CW STED nanoscopy with a Ti:Sapphire oscillator

Fluorescence microscopy has become an essential tool to study biological molecules, pathways and events in living cells, tissues and animals. Meanwhile, the conventional optical microscopy is limited by the wavelength of the light. Even the most advanced confocal microscopy or multiphoton microscopy can only yield optical resolution approaching the diffraction limit of ~200 nm. This is still larger than many subcellular structures, which are too small to be resolved in detail. These limitations have driven the development of super-resolution optical imaging methodologies over the past decade. The stimulated emission depletion (STED) microscopy was the first and most direct approach to overcoming the diffraction limit for far-field nanoscopy. Typically, the excitation focus is overlapped by an intense doughnut-shaped spot to instantly de-excite markers from their fluorescent state to the ground state by stimulated emission. This effectively eliminates the periphery of the Point Spread Function (PSF), resulting in a narrower focal region, or super-resolution. Scanning a sharpened spot through the specimen renders images with sub-diffraction resolution. Multi-color STED imaging can present important structural and functional information for protein-protein interaction. In this work, we presented a dual color, synchronization-free STED stimulated emission depletion (STED) microscopy with a Ti:Sapphire oscillator. The excitation wavelengths were 532nm and 635nm, respectively. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. We also imaged 200 nm nanospheres as well as all three cytoskeletal elements (microtubules, intermediate filaments, and actin filaments), clearly demonstrating the super-resolution resolving power over conventional diffraction limited imaging. It also allowed us to discover that, Dylight 650, exhibits improved performance over ATTO647N, a fluorophore frequently used in STED. Furthermore, we applied synchronization-free STED to image fluorescently-labeled intracellular viral RNA granules, which otherwise cannot be differentiated by confocal microscopy. Thanks to the widely available Ti:Sapphire oscillators in multiphoton imaging system, this work suggests easier access to setup super-resolution microscope via the synchronization-free STED A series of biological specimens were imaged with our dual-color STED. © Copyright SPIE

Molecular mechanisms underlying altered neurobehavioural development of female offspring of mothers with polycystic ovary syndrome: FOS-mediated regulation of neurotrophins in placenta

Background: This study explored the mechanisms underlying altered neurobehavioural development of female offspring born to mothers with polycystic ovary syndrome (PCOS). / Methods: In total, 20 women with PCOS and 32 healthy women who underwent caesarean deliveries with a single female foetus were recruited. Infants were assessed with Dubowitz scoring. Swan71 cell line with stable FOS overexpression was used to verify the regulatory effects of FOS on brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) expression. Learning and memory in female first-generation (F1) and second-generation (F2) offspring in a rat model of PCOS was tested using the Morris water maze at puberty and adulthood. Transcriptome analysis of pubertal hippocampi and hypothalami of female F1 offspring was conducted. / Findings: Total score and behaviour subscales of Dubowitz scoring were significantly lower in female infants of women with PCOS. FOS and NGF protein levels were downregulated in placental villi of the PCOS group. FOS played a key role in BDNF inhibition and enhancing NGF in Swan71 cells. PCOS female F1 rats exhibited lower target crossing times during puberty when compared to controls. Transcriptome analysis revealed significant changes in hippocampal and hypothalamic neuronal pathways in female F1 rats at puberty. / Interpretation: FOS regulation of neurotrophins in the placenta negatively affects neurobehavioural development of female offspring of PCOS mothers

DeepEthnic: Multi-Label Ethnic Classification from Face Images

Ethnic group classification is a well-researched problem, which has been pursued mainly during the past two decades via traditional approaches of image processing and machine learning. In this paper, we propose a method of classifying an image face into an ethnic group by applying transfer learning from a previously trained classification network for large-scale data recognition. Our proposed method yields state-of-the-art success rates of 99.02%, 99.76%, 99.2%, and 96.7%, respectively, for the four ethnic groups: African, Asian, Caucasian, and Indian

Mirror-enhanced super-resolution microscopy

Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of similar to 100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen. With no additional complexity, the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED, which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments. The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens, which cannot tolerate high laser power.National Instrument Development Special Program [2013YQ03065102]; '973' Major State Basic Research Development Program of China [2011CB809101]; Natural Science Foundation of China [31327901, 61475010, 61428501]; Australian Research Council Centre of Excellence for Nanoscale BioPhotonics [CE140100003]; National Institute of Health [GM094198]SCI(E)PubMed中国科技核心期刊(ISTIC)[email protected]