Beyond the diffraction barrier: Lighting nanoscale architecture of nuclear pores
The nuclear pore complex (NPC) is a remarkable molecular machine that plays a critical role in mediating nucleocytoplasmic transport and regulating gene expression.
Its intricate structure, composed of multiple proteins, has long presented a challenge for traditional imaging techniques. In particular, in electron tomography, the reconstruction of individual proteins in the complex is unclear. SMLM addresses this problem by labeling each protein with specific fluorophores and acquiring very high-resolution images at the nanoscale. Combining SMLM imaging together with single-particle averaging leads the precision well below 1 nm.
Specific analysis can then be performed to extract the most relevant parameters.
Here by specifically extracting large cavities, the clathrin coated-pit structure can almost be resolved (Figure .c), a feature that is normally only accessible through electron microscopy.
In other works, the precise geometry of clathrin coated pits at endocytic sites were also investigated through SMLM and statistical analysis [3].
Thus by combining SMLM with clustering, we can distinguish and analyze clathrin structures independently, a process which is also applicable to various samples, such as protein clusters, viruses, bacteria and cells. The relevance of these statistical data is strengthened by Abbelight’s ASTER uniform excitation scheme, which guarantees uniformity between data along the field of view.
