One-Shot Dual-Detection-Based Single-Molecule Super-Resolution Imaging Method for Real-Time Observation of Spatiotemporal Catalytic Activity Variations on the Plasmonic Gold Nanoparticle Surface.

Understanding the relationship between the surface properties of a single plasmonic nanoparticle and its catalytic performance is critical for developing highly efficient nanocatalysts. In this study, a one-shot dual-detection-based single-molecule super-resolution imaging method in the evanescent field was developed to observe real-time spatiotemporal catalytic activity on a single plasmonic gold nanoparticle (AuNP) surface. The scattering intensity of AuNPs and the fluorescence of resorufin molecules produced on the AuNP surface were obtained simultaneously to investigate the relationship between nanoparticles and catalytic reactions at a single-molecule level. Chemisorbed adsorbates (i.e., catalytic product and resorufin) changed the electron density of individual AuNPs throughout the catalytic cycle, resulting in the fluctuation of the scattering intensity of individual AuNPs, which was attributed to the electron transfer between reactant resazurin molecules and AuNPs. The increase in the electron density of individual AuNPs affected the catalytic reaction rate. Furthermore, sequential mapping of individual catalytic events at the subdiffraction limit resolution was completed for real-time surface dynamics and spatiotemporal activity variations on the single AuNP surface. The developed method can aid in understanding surface-property-dependent catalytic kinetics and facilitate the development of nanoparticle-based heterogeneous catalysts at subdiffraction limit resolution.

MicroRNA-765 is upregulated in myelodysplastic syndromes and induces apoptosis via PLP2 inhibition in leukemia cells.

Background: Epigenetic studies, particularly research on microRNA (miRNA), have flourished. The abnormal expression of miRNA contributes to the development of hematologic malignancies. miR-765 has been reported to inhibit cell proliferation by downregulating proteolipid protein 2 (PLP2), which causes apoptosis. We investigated miR-765 dysregulation in myelodysplastic syndromes (MDS). Methods: We compared the expression profiles of miR-765 in 65 patients with MDS and 11 controls. Cell proliferation and apoptosis assays were performed to determine the in vitro effects of miR-765 on leukemia cells transfected with the miR-765 mimic. Reverse transcription quantitative PCR (RT-qPCR) and western blotting were performed to examine the targets of miR-765. Results: We found that miR-765 levels were upregulated 10.2-fold in patients with MDS compared to controls. In refractory cytopenia with multilineage dysplasia, the percentage of patients with elevated miR-765 levels was significantly higher than in other forms of MDS. Experiments with leukemia cells revealed that transfection with a miR-765 mimic inhibited cell proliferation and induced apoptosis. RT-qPCR and western blotting demonstrated that the target of miR-765 was PLP2. Conclusion: These findings imply that upregulation of miR-765 induces apoptosis via downregulation of PLP2 and may have a role in MDS pathogenesis.

Wavelength-Dependent Metal-Enhanced Fluorescence Biosensors via Resonance Energy Transfer Modulation.

Fluorescence can be enhanced or quenched depending on the distance between the surface of a metal nanoparticle and the fluorophore molecule. Fluorescence enhancement by nearby metal particles is called metal-enhanced fluorescence (MEF). MEF shows promising potential in the field of fluorescence-based biological sensing. MEF-based biosensor systems generally fall into two platform categories: (1) a two/three-dimensional scaffold, or (2) a colloidal suspension. This review briefly summarizes the application studies using wavelength-dependent carbon dots (UV-VIS), noble metals (VIS), and upconversion nanoparticles (NIR to VIS), representative nanomaterials that contribute to the enhancement of fluorescence through the resonance energy transfer modulation and then presents a perspective on this topic.

Supersensitive detection of single-histamine molecule on nanoplates by turn-on small molecule fluorescence sandwich immunoassay.

