Comment on “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics”

Abstract

Li et al. (Research Articles, 28 August 2015, aab3500) purport to present solutions to longstanding challenges in live-cell microscopy, reporting relatively fast acquisition times in conjunction with improved image resolution. We question the methods' reliability to visualize specimen features at sub-100-nanometer scales, because the mandatory mathematical processing of the recorded data leads to artifacts that are either difficult or impossible to disentangle from real features. We are also concerned about the chosen approach of subjectively comparing images from different super-resolution methods, as opposed to using quantitative measures.(1) extend structured illumination microscopy (SIM) (2, 3) by exploiting a higher numerical aperture (NA) lens and using standing light waves to switch reversibly switchable fluorescent proteins (RSFPs). The patterned on-switching of RSFPs yields a spatially controlled distribution of fluorophores in the fluorescent "on" state, adding spatial frequencies of higher order to the fluorescence emission. These higher-order frequencies improve SIM resolution, provided that they can be disentangled from the low-frequency signals. According to Li et al., even moderate illumination intensities lead to higher frequencies of enhanced amplitudes, making their method more applicable to living cells than other super-resolution approaches. Employing Skylan-NS, an apparently quite photostable-but undisclosed-RSFP, recordings of a few tens of images were made. The resolution was reportedly improved to nominally 84 nm by SIM with an NA 1.7 lens and improved to nominally 62 nm when performing the RSFP switching with sinusoidal on-switching and readout patterns. By saturating the on-switching of the fluorescent proteins, Li et al. extended the resolution to nominally 45 nm.

Optimal 2D-SIM reconstruction by two filtering steps with Richardson-Lucy deconvolution

“…4b,e). Similar artifacts have been previously reported and associated to raw images ( O j,m ) with low signal-to-noise ratio and strong out-of-focus signal3412.…”

“…Therefore, further improvement of the SIM reconstruction can be achieved by adjusting the weight of certain frequencies. This step is usually performed by multiplying the spectrum by an apodization function to tune the final appearance of the reconstruction356 (Supplementary Material Figures S3 and S5). In our reconstruction approach we simply deconvolve with an effective point spread function ( PSF SIM ), to equalize the weights of the high frequency and low frequency contents in the final image.…”

“…The out-of-focus background generates periodic patterns in the image background34, while ripples in the image spectrum spuriously highlights or attenuates certain frequencies that translate into side lobe artifacts that diminish the quality of the super-resolved image . Our analysis leads to a reconstruction approach where we implement a pre- and a post-reconstruction filtering steps to minimize the artifacts.…”

“…The apodization function has evolved into a parametrized function whose shape is empirically tuned to enhance the reconstruction and eliminate artifacts. In this sense, the reconstruction has become reliant on the appointed apodization function and is still a heavily debated issue23456789. In our method, we perform two filtering steps with the Richardson-Lucy (RL) deconvolution.…”

Three-dimensional live multi-label light-sheet imaging with synchronous excitation-multiplexed structured illumination

“…When out-of-focus emission or tissue scattering is strong, the residue could generate strip-shape artifacts in final spatial domain images. The phenomenon has been experimentally observed in both superresolution NSIM [22,23] and light-sheet NSIM [10]. Fourier imageR n (k x , k y ), the desired in-focus and un-scattered signal is shifted in frequency, but residue of the background signal remains near the zero frequency.…”