Biology subjects

Hung, S.-T.

Publications and source records attributed to Hung, S.-T..

4 recordsLinked to original sources

bioRxiv2025

Patterned illumination enables denser deep-learning based single-molecule localization microscopy

Single-molecule localization microscopy (SMLM) improves resolution beyond diffraction limits by imaging cellular structures at the nanometer scale. Recent advances include using modulation patterns to improve localization precision, and deep learning to accurately process high-density samples with overlapping fluorophore emissions, thus improving imaging speed. A method combining these two advances, SIMCODE, is presented here, allowing high-density modulation-enhanced SMLM. SIMCODE achieved resolution improvements in high-density areas compared to SMLM, deep learning-based SMLM (DECODE), and modulation-enhanced SMLM alone (SIMFLUX). In DNA-PAINT imaging of COS-7 cells, SIMCODE showed improvements in the Fourier Ring Correlation and resolution-scaled Pearson coefficient, with overall improvement increasing as imaging buffer concentration increased five-fold. Modulation-enhanced localization microscopy combined with deep learning thus produced higher quality reconstructions at higher emitter densities (i.e., [~]3x the number of detected spots). This will enable faster imaging, higher labeling densities, and more flexibility in fluorophore choice, which are important for studying dynamic processes and densely labeled structures.

biophysics↗

bioRxiv2024

Using splines for point spread function calibration atnon-uniform depths in localization microscopy

Single-molecule localization microscopy methods extensively leverage the microscope point spread function (PSF) for fitting the molecules. Calibrating an accurate PSF model is especially difficult in the presence of depth-dependent aberrations which alter the PSF shape depending on the imaging depth. The aberrations at depths of a few micrometers become substantial enough to considerably impoverish the conventional calibration methods performance. In our work, we propose a novel spline model which enables the depth-dependent PSF model calibration by interpolating between the beads at arbitrary depths. We show that diffspline reduces the PSF intensity overestimation by 67.8 percentage points and underestimation by 21.8 percentage points. Moreover, it eliminates the depth-dependent bias and improves the localization precision two-fold compared to previous approaches.

biophysics↗

bioRxiv2023

Theoretical minimum uncertainty of modulation enhanced spinning disk confocal microscopy

Modulation enhanced single-molecule localization microscopy (meSMLM), where emitters are sparsely activated with patterned illumination, increases the localization precision over SMLM. Furthermore, meSMLM improves the resolution over structured illumination microscopy while reducing the required amount of illumination patterns. These factors motivate enabling meSMLM in existing systems which employ patterned illumination intensity. Here, we introduce SpinFlux: modulation enhanced localization for spinning disk confocal microscopy. SpinFlux uses a spinning disk with pinholes in its illumination and emission paths, to illuminate select regions in the sample during each measurement. The resulting intensity-modulated emission signal is analyzed to localize emitters with improved precision. We derive a statistical image formation model for SpinFlux and we quantify the theoretical minimum uncertainty, in terms of the Cramer-Rao lower bound, for various illumination pattern configurations. We find that SpinFlux requires multiple patterns to improve the localization precision over SMLM, with the maximum improvement being 1.17 when using a single pattern. When using two pinholes on opposing sides of the emitter position, the x-localization precision can locally be improved 2.62-fold over SMLM, whereas the y-precision is improved by maximally a factor 1.12. When using pinholes in a triangular configuration around the emitter position, the localization precision is balanced over the xand y-directions at approximately a twofold local improvement over SMLM, at the cost of suboptimal precision in each individual direction. When doughnut-shaped illumination patterns, created with a phase mask in the illumination and emission paths, are used for SpinFlux, the local precision improvement over SMLM is increased 3.5-fold in the x- and y-directions. While localization on ISM data ideally results in an average global improvement of 1.48 over SMLM, or 2.10 with Fourier reweighting, SpinFlux is the method of choice for local refinements of the localization precision. Why it matters: One of the main objectives of singlemolecule localization microscopy (SMLM) is to improve the precision with which single molecules can be localized. This has been successfully achieved through modulation enhanced SMLM, which uses patterned illumination to increase the information content of signal photons. However, this technique relies on setups with increased technical complexity over SMLM. With SpinFlux, we locally enable a twoto 3.5-fold precision improvement over singlemolecule localization microscopy, which can be achieved with only minor modifications to existing spinning disk confocal microscopy setups (e.g. a phase mask in the illumination and emission paths). In addition, our modeling framework enables evaluation of a wide variety of spinning disk setups and therefore paves the way for optimal spinning disk design.

biophysics↗

bioRxiv2021

Non-Coding Genetic Analysis Implicates Interleukin 18 Receptor Accessory Protein 3'UTR in Amyotrophic Lateral Sclerosis

The non-coding genome is substantially larger than the protein-coding genome but is largely unexplored by genetic association studies. Here, we performed region-based burden analysis of >25,000 variants in untranslated regions of 6,139 amyotrophic lateral sclerosis (ALS) whole-genomes and 70,403 non-ALS controls. We identified Interleukin-18 Receptor Accessory Protein (IL18RAP) 3'UTR variants significantly enriched in non-ALS genomes, replicated in an independent cohort, and associated with a five-fold reduced risk of developing ALS. Variant IL18RAP 3'UTR reduces mRNA stability and the binding of RNA-binding proteins. Variant IL18RAP 3'UTR further dampens neurotoxicity of human iPSC-derived C9orf72-ALS microglia that depends on NF-{kappa}B signaling. Therefore, the variant IL18RAP 3'UTR provides survival advantage for motor neurons co-cultured with C9-ALS microglia. The study reveals direct genetic evidence and therapeutic targets for neuro-inflammation, and emphasizes the importance of non-coding genetic association studies. One Sentence SummaryNon-coding genetic variants in IL-18 receptor 3UTR decrease ALS risk by modifying IL-18-NF-{kappa}B signaling in microglia.

neuroscience↗