We develop a supersensitive "turn-on format" fluorescence sandwich immunoassay for detecting small single molecules. Gold nanoplate-based biotin antibodies and streptavidin-fluorophores were used instead of streptavidin-horseradish peroxidase reacting with a biotin tracer in a microplate-based competitive enzyme-linked immunosorbent assay (ELISA). Our platform showed a low detection limit of 5 zeptomolar (5 × 10-21 M), 5.4 × 1010 times higher detection sensitivity than the conventional tune-off format ELISA.

Establishment of Korean Pediatric Reference Intervals for Estradiol using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) is a reliable and accurate method for measuring steroid hormone levels. There is an increasing need for sensitive and precise methods to measure estradiol in pediatric patients. Here, we established reference intervals for estradiol in healthy children using a UHPLC-MS/MS-based method for the first time in South Korea. METHODS: Serum estradiol was measured using a Sciex Triple QuadTM 6500 + UHPLC-MS/MS (Sciex, Framingham, MA, USA). Reference intervals for estradiol were established according to the CLSI document EP28-A3c:2008. The reference intervals were validated using serum samples from 634 pediatric patients, including neonates, children, and adolescents. Among them, 389 specimens were used in analysis of the specimen acceptance time. Statistical analysis was performed using MedCalc (MedCalc, Ostend, Belgium) and Analyse-it (Analyse-it Software Ltd., Leeds, United Kingdom) software. RESULTS: Reference intervals for boys (n = 297) were <16.6, <7.3, <19.0, <30.5, 7.6-96.5, and 10.6-134.4 pmol/L among those aged <1, 1-5, 6-9, 10-11, 12-14, and 15-17 years, respectively. Reference intervals for girls (n = 337) were <114.7, <24.2, <34.8, 8.0-177.0, 10.4-480.5, and 9.1-486.7 pmol/L among those aged <1, 1-5, 6-9, 10-11, 12-14, and 15-17 years, respectively. Overall, there was no effect of specimen acceptance time on estradiol measurements in boys or girls, except for that in the group aged 10-11 years. CONCLUSIONS: The reference intervals for healthy children were validated using a UHPLC-MS/MS-based method. The highly analytical sensitive UHPLC-MS/MS method may be useful for estradiol determination in pediatric patients.

Real-time six-dimensional spatiotemporal tracking of single anisotropic nanoparticles in live cells by integrated multifunctional light-sheet nanoscopy.

An integrated multifunctional light-sheet nanoscopy (iMLSN) combined with differential interference contrast, total internal reflection, epifluorescence, a super-resolution radial fluctuation-stream module, and a wavelength-dependent light sheet was developed to simultaneously realize the six-dimensional (6D) vector-valued (three coordinates + rotational dynamics (azimuth and elevation angles) + transport speed) tracking of anisotropic nanoparticles in single living cells. The wavelength-dependent asymmetric scattering of light by gold nanorods was used to trigger signals depending on the polarizer angle, and real-time photo-switching was achieved by turning the polarizer, obtaining a series of super-resolution images, and tracking using different polarization directions and two channels. This technique was employed to directly observe native gold nanorods (AuNRs; 5 nm diameter × 15 nm length) and surface-functionalized AuNRs during their endocytosis and transport at the upper and attaching side membrane regions of single living cells, revealing that the AuNRs bound to the membrane receptors. The nanorods were subsequently internalized and transported away from the original entry spots. Detailed dynamic information regarding the rotation properties and endocytosis speed during the transmembrane process was also acquired for each region. The developed technique can be considered useful for the real-time monitoring of intracellular transport at various regions in single living cells, as well as for 6D vector-valued non-fluorescence super-resolution imaging and tracking.

Simultaneous quantification of thyroid hormones using an ultrasensitive single-molecule fourplex nanoimmunosensor in an evanescent field.

