Mirror-enhanced super-resolution microscopy

Abstract

Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~ 100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen. With no additional complexity, the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED, which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments. The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens, which cannot tolerate high laser power.

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

“…Adopting an approach similar to ISO, Xi and colleagues utilized a simpler system to obtain the similar PSF‐shrinking effect; they referred to this technique as mirror‐enhanced axial‐narrowing super‐resolution microscopy (MEANS) (Figure g) . Unlike ISO, it is not necessary to shape the incident light to form two foci in MEANS, and the mirror is placed just at the focus of the objective lens.…”

“…Moreover, the intensity of the depletion focus generated by MEANS method is also 3.6 times higher than that of conventional STED method. According to the STED resolution formula, the lateral resolution of MEANS‐STED is improved approximately twofold (≈19 nm), which is useful for observing the inner ring structure of the nuclear pore complex and the tubular structure of human respiratory syncytial virus viral filaments; these structures are difficult to be distinguished even using conventional STED and STORM . Further, this technique can be combined with nearly all point‐scanning techniques, such as spinning disk and two‐photon microscopy, to break the axial diffraction limit by replacing the usual glass slide with a mirror (this technique is simpler than 4Pi and ISO).…”

Photon Upconversion Kinetic Nanosystems and Their Optical Response

“…The development of this luminescence kinetics‐based optical encoding strategy is also instructive for developing multiplexing techniques using other cascade luminescent systems that inherently lack multi‐spectral channels, such as triplet‐triplet annihilation molecule pairs . In addition, this multiplexing strategy can potentially benefit applications where non‐spectral “multicolor” strategies are preferred, e.g., in stimulated emission depletion (STED) microscopy, due to the challenge of aligning multiple laser beams …”

Super-resolution microscopy and its applications in neuroscience

“…Recently, Xi et al narrowed the axially PSF of STED by employing the interference between the PSF and its re°ection from a mirror placed behind the specimen. 27 Despite its short time from the invention, STED microscopy has made many contributions to the observation of subcellular structures such as endoplasmic reticulum, 28 mitochondria 29,30 and cytoskeletons. 31,32 Because of complicated components involved in signal transduction between cells, dualcolor STED [33][34][35] and even multi-color STED 36,37 have been established to study several active complexes at the same time.…”