Abstract
Mean-Shift Super Resolution (MSSR) is a principle based on the Mean Shift theory that extends spatial resolution in fluorescence images, beyond the diffraction limit. MSSR works on low- and high-density fluorophore images, is not limited by the architecture of the detector (EM-CCD, sCMOS, or photomultiplier-based laser scanning systems) 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 series, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other analytical super resolution image approaches. Altogether, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
The changes to the manuscript are the following: Main manuscript New figures: - Figure 3: sf-MSSRn extends spatial resolution in confocal microscopy, - Figure 4: sf-MSSRn enhances the resolution and contrast of Airy scan and SIM reconstructions. - Figure 6: sf-MSSRn enhances spatial resolution in STED microscopy. - Figure 9: Showcase of MSSR wide-range fluorescence microscopy applications. Other figures have been updated. Supplementary material A description of Fourier interpolation and its comparison with bicubic interpolation (see section 6.2). otencial adaptation of MSSR to 3D images has been added to Supplementary (see section 11). New supplementary figures: - Supplementary Figure S1: Behavior of ks according to the distance in a neighborhood. - Supplementary Figure S8: Selection of spatial parameter hs related to FWHM. - Supplementary Figure S13: Thresholding and normalizing processes are not enough to overcome the Sparrow limit. - Supplementary Figure S14: Resolution increase provided by deconvolution methods, radiality maps and sf-MSSRn. - Supplementary Figure S23: Fourier interpolation in 2D. - Supplementary Figure S24: sf-MSSR processing times for bicubic and Fourier interpolations. - Supplementary Figure S25: Comparison between Fourier and bicubic interpolation for temporal analysis on experimental dataset. - Supplementary Figure S37: Nanoscopic, single-frame, live-cell imaging of microtubule dynamics in LLC-PK1 cells. - Supplementary Figure S41: Comparison DL images of sf-MSSR reconstruction of epifluorescence BPAE cells for 2D and 3D images. - Supplementary Figure S44: Temporal fluctuation characteristics of two GattaPaint Nanorulers. Most of the figures have been updated with the mpl-infierno LUT using FIJI/ImageJ. Manual Figures 3, 5, 6, 8 and 9 have been updated. Movies New movies: - Supplementary Movie 10: sf-MSSR video of live LLC-PK1 cells expressing mEmerald-EB3. - Supplementary Movie 11: sf-MSSR video of an apoptotic LLC-PK1 cell expressing mEmerald-EB3. - Supplementary Movie 13: DL reconstruction of epifluorescence BPAE cells for 2D and 3D images. - Supplementary Movie 14: sf-MSSR1 reconstruction of epifluorescence BPAE cells for 2D and 3D images. - Other movies have been updated. MSSR Plugin Fiji/ImageJ and other codes With the new changes MSSR Plugin has been updated to version 2.0.0. "PSFp" parameter has been redefined as "FWHM of PSF". An option to select the type of interpolation has been added. An option related to perform intensity normalization on sequence of images has been added. Codes for Matlab, R, and Python have been updated according to the new changes on the MSSR theory.