The thyroid gland, which regulates the metabolism of the human body, has a sophisticated feedback system that induces the secretion of thyroid-stimulating hormone (TSH) to regulate the levels of triiodothyronine (T3) and thyroxine (T4). In this study, a single-molecule fourplex nanoimmunosensor was developed for the simultaneous quantitative analysis of TSH, T3, and T4. The three thyroid hormones were detected with a high signal-to-noise ratio in an evanescent field using laser-induced total internal reflection fluorescence. Additionally, the use of gold nanoislands for the detection of molecular interactions between thyroid hormones and antibodies labeled with quantum dots minimized the background noise from the substrate compared with the use of microislands or microwells. The nanoimmunosensor exhibited excellent detection limits of 114-193 yM (yoctomolar = 10-24 M) for thyroid hormones. The detection sensitivity was approximately 1015-fold higher than that of the conventional enzyme-linked immunosorbent assay. Paired Student's t-test of the human blood samples revealed that the difference between the two methods was insignificant at the 98% confidence level. Therefore, the proposed single-molecule fourplex nanoimmunosensor can be used for early diagnosis and prognosis monitoring at the single-molecule level because it can accurately, rapidly, and simultaneously diagnose various thyroid diseases, such as hyperthyroidism and hypothyroidism.

Minimizing the Optical Illusion of Nanoparticles in Single Cells Using Four-Dimensional Cuboid Multiangle Illumination-Based Light-Sheet Super-Resolution Imaging.

Although light-sheet-based super-resolution microscopy is an excellent detection technique for biological samples because of minimal photodamage, uneven light paths due to solid-angle illumination limits it, resulting in an optical illusion. Furthermore, the optical illusion limits the observations of individual molecules in diffraction. In this study, a four-dimensional cuboid multiangle illumination-based light-sheet super-resolution (4D CMLS) imaging system was developed to minimize optical illusions in cells. The lab-built 4D CMLS imaging system was integrated with total internal reflection fluorescence and a differential interference contrast microscope. A specially designed rotatable cuboid prism simply overcame the optical illusion by rotating a specimen on the prism to change the direction of light coming from an illumination lens. 4D CMLS reconstructed images of nanoparticles of different sizes were acquired in multi-illumination angles of 0°, 90°, 180°, and 270°. Additionally, a 4D multiangle illumination-based algorithm was created to select the optimal illumination angle by combining three-dimensional super-resolution imaging with multiangle observation, even in the presence of obstacles. The 4D CMLS imaging method demonstrates the in-depth 4D observation of samples at an optimum angle that can be used in various applications, such as single-molecule and subcellular organelle observations in single cells at subdiffraction limit resolutions that describe the scenario of nature.

Upregulation of microRNA-597 in myelodysplastic syndromes induces apoptosis through FOSL2 inhibition.

INTRODUCTION: Dysregulation of microRNAs (miRNAs) has been associated with the pathophysiology of myelodysplastic syndrome (MDS). Trisomy 8 is the most frequent chromosomal abnormalities in Korean patients with MDS. We investigated the dysregulation of miR-597-5p, located on chromosome 8, which is reported to induce cell death in numerous cancers. MATERIALS AND METHODS: We compared the expression profiles of miR-597-5p among 65 MDS patients and 11 controls, and analyzed the in vitro effects of miR-597 on leukemic cells using an acute myeloid leukemia cell line transfected with miR-597. RESULTS: We found that miR-597-5p levels were upregulated 4.05-fold in MDS patients compared to those in controls. In vitro study results demonstrated that transfection with a miR-597 mimic induced apoptosis through downregulation of FOS like 2 (FOSL2). CONCLUSION: These findings suggest that upregulation of miR-597 induces apoptosis and that miR-597 has a possible role in the pathophysiology of MDS.

Ultrasensitive Hypoxia Sensing at the Single-Molecule Level via Super-Resolution Quantum Dot-Linked Immunosandwich Assay.

