Ying Yang

Super‐resolution microscopy has advanced cellular imaging by enabling nanoscale resolution, while reliable multicolor strategies remain crucial for resolving multiple targets in complex cellular samples. Here, we demonstrate a two‐color imaging strategy using electrochemically controlled stochastic optical reconstruction microscopy (EC‐STORM), which allows precise control over the switching behavior of two cyanine dyes, Alexa 647 and CF680. An oscillating electrochemical potential enables control of emitter density and molecule ON time. This minimizes the spatial and temporal overlap of emitters, enabling effective spectral demixing for two‐color EC‐STORM imaging. We further combined spectral demixing with a biplane detection configuration, enabling additional 3D localization of Alexa 647, where axial coordinates are extracted from the photon‐intensity ratio between the two detection channels instead of point spread function width. This integrated approach offers a practical solution for high‐density multicolor 3D STORM imaging.

Super‐resolution microscopy has advanced cellular imaging by enabling nanoscale resolution, while reliable multicolor strategies remain crucial for resolving multiple targets in complex cellular samples. Here, we demonstrate a two‐color imaging strategy using electrochemically controlled stochastic optical reconstruction microscopy (EC‐STORM), which allows precise control over the switching behavior of two cyanine dyes, Alexa 647 and CF680. An oscillating electrochemical potential enables control of emitter density and molecule ON time. This minimizes the spatial and temporal overlap of emitters, enabling effective spectral demixing for two‐color EC‐STORM imaging. We further combined spectral demixing with a biplane detection configuration, enabling additional 3D localization of Alexa 647, where axial coordinates are extracted from the photon‐intensity ratio between the two detection channels instead of point spread function width. This integrated approach offers a practical solution for high‐density multicolor 3D STORM imaging.

In superresolution optical fluctuation imaging (SOFI), molecules spaced closer than the diffraction limit can be separated through spatial and temporal correlation analysis of the fluorescence intensity fluctuations. The resolution and speed of SOFI imaging greatly depend on the characteristics of these fluorescence fluctuations. Fluorophores with stochastic and rapid fluorescence fluctuations are favorable for improving SOFI imaging resolution and speed, especially in high-order cumulant analysis. Stochastic blinking of organic dyes in oxygen scavenging and thiol reducing buffers are well suited for SOFI due to their high brightness. However, the blinking rates can be too slow and nonuniform, which introduces nonlinearity in SOFI image. To address these challenges, we introduce electrochemically controlled dye switching for SOFI (EC-SOFI). By applying an oscillating electrochemical potential to a transparent electrode surface, we increase the overall dye switching rate and uniformity. Using Alexa 647 dye as an example, EC-SOFI reduces the average ON time by over threefold and switching variance by more than twofold compared to conventional photochemical switching, achieving ~60 nm spatial resolution in 6th order EC-SOFI image. We further demonstrate that EC-SOFI achieves ~130 nm and ~80 nm resolution with 100 and 300 frames, respectively, enabling fast, large-area tile-scan superresolution imaging. This advancement in EC-SOFI significantly enhances the practical potential of the SOFI technique.

Multicolour fluorescence imaging is crucial to simultaneously visualize multiple targets in cells, enabling the study of complicated cellular processes. Common multicolour methods rely on using fluorophores with sufficiently different spectral or lifetime characteristics. Here we present a new multicolour imaging strategy on a standard fluorescence microscope, where up to four fluorophores with high spectral overlap can be resolved using a single-colour optical configuration. We find that under electrochemical modulation, the fluorophores are regulated between the bright and dim states, with each displaying a distinct fluorescence response pattern. These unique fluorescence potential profiles enable the effective separation of different fluorophores through linear unmixing. We also demonstrate that electrochemical fluorescence switching is readily applicable to four-colour STED imaging. With no modification to the optical setups and easy adaptation to different microscopes, we anticipate that colour unmixing based on electrochemical fluorescence switching will provide an easily accessible multicolour imaging pathway for discoveries in diverse fields.

We demonstrate how to effectively modulate the fluorescence intensity of organic dye-labelled cell samples on an indium tin oxide surface using electrochemistry with redox-active mediators present in an oxygen scavenger buffer.

