Breaking the Axial Diffraction Limit: A Guide to Axial Super‐Resolution Fluorescence Microscopy

Abstract

Optical microscopy is a powerful tool for understanding the fundamentals of the microscopic world. However, for centuries its resolving ability remained limited by the optical diffraction limit. Super‐resolution fluorescence microscopy (SRFM) has been introduced to break the diffraction limit and significantly expand the fields in which optical microscopy can be applied. Unfortunately, SRFM contributes little towards axial resolution enhancement, rendering observation of the axial and three‐dimensional structures of biological tissues difficult; this may yield a misunderstanding of intracellular interactions. Based on the existing literature, the development of axial SRFM is still behind that of lateral SRFM. In light of this, this Review presents a comprehensive summary of the principles, development, characteristics, and applications of existing techniques for improving the axial resolution. This Review will provide a guide to researchers and promote further development of related technology.

Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy

“…Several techniques have been devised to mitigate this problem, such as 4π detection [4], bi-plane imaging [5,6], phase detection [7], photometry [8] or methods based on PSF engineering [9,10], where the shape of the PSF is altered to provide higher z-precision. An overview of different far-field methods for 3D localization is for instance provided in [11] and a more general overview of axial super-resolution in fluorescence microscopy in [12].…”

Bloch Surface Wave Assisted Structured Illumination Microscopy for Sub-100 nm Resolution

“…For BSW with a transverse wavevector of 1.825k0 in aqueous solutions, the evanescent field penetration depth is about 68 nm, which is smaller than that of TIRF microscopy. This means the optical sectioning [30] capability near the BSW illumination surface could be achieved. This good property makes BSW SIM suitable for studying the dynamic molecular behaviors of proteins bounded by plasma membrane and the labelled biomolecules on plasma membrane in vivo, such as vesicle endocytosis and exocytosis [31,32], plasma membrane heterogeneity [33], protein stoichiometry [34], neuron structure [35] and cell growth [36].…”

A Review on Dual-Lens Fluorescence Microscopy for Three-Dimensional Imaging

“…Specifically, the dual-lens axial SRFM methods have superior axial super resolution but are sensitive to the aberrations induced by the refractive index inhomogeneities within the sample, so they are limited to image fine intracellular structures of relatively thin biological samples like mammalian cells, and the dual-lens LSFM methods have an inferior axial resolution but inherent optical sectioning capability arising from selective plane illumination, and thus can be used for 3D imaging thicker biological samples like mammalian/model animal tissues and embryos. There are other dual-lens fluorescence microscopy methods [67] besides the ones reviewed here, for example, confocal theta fluorescence microscopy [68,69], whose angle between illumination axis and detection axis is typically set to be 90°to improve the axial resolution and thus obtain isotropic 3D resolution. However, its axial resolution can only be increased by 3-4 times, and its lateral resolution is slightly reduced because the effective NA of the two objective lenses is less than that of a single objective lens.…”