Activated hypoxia-inducible factor-1alpha (HIF-1α) plays an important role in the adaptive response of tumor cells to oxygen changes through the transcriptional activation of genes that regulate important biological processes required for tumor survival and progression. In this study, we developed an ultrasensitive hypoxia sensor based on read-out with quantum dots on a gold nanodisc (quantum dot-linked immunosandwich assay, QLISA) with excellent selectivity for HIF-1α. The immunoassay platform was established by comparing the immune response results using Qdot525 as a detection nanoprobe instead of a fluorescent dye (Alexa488) (fluorescent-linked immunosandwich assay, FLISA). In addition, using three-dimensional total internal reflection fluorescence microscopy, the platform was optically sectioned along the z-axis at 10 nm intervals to compare the height difference between the nanodisc and the nanoprobe following the QLISA and FLISA procedures and to localize the target location. Here, the super-resolution QLISA (srQLISA)-based hypoxia sensor exhibited high accuracy and precision for the detection of HIF-1α-extracted samples in cancer spheroids compared with the super-resolution FLISA (srFLISA) method. The developed nanobiosensor method demonstrated a wide dynamic linear detection range of 32.2 zM-8.0 pM with a limit of detection of 16 zM under optimal experimental conditions for HIF-1α, an approximate 106-fold enhanced detection sensitivity compared with the conventional enzyme-linked immunosorbent assay method based on absorbance. The detection of HIF-1α using the newly developed srQLISA sensor allows for independently predicting tumor progression and early cancer onset increases in the microvasculature density of tumor lesions.

Accuracy of ASTA MicroIDSys, a new matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system, for the identification of Korean reference and clinical bacterial and yeast strains.

This study compared the accuracy of a new MALDI-TOF mass spectrometry system, ASTA MicroIDSys system, with that of MALDI Biotyper system for the identification of reference and clinical bacterial and yeast strains. The identification accuracy of the 2 systems was compared using a total of 406 strains comprising 142 aerobic and 180 anaerobic bacterial strains and 84 yeast strains. The genus and species identification rates were 98.0% and 89.4% using MicroIDSys and 96.1% and 89.4% using Biotyper, respectively. The species identification rates of MicroIDSys and Biotyper for aerobic bacteria were 93.0% and 97.2%, respectively, and those for anaerobic bacteria were 85.6% and 81.7%, respectively. The accuracy of yeast identification at the species level was 91.7% using MicroIDSys and 92.9% using Biotyper. These findings indicate that MicroIDSys could be useful for the accurate identification of bacteria and yeast in clinical microbiology laboratories.

Quantifying intracellular trafficking of silica-coated magnetic nanoparticles in live single cells by site-specific direct stochastic optical reconstruction microscopy.

BACKGROUND: Nanoparticles have been used for biomedical applications, including drug delivery, diagnosis, and imaging based on their unique properties derived from small size and large surface-to-volume ratio. However, concerns regarding unexpected toxicity due to the localization of nanoparticles in the cells are growing. Herein, we quantified the number of cell-internalized nanoparticles and monitored their cellular localization, which are critical factors for biomedical applications of nanoparticles. METHODS: This study investigates the intracellular trafficking of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)] in various live single cells, such as HEK293, NIH3T3, and RAW 264.7 cells, using site-specific direct stochastic optical reconstruction microscopy (dSTORM). The time-dependent subdiffraction-limit spatial resolution of the dSTORM method allowed intracellular site-specific quantification and tracking of MNPs@SiO2(RITC). RESULTS: The MNPs@SiO2(RITC) were observed to be highly internalized in RAW 264.7 cells, compared to the HEK293 and NIH3T3 cells undergoing single-particle analysis. In addition, MNPs@SiO2(RITC) were internalized within the nuclei of RAW 264.7 and HEK293 cells but were not detected in the nuclei of NIH3T3 cells. Moreover, because of the treatment of the MNPs@SiO2(RITC), more micronuclei were detected in RAW 264.7 cells than in other cells. CONCLUSION: The sensitive and quantitative evaluations of MNPs@SiO2(RITC) at specific sites in three different cells using a combination of dSTORM, transcriptomics, and molecular biology were performed. These findings highlight the quantitative differences in the uptake efficiency of MNPs@SiO2(RITC) and ultra-sensitivity, varying according to the cell types as ascertained by subdiffraction-limit super-resolution microscopy.

