bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/158279

NanoJ-SQUIRREL: quantitative mapping and minimisation of super-resolution optical imaging artefacts

Culley, S.·Albrecht, D.·Jacobs, C.·Pereira, P. M.·Leterrier, C.·Mercer, J.·Henriques, R.
View original source ↗Export metadata ↓

Abstract

Most super-resolution microscopy methods depend on steps that contribute to the formation of image artefacts. Here we present NanoJ-SQUIRREL, an ImageJ-based analytical approach providing a quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same focal volume, this approach generates a quantitative map of super-resolution defects, as well as methods for their correction. To illustrate its broad applicability to super-resolution approaches we apply our method to Localization Microscopy, STED and SIM images of a variety of in-cell structures including microtubules, poxviruses, neuronal actin rings and clathrin coated pits. We particularly focus on single-molecule localisation microscopy, where super-resolution reconstructions often feature imperfections not present in the original data. By showing the quantitative evolution of data quality over these varied sample preparation, acquisition and super-resolution methods we display the potential of NanoJ-SQUIRREL to guide optimization of superresolution imaging parameters.

Source connections

Explore related subjects

biophysicsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Culley, S., Albrecht, D., Jacobs, C., Pereira, P. M., Leterrier, C., Mercer, J., Henriques, R.. 2017-07-02. NanoJ-SQUIRREL: quantitative mapping and minimisation of super-resolution optical imaging artefacts. https://doi.org/10.1101/158279

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Low-pass filtering revisited: ramped phase randomization for cryo-EM map comparison and data augmentation

Conventional low-pass filters attenuate or remove high-frequency Fourier amplitudes but leave the phases intact wherever the amplitude response stays above zero. As a result, fine-scale detail of the original signal can persist beyond the target resolution. A sharp cutoff removes this detail but produces ringing artifacts. To address both limitations, this work introduces ramped phase randomization, which extends a conventional low-pass filter by adding random offsets to Fourier phases that increase smoothly with spatial frequency. For cryo-EM maps, the correlation between a map at the target resolution and an ideal, noiseless map sets the levels of the phase offsets, with a correction for the finite resolution of the input map. On three published map pairs, ramped phase filtering reduced the higher resolution parent's fine detail to a level similar to that of independently determined maps at the lower target resolution, whereas amplitude-only filtering kept the parent's sharp features. The FSCs between the filtered maps and their parents closely resembled those of the experimental maps. The same filter also serves as a data augmentation approach for neural network-based machine learning. With ramped phase randomization, each augmented copy keeps the power spectrum and low-frequency content of the original example but has a different high-frequency pattern. For simulated CryoBench particles of 16 ribosome assembly states, phase augmentation raised mean test accuracy in all 20 combinations of training-set size and latent dimension, by 20.8 percentage points on average (range from 3.3 points to 44.2 points). On two general deep learning benchmarks, mean test accuracy rose from 92.15% to 96.99% with 60 labelled UCI Synthetic Control traces and from 53.44% to 74.27% with 500 labelled noisy MNIST images. The ramped phase randomization is implemented in an open-source Python package, with a companion browser application for interactive visualization of cryo-EM maps and filtering effects.

biophysics↗
bioRxiv2026

Kinetic gating by an iron sulfur chain modulates flavin radical-pair spin chemistry in mitochondrial Complex I

Mitochondrial Complex I generates reactive oxygen species at its flavin cofactor, where spin dependent reaction pathways have been proposed. Models of this chemistry treat the flavin oxygen radical pair in isolation, although the flavin is wired to a chain of eight iron sulfur clusters. Here we model the pair with this chain using the human Complex I structure and open quantum system models of spin coupling and electron transfer. In the simulated network, spin coherence decays rapidly along the chain, whereas proximal clusters alter the calculated radical-pair singlet yield. Their influence is kinetically gated: a downstream cluster affects the modelled yield when its electron-transfer rate competes with radical pair recombination, a threshold robust to driving force and to the transfer model. Modelling does set the sign: one directional spin hops predict suppression and inflate coherence transport, whereas charge resolved transfer drives the singlet yield towards its spin-randomized limit unless triplet recombination dominates. Detectable spin dependent effects in this framework require superoxide spin relaxation to be substantially slower than in free solution, a condition not yet established for Complex I.

biophysics↗
bioRxiv2026

Semidilute Polymer Scaling Controls PEG-induced Protein Phase Separation

When does polyethylene glycol (PEG) mass concentration provide a common measure of crowding strength across polymer chain lengths? Here we use bovine serum albumin (BSA) and PEG as a controlled depletion system and measure both coexisting phases to determine binodals, tie lines, and critical points across PEG molecular weights of 4--100 kDa. Phase diagrams for PEG 20, 35, and 100 kDa approximately collapse in mass-concentration coordinates, consistent with semidilute polymer scaling. Generalized free volume theory (GFVT) describes the BSA binodals and tie lines. It also approximately describes binodals for three additional globular proteins in PEG 35K, extending the comparison across proteins spanning 7.2--66.4 kDa. For the binary PEG mixtures tested, GFVT approximates the binodals using an effective polymer size corresponding to the weight-average molecular weight. These results identify a regime in which PEG mass concentration (g/L) provides a transferable measure of depletion-induced crowding.

biophysics↗