoactivatable fluorophores into the imaging probes of choice for the investigation of live cells.
光学衍射从根本上限制了传统荧光图像的空间分辨率，使其至少比单个分子的尺寸长 2 个数量级。因此，非常需要开发创新的探针和成像方案来克服衍射，以能够在分子水平上研究调节细胞功能的基本因素。在这种情况下，具有光活化荧光的分子结构的化学合成以及标记活细胞亚细胞成分的能力具有变革性的意义。实际上，所得组装体的荧光可以通过时空控制来激活，即使在细胞内环境中也是如此，从而可以精确地在纳米级水平上对单个发射标记进行顺序定位，并以亚衍射分辨率逐渐重建图像。然而，如果要实现这些亚衍射成像的操作原理，则只有满足苛刻的光化学和光物理要求以实现光活化和定位以及严格的结构要求以允许在活细胞中对细胞内靶标进行共价标记的情况下才有可能。由于这些复杂性，迄今为止，仅开发出了少数具有适当性能的用于纳米级活细胞成像的合成光活化荧光染料。为了进一步推进这一很有前途的研究领域并将光活化荧光染料转化为研究活细胞的首选成像探针，仍需要进行大量的合成工作以及光谱分析。