Silica-coated magnetic-nanoparticle-induced cytotoxicity is reduced in microglia by glutathione and citrate identified using integrated omics.

BACKGROUND: Nanoparticles have been utilized in brain research and therapeutics, including imaging, diagnosis, and drug delivery, owing to their versatile properties compared to bulk materials. However, exposure to nanoparticles leads to their accumulation in the brain, but drug development to counteract this nanotoxicity remains challenging. To date, concerns have risen about the potential toxicity to the brain associated with nanoparticles exposure via penetration of the brain blood barrier to address this issue. METHODS: Here the effect of silica-coated-magnetic nanoparticles containing the rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)] were assessed on microglia through toxicological investigation, including biological analysis and integration of transcriptomics, proteomics, and metabolomics. MNPs@SiO2(RITC)-induced biological changes, such as morphology, generation of reactive oxygen species, intracellular accumulation of MNPs@SiO2(RITC) using transmission electron microscopy, and glucose uptake efficiency, were analyzed in BV2 murine microglial cells. Each omics data was collected via RNA-sequencing-based transcriptome analysis, liquid chromatography-tandem mass spectrometry-based proteome analysis, and gas chromatography- tandem mass spectrometry-based metabolome analysis. The three omics datasets were integrated and generated as a single network using a machine learning algorithm. Nineteen compounds were screened and predicted their effects on nanotoxicity within the triple-omics network. RESULTS: Intracellular reactive oxygen species production, an inflammatory response, and morphological activation of cells were greater, but glucose uptake was lower in MNPs@SiO2(RITC)-treated BV2 microglia and primary rat microglia in a dose-dependent manner. Expression of 121 genes (from 41,214 identified genes), and levels of 45 proteins (from 5918 identified proteins) and 17 metabolites (from 47 identified metabolites) related to the above phenomena changed in MNPs@SiO2(RITC)-treated microglia. A combination of glutathione and citrate attenuated nanotoxicity induced by MNPs@SiO2(RITC) and ten other nanoparticles in vitro and in the murine brain, protecting mostly the hippocampus and thalamus. CONCLUSIONS: Combination of glutathione and citrate can be one of the candidates for nanotoxicity alleviating drug against MNPs@SiO2(RITC) induced detrimental effect, including elevation of intracellular reactive oxygen species level, activation of microglia, and reduction in glucose uptake efficiency. In addition, our findings indicate that an integrated triple omics approach provides useful and sensitive toxicological assessment for nanoparticles and screening of drug for nanotoxicity.

Performance assessment of ASTA MicroIDSys, a new matrix assisted laser desorption ionization-time of flight mass spectrometry system, for identification of viridans group streptococci.

The performance of the ASTA MicroIDSys system (ASTA), a new matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) system, was evaluated for the identification of viridans group streptococci (VGS) and compared with the results obtained with the Bruker Biotyper system (Bruker Daltonics). A total of 106 Streptococcus reference strains belonging to 24 species from the bacterial strain bank was analyzed using the two MALDI-TOF MS systems. Of the 106 reference strains tested, ASTA MicroIDSys and Bruker Biotyper correctly identified 84.9% and 81.1% at the species level, 100% and 97.2% at the group level and 100% and 98.1% at the genus level, respectively. The difference between the two systems was not statistically significant (P = 0.289). Out of 24 species, 13 species were accurately identified to the species level with 100% accurate identification rates with both systems. The accurate identification rates at the species level of ASTA MicroIDSys and Bruker Biotyper were 100% and 87.5% for the S. anginosus group; 78.4% and 73.5% for the S. mitis group; 91.7% and 91.7% for the S. mutans group; and 100% and 100% for the S. salivarius group, respectively. The ASTA MicroIDSys showed an identification performance equivalent to that of the Bruker Biotyper for VGS. Therefore, it would be useful for the identification of VGS strains in clinical microbiology laboratories.

Cubic spline-based depth-dependent localization of mitochondria-endoplasmic reticulum contacts by three-dimensional light-sheet super-resolution microscopy.