Stochastic optical reconstruction microscopy (STORM) allows wide-field imaging with single-molecule resolution by calculating the coordinates of individual fluorophores from the separation of fluorophore emission in both time and space. Such separation is achieved by photoswitching the fluorophores between a long-lived OFF state and an emissive ON state. Although STORM can image single molecules, molecular counting remains challenging due to undercounting errors from photobleached or overlapping dyes and overcounting artefacts from the repetitive random blinking of dyes. Here we show that fluorophores can be electrochemically switched for STORM imaging (EC-STORM), with excellent control over the switching kinetics, duty cycle and recovery yield. Using EC-STORM, we demonstrate molecular counting by using electrochemical potential to control the photophysics of dyes. The random blinking of dyes is suppressed by a negative potential but the switching-ON event can be activated by a short positive-potential pulse, such that the frequency of ON events scales linearly with the number of underlying dyes. We also demonstrate EC-STORM of tubulin in fixed cells with a spatial resolution as low as ~28 nm and counting of single Alexa 647 fluorophores on various DNA nanoruler structures. This control over fluorophore switching will enable EC-STORM to be broadly applicable in super-resolution imaging and molecular counting.

In order to develop immune tolerant to the fetal, maternal immune system will have some modification comparing to the time before pregnancy. Immune tolerance starts and develops at the maternal placental interface. In innate immunity, decidual natural killer (dNK) cells, macrophages and dendritic cells play a key role in immue tolerance. In adaptive immunity, a moderate increase of number and immune inhibition function of regulatory T cells (Treg) are essential for immune tolerance. The trophoblast cells and immune cells expressing indoleamine 2,3-dioxygenase (IDO), the trophoblast cells expressing HLA-G, and Th1/Th2 shifting to Th2 dominant and Th17/Treg shifting to Treg domiant are in favor of maternal fetal immune tolerance. Steroids (estrogen and progesterone) and human chorionic gonadotropin (HCG) also participate in immune tolerance by inducing Treg cells or upregulating immunosuppressive cytokines. Most of the patients with chronic HBV infection are in the “HBV immune tolerance period” before pregnancy, and the liver disease is relatively stable during pregnancy. In chronic HBV infection women, after delivery, the relative immunosuppression in vivo is reversed, and Th1 is dominant in Th1/Th2 and Th17 is dominant in Th17/Treg balance. After delivery, the number of Treg decrease and NK cells increase in quantity and cytotoxicity in peripheral blood. Liver NK cells may cause liver inflammation through a non-antigen specific mechanism. After delivery, the number of CD8⁺ T cells will increase and HBV specific T cell response recovers from the disfunction in pregnancy. Under the background of postpartum inflammation, the rapid decrease of cortisol after delivery, and especially the enhancement of HBV specific T cell response induced by HBV DNA and cytokines, are the main reasons for postpartum hepatitis. HBeAg positive, especially HBeAg3-5Log₁₀IU/ml are risk factors for postpartum hepatitis. Antiviral treatment in late pregnancy can reduce the incidence of mother to child transmission (MTCT) in chronic HBV infection women. Chronic HBV infection women have hepatitis both during pregnancy and more often in 12 weeks postpartum. It is generally agreed that postpartum hepatitis is mild symptoms and self-limited. Delaying drug withdrawal to 48 weeks can increase the seroconversion rate of HBeAg in delivery women with elevated alanine aminotransferase (ALT) in pregnancy.

To explore the genetic basis for two unrelated patients with global developmental delay and coarse facial features. Clinical data and family history of the two pedigrees were collected. Whole exome sequencing and Sanger sequencing were carried out to detect potential variants. The two patients have presented with global developmental delay, coarse facies, muscular hypotonia, congenital heart disease, and pectus excavatum, and were found to harbor two de novo loss-of-function variants of the ARID1B gene, namely c.3586delC (p.Gln1196Serfs*15) and c.4954_4957delACGT (p.Thr1652Glyfs*31). Both variants were unreported previously. The nonsense variants of the ARID1B gene probably underlay the etiology in these patients. Above finding has enriched the genotypic and phenotypic spectrum of the disease and provided a basis for prenatal diagnosis.

Prader-Willi syndrome (PWS) is a complex and multisystem neurobehavioral disease, which is caused by the lack of expression of paternally inherited imprinted genes on chromosome15q11.2-q13.1. The clinical manifestations of PWS vary with age. It is characterized by severe hypotonia with poor suck and feeding difficulties in the early infancy, followed by overeating in late infancy or early childhood and progressive development of morbid obesity unless the diet is externally controlled. Compared to Western PWS patients, Chinese patients have a higher ratio of deletion type. Although some rare disease networks, including PWS Cooperation Group of Rare Diseases Branch of Chinese Pediatric Society, Zhejiang Expert Group for PWS, were established recently, misdiagnosis, missed diagnosis and inappropriate intervention were usually noted in China. Therefore, there is an urgent need for an integrated multidisciplinary approach to facilitate early diagnosis and optimize management to improve quality of life, prevent complications, and prolong life expectancy. Our purpose is to evaluate the current literature and evidences on diagnosis and management of PWS in order to provide evidence-based guidelines for this disease, specially from China.