Cellular responses to external threats probed by super-resolution microscopy

Fluorescence microscopy has been an essential tool in biology. However, its imaging resolution has been limited around >200 nm in lateral dimensions, and >500 nm in axial dimension, leaving many biological structures too small to study. However the recent developments of several super-resolution techniques, this limit has been overcome. Here we present our approach to this matter, using our three-dimensional (3D) multicolor super-resolved single fluorophore microscopy. Especially we report imaging of a near-IR dye for imaging thick and dense structures for multicolor colocalization studies. Then we present three applications of the super-resolution imaging technique to the following three biological systems. Viral infection in mammalian cells triggers the immune response, a cascade of antiviral signaling proteins. For double stranded RNA (dsRNA) virus, the innate immune response starts from retinoic acid-inducible gene-I (RIG-I) protein which is a dsRNA sensor. For certain types of viruses, RIG-I localizes in cytoplasmic granular aggregates called as antiviral granules (AVGs). We infected/transfected cells by influenza virus lacking NS1 (IAVΔNS1) or polyinosinic:polycytidylic acid (poly I:C) to induce AVGs. We found in AVGs, RIG-I forms clusters of around 110nm in diameter. Then we treated intact cells with various stress conditions to from stress granules (SGs) and could also observe RIG-I clusters in SGs. RIG-I was also clustered in intact cells, but the clustering percentage and diameter were small than in AVGs or SGs, so we conclude that the intrinsically clustered RIG-I relocalizes into granular structures upon external stimuli, and its clustering is enhanced. To verify RIG-I clustering, we imaged TIAR, a marker for SGs, which showed much less clustering. Also we conducted various tests on our clustering algorithm, as well as structural illumination microscopy (SIM) imaging that all support the idea of the clustering of RIG-I. In bacterial cells, upon vast global DNA damage, an error-prone DNA repair response called SOS response occurs. SOS response is initiated by RecA, a protein essential for maintenance of DNA. It was reported that RecA forms a bundle to connect uncut and cut locus of in the event of double strand break, supporting the idea that RecA mediates homology search. We report that RecA also forms bundles in the SOS response, and these bundles are ribbon-like structures, i.e. flat at one side and wide at the other side, hypothetically wrapping around DNA damaged sites. The conventional approach of applying broad-spectrum antibiotics to treat bacterial infections contributes to the emerging of antibiotic resistance. To cope with this, species specific and narrow-spectrum antibiotics draw attention. Plantazolicin (PZN) is a natural antibiotic that is highly specific against B. anthracis which is the agent of anthrax and a category A priority pathogen, but the mechanism of how PZN kills B. anthracis has been unknown. Recent investigation showed PZN depolarizes B. anthracis membrane, and it was supported by the super-resolution imaging that PZN foci localize on the membrane of cells

Robust nonparametric quantification of clustering density of molecules in single-molecule localization microscopy

We report a robust nonparametric descriptor,&nbsp;J&prime;(r), for quantifying the density of clustering molecules in single-molecule localization microscopy.&nbsp;J&prime;(r), based on nearest neighbor distribution functions, does not require any parameter as an input for analyzing point patterns. We show that&nbsp;J&prime;(r) displays a valley shape in the presence of clusters of molecules, and the characteristics of the valley reliably report the clustering features in the data. Most importantly, the position of the&nbsp;J&prime;(r) valley () depends exclusively on the density of clustering molecules (&rho;c). Therefore, it is ideal for direct estimation of the clustering density of molecules in single-molecule localization microscopy. As an example, this descriptor was applied to estimate the clustering density of&nbsp;ptsG&nbsp;mRNA in&nbsp;E. coli&nbsp;bacteria.</p

Optical manipulation of a dielectric particle along polygonal closed-loop geometries within a single water droplet

This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea government (MSIT) (No. 2019R1A2C2011293) and the UK Engineering and Physical Sciences Research Council (Grant EP/P030017/1).We report a new method to optically manipulate a single dielectric particle along closed-loop polygonal trajectories by crossing a suite of all-fiber Bessel-like beams within a single water droplet. Exploiting optical radiation pressure, this method demonstrates the circulation of a single polystyrene bead in both a triangular and a rectangle geometry enabling the trapped particle to undergo multiple circulations successfully. The crossing of the Bessel-like beams creates polygonal corners where the trapped particles successfully make abrupt turns with acute angles, which is a novel capability in microfluidics. This offers an optofluidic paradigm for particle transport overcoming turbulences in conventional microfluidic chips.Publisher PDFPeer reviewe

Cdc42-dependent modulation of rigidity sensing and cell spreading in tumor repopulating cells

Recently, a robust mechanical method has been established to isolate a small subpopulation of highly tumorigenic tumor repopulating cells (TRCs) from parental melanoma cells. In order to characterize the molecular and mechanical properties of TRCs, we utilized the tension gauge tether (TGT) single-molecule platform and investigated force requirements during early cell spreading events. TRCs required the peak single molecular tension of around 40 pN through integrins for initial adhesion like the parental control cells, but unlike the control cells, they did not spread and formed very few mature focal adhesions (FAs). Single molecule resolution RNA quantification of three Rho GTPases showed that downregulation of Cdc42, but not Rac1, is responsible for the unusual biophysical features of TRCs and that a threshold level of Cdc42 transcripts per unit cell area is required to initiate cell spreading. Cdc42 overexpression rescued TRC spreading through FA formation and restored the sensitivity to tension cues such that TRCs, like parental control cells, increase cell spreading with increasing single-molecular tension cues. Our single molecule studies identified an unusual biophysical feature of suppressed spreading of TRCs that may enable us to distinguish TRC population from a pool of heterogeneous tumor cell population