The contact distance between mitochondria (Mito) and endoplasmic reticulum (ER) has received considerable attention owing to their crucial function in maintaining lipid and calcium homeostasis. Herein, cubic spline algorithm-based depth-dependent fluorescence-free three-dimensional light-sheet super-resolution microscopy (3D LSRM) with dual-wavelength illumination sources was investigated to study the distance of Mito-ER contacts in various live cells. To detect wavelength-dependent scattering, 12 nm gold nanoparticles (AuNPs) and 20 nm silver nanoparticles (AgNPs) as fluorescence-free nanoprobes were conjugated with Mito and ER. The cubic spline algorithm-based method showed improved localization precision in lateral and axial directions compared with that for previously used least squares and least cubic algorithms. The cubic spline-based depth-dependent localization was applied to the spatial localization of nanoprobes in super-resolution images, in which the average distance of Mito and ER was 22.4 nm in HeLa cells, 22.2 nm in RAW264.7 macrophage cells, 21.9 nm in AGS cells, 21.4 nm in HT29 cells, and 21.3 nm in HEK293 cells. The distances were ∼12% larger than those previously determined by electron microscopy, which demonstrated that this method was accessible and reliable for studying the intracellular structures of various live cells at the subdiffraction limit resolution.

Genome wide CRISPR screening reveals a role for sialylation in the tumorigenesis and chemoresistance of acute myeloid leukemia cells.

Aberrant activation of cytokine and growth factor signal transduction pathways confers enhanced survival and proliferation properties to acute myeloid leukemia (AML) cells. However, the mechanisms underlying the deregulation of signaling pathways in leukemia cells are unclear. To identify genes capable of independently supporting cytokine-independent growth, we employed a genome-wide CRISPR/Cas9-mediated loss-of-function screen in GM-CSF-dependent human AML TF-1 cells. More than 182 genes (p < 0.01) were found to suppress the cytokine-independent growth of TF-1 cells. Among the top hits, genes encoding key factors involved in sialylation biosynthesis were identified; these included CMAS, SLC35A1, NANS, and GNE. Knockout of either CMAS or SLC35A1 enabled cytokine-independent proliferation and survival of AML cells. Furthermore, NSG (NOD/SCID/IL2Rγ-/-) mice injected with CMAS or SLC35A1-knockout TF-1 cells exhibited a shorter survival than mice injected with wild-type cells. Mechanistically, abrogation of sialylation biosynthesis in TF-1 cells induced a strong activation of ERK signaling, which sensitized cells to MEK inhibitors but conferred resistance to JAK inhibitors. Further, the surface level of α2,3-linked sialic acids was negatively correlated with the sensitivity of AML cell lines to MEK/ERK inhibitors. We also found that sialylation modulated the expression and stability of the CSF2 receptor. Together, these results demonstrate a novel role of sialylation in regulating oncogenic transformation and drug resistance development in leukemia. We propose that altered sialylation could serve as a biomarker for targeted anti-leukemic therapy.

Relationships between Relative Expression of RND Efflux Pump Genes, H33342 Efflux Activity, Biofilm-Forming Activity, and Antimicrobial Resistance in Acinetobacter baumannii Clinical Isolates.

Various mechanisms underlying antimicrobial resistance in Acinetobacter baumannii have been reported. However, the relationships between efflux pump activity, biofilm formation, and antimicrobial resistance in A. baumannii is controversial. In this study, we investigated the relative expression of RND efflux pump genes, H33342 efflux activity, and biofilm-forming activity in 120 A. baumannii clinical isolates, examined their potential relationships with each other, and statistically analyzed their effects on antibiotic resistance. High adeB expression and high H33342 efflux activity were correlated with low biofilm-forming activity. High adeB expression was significantly correlated with resistance to tigecycline and cefotaxime, but not with the multidrug resistance (MDR) phenotype. Importantly, only high adeJ expression was significantly correlated with the MDR phenotype and was correlated with resistance to various antibiotics. However, we found no significant correlation between adeJ expression and biofilm-forming activity. Furthermore, adeG expression was not correlated with antibiotic resistance and biofilm-forming activity. The results of multivariate analysis showed that adeB overexpression and high H33342 efflux activity were related to biofilm-forming activity, and only adeJ overexpression was significantly associated with the MDR phenotype, highlighting the importance of adeJ overexpression.

