Extending resolution within a single imaging frame

Abstract

The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.

Reorganization of the Flagellum Scaffolding Induces a Sperm Standstill During Fertilization

“…In the negative control (no acrosomal exocytosis), the diameter remained unchanged. This phenomenon was also observed using mean shift super-resolution (MSSR) (18). To further support this observation, other plasma membrane probes of different molecular structure, such as Memglow 700 (Figure S2A), Bodipy-GM1 (Figure S2B), and FM1-43 (Figure S2C), were used.…”

Visualizing proteins by expansion microscopy

“…The initial implementation of SRRF resulted in a 50-70 nm resolution 6 , leading to the impression that this is the best achievable resolution for this technique, as it is implied by some subsequent works (e.g. 32 ). This is not the case, as demonstrated in our work on nanorulers ( Supplementary Figs.…”

“…100-300 frames), thus rendering it a method of choice for live-cell SRRF 6, 27 . TRA is heavily dependent on the distance between the fluorophores, and performs best when the different fluorescent objects are separated by more than 70% of the full width at half maximum (FWHM) of the point-spread-function (PSF 32 ). This implies that this procedure is not intended to produce a very high resolution, unlike the TRAC analyses.…”

“…This, however, will result in a lower signal-to-noise ratio (SNR), which is an essential parameter for all fluctuation-based analyses. Even when applied to low numbers of frames, SRRF provided excellent images when the signal-to-noise ratio surpassed 10-15 6, 32 . Below these values, SRRF will perform more poorly than many other related methods, as MUSICAL or ESI 32 , implying that users should consider carefully the noise levels of their images, as explained in Supplementary Fig.…”

“…Working with low frame numbers reduces the achievable resolution, even when working with ExM gels 20, 31 . Second, the signal-to-noise ratio needs to be optimized carefully 32 , as we also demonstrate in Supplementary Fig. 4 .…”