Separation of Zr from Zr-2.5Nb by Electrorefining in LiCl-KCl for Volumetric Decontamination of CANDU Pressure Tube

This study presents an experimental investigation on Zr separation from Zr-2.5Nb by anode potentiostatic electrorefining in LiCl-KCl-ZrCl4 0.5 wt. % at 773 K for irradiated CANDU pressure tube decontamination. By the ORIGEN-2 code calculation, radioactive characteristics were investigated to show that Nb-94 was the most significant radionuclide with an aspect of waste level reduction by electrorefining. Three electrorefining tests were performed by fixing the applied potential as -0.9 V (vs. Ag/AgCl 1 wt. %) at the anode to dissolve only Zr. A cathode basket was installed to collect detached deposits from the cathode. Electrorefining results showed Zr was deposited on the cathode with a small amount of Nb and other alloying elements. The chemical form of the cathode deposits was shown to be only Zr metal or a mixture of Zr metal and ZrCl, depending on the experimental conditions related to the surface area ratio of the cathode to the anode. It was determined that the Zr metal reduction at the cathode was attributed to the two-step reduction reaction of Zr4+/ZrCl and ZrCl/Zr

Narrowband THz Emission from a Plasma Oscillator Imbedded in a Plasma Density Gradient

A novel method is presented for generating radiation using the beat wave associated with a bi-frequency laser pulse, to excite plasma oscillations in a plasma slab with a density gradient. By resonantly exciting a plasma wave, it can be localised and transformed into a plasma oscillator that produces a beam of radially polarised terahertz radiation. Particle-in-cell simulations and analytic theory are used to demonstrate its main characteristics, which includes narrow bandwidth. The radiator should have useful applications such as terahertz-band particle accelerators and pump-probe experiments

Enhanced Mechanical and Antibacterial Properties of Nanocomposites Based on Poly(vinyl Alcohol) and Biopolymer-Derived Reduced Graphene Oxide

Functionalized graphene-polymer nanocomposites have gained significant attention for their enhanced mechanical, thermal, and antibacterial properties, but the requirement of multi-step processes or hazardous reducing agents to functionalize graphene limits their current applications. Here, we present a single-step synthesis of thermally reduced graphene oxide (TrGO) based on shellac, which is a low-cost biopolymer that can be employed to produce poly(vinyl alcohol) (PVA)/TrGO nanocomposites (PVA-TrGO). The concentration of TrGO varied from 0.1 to 2.0 wt.%, and the critical concentration of homogeneous TrGO dispersion was observed to be 1.5 wt.%, below which strong interfacial molecular interactions between the TrGO and the PVA matrix resulted in improved thermal and mechanical properties. At 1.5 wt.% filler loading, the tensile strength and modulus of the PVA-TrGO nanocomposite were increased by 98.7% and 97.4%, respectively, while the storage modulus was increased by 69%. Furthermore, the nanocomposite was 96% more effective in preventing bacterial colonization relative to the neat PVA matrix. The present findings indicate that TrGO can be considered a promising material for potential applications in biomedical devices

Parallelized Seeded Region Growing Using CUDA

This paper presents a novel method for parallelizing the seeded region growing (SRG) algorithm using Compute Unified Device Architecture (CUDA) technology, with intention to overcome the theoretical weakness of SRG algorithm of its computation time being directly proportional to the size of a segmented region. The segmentation performance of the proposed CUDA-based SRG is compared with SRG implementations on single-core CPUs, quad-core CPUs, and shader language programming, using synthetic datasets and 20 body CT scans. Based on the experimental results, the CUDA-based SRG outperforms the other three implementations, advocating that it can substantially assist the segmentation during massive CT screening tests

Naturally occurring radioactive materials (NORM) in the groundwater of two islands with various geologic settings in South Korea

Since 2007, Naturally Occurring Radioactive Materials (NORM) such as uranium-238 and radon-222 etc. in groundwater from the Community Water-supply Systems (CWS) in two islands have been studied in South Korea. In 71 samples from Ganghwa (G) Island, the maximum value of uranium-238 concentration is 72.21 μg/L. 3 CWSs (4.2%) exceeded the Maximum Contaminant Level (MCL) of 30 μg/L for uranium-238. The maximum value of radon-222 activity is 614 Bq/L. 28 CWSs (39.4 %) did not meet the United States Environment Protection Agency (US EPA) proposed Alternative Maximum Contaminant Level (AMCL) of 148 Bq/L for radon-222. At all CWS that did not meet the US EPA’s MCL or AMCL, some appropriate actions were taken such as water treatment, alternative well development, mixing water of different origins, and so forth. In the 52 CWSs of Jeju (J) Island, the maximum value of uranium-238 and radon-222 concentrations are 1.37 μg/L and 94.83 Bq/L, respectively. All values for uranium-238, gross alpha, and radon-222 meet MCL and proposed AMCL of US EPA drinking water standard. The two islands have different geological settings that are believed to be the causes of the big differences in the NORM levels. Geologically an old island has much higher NORM values than a young island formed in the Quaternary Period due to hydrogeological factors such as recharge and infiltration rates of precipitation. The residence times in the aquifers for water-rock (mineral) interactions are very different from each other.</p