Decrease in membrane fluidity and traction force induced by silica-coated magnetic nanoparticles.

BACKGROUND: Nanoparticles are being increasingly used in biomedical applications owing to their unique physical and chemical properties and small size. However, their biophysical assessment and evaluation of side-effects remain challenging. We addressed this issue by investigating the effects of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate [MNPs@SiO2(RITC)] on biophysical aspects, such as membrane fluidity and traction force of human embryonic kidney 293 (HEK293) cells. We further extended our understanding on the biophysical effects of nanoparticles on cells using a combination of metabolic profiling and transcriptomic network analysis. RESULTS: Overdose (1.0 µg/µL) treatment with MNPs@SiO2(RITC) induced lipid peroxidation and decreased membrane fluidity in HEK293 cells. In addition, HEK293 cells were morphologically shrunk, and their aspect ratio was significantly decreased. We found that each traction force (measured in micropillar) was increased, thereby increasing the total traction force in MNPs@SiO2(RITC)-treated HEK293 cells. Due to the reduction in membrane fluidity and elevation of traction force, the velocity of cell movement was also significantly decreased. Moreover, intracellular level of adenosine triphosphate (ATP) was also decreased in a dose-dependent manner upon treatment with MNPs@SiO2(RITC). To understand these biophysical changes in cells, we analysed the transcriptome and metabolic profiles and generated a metabotranscriptomics network, which revealed relationships among peroxidation of lipids, focal adhesion, cell movement, and related genes and metabolites. Furthermore, in silico prediction of the network showed increment in the peroxidation of lipids and suppression of focal adhesion and cell movement. CONCLUSION: Taken together, our results demonstrated that overdose of MNPs@SiO2(RITC) impairs cellular movement, followed by changes in the biophysical properties of cells, thus highlighting the need for biophysical assessment of nanoparticle-induced side-effects.

Toward Sub-Diffraction Imaging of Single-DNA Molecule Sensors Based on Stochastic Switching Localization Microscopy.

The natural characteristics of deoxyribonucleic acid (DNA) enable its advanced applications in nanotechnology as a special tool that can be detected by high-resolution imaging with precise localization. Super-resolution (SR) microscopy enables the examination of nanoscale molecules beyond the diffraction limit. With the development of SR microscopy methods, DNA nanostructures can now be optically assessed. Using the specific binding of fluorophores with their target molecules, advanced single-molecule localization microscopy (SMLM) has been expanded into different fields, allowing wide-range detection at the single-molecule level. This review discusses the recent progress in the SR imaging of DNA nano-objects using SMLM techniques, such as direct stochastic optical reconstruction microscopy, binding-activated localization microscopy, and point accumulation for imaging nanoscale topography. Furthermore, we discuss their advantages and limitations, present applications, and future perspectives.

Characterizing the non-paraxial Talbot effect of two-dimensional periodic arrays of plasmonic gold nanodisks by differential interference contrast microscopy.

Exploiting the working principle of conventional differential interference contrast (DIC) microscopy, we experimentally investigate the non-paraxial Talbot effect of two-dimensional periodic arrays of gold nanodisks (AuNDs) with a periodicity ao comparable to the excitation wavelength λ. In the non-paraxial regime, strongly contrasting self-image patterns at the Talbot and fractional Talbot distances appeared perpendicularly to the AuND array. The experimental self-image distances were comparable to the calculated non-paraxial Talbot distances at two excitation wavelengths (540 nm and 600 nm). Beyond the paraxial limit, Talbot distances were observed at positions smaller than the paraxial Talbot distance.