Fourier Channel Attention Powered Lightweight Network for Image Segmentation.

The accuracy of image segmentation is critical for quantitative analysis. We report a lightweight network FRUNet based on the U-Net, which combines the advantages of Fourier channel attention (FCA Block) and Residual unit to improve the accuracy. FCA Block automatically assigns the weight of the learned frequency information to the spatial domain, paying more attention to the precise high-frequency information of diverse biomedical images. While FCA is widely used in image super-resolution with residual network backbones, its role in semantic segmentation is less explored. Here we study the combination of FCA and U-Net, the skip connection of which can fuse the encoder information with the decoder. Extensive experimental results of FRUNet on three public datasets show that the method outperforms other advanced medical image segmentation methods in terms of using fewer network parameters and improved accuracy. It excels in pathological Section segmentation of nuclei and glands.

Glucose increases the length and spacing of the lattice structure of the axon initial segment.

The axon initial segment (AIS) plays an important role in maintaining neuronal polarity and initiating action potentials (APs). The AIS adapts to its environment by changing its length and distance from the cell body, resulting in modulation of neuronal excitability, which is referred to as AIS plasticity. Previous studies found an ~200 nm single periodic distribution of the key AIS components ankyrinG (AnkG), Nav 1.2, and ßIV-spectrin, while it remains unclear how the lattice structure is altered by AIS plasticity. In this study, we found that the length of the AIS significantly increased, resulting in increased neuronal excitability, with high-concentration glucose treatment. Structured illumination microscopy (SIM) images of the lattice structure showed a dual-spacing periodic distribution (~200 nm and ~260 nm) of AnkG, Nav 1.2, and ßIV-spectrin. Moreover, 480-kDa AnkG was crucial for AIS plasticity and increased lattice structure spacing. The discovery of new regulators for modulating AIS plasticity will help us to understand and manipulate the structure and function of the AIS. Glucose triggers axon initial segment (AIS) plasticity of cultured neurons. AIS lattice structure under glucose treatment shows an increased spacing by structured illumination microscopy imaging. 480-kDa AnkG contributes to AIS plasticity.

Polarization modulation with optical lock-in detection reveals universal fluorescence anisotropy of subcellular structures in live cells.

The orientation of fluorophores can reveal crucial information about the structure and dynamics of their associated subcellular organelles. Despite significant progress in super-resolution, fluorescence polarization microscopy remains limited to unique samples with relatively strong polarization modulation and not applicable to the weak polarization signals in samples due to the excessive background noise. Here we apply optical lock-in detection to amplify the weak polarization modulation with super-resolution. This novel technique, termed optical lock-in detection super-resolution dipole orientation mapping (OLID-SDOM), could achieve a maximum of 100 frames per second and rapid extraction of 2D orientation, and distinguish distance up to 50 nm, making it suitable for monitoring structural dynamics concerning orientation changes in vivo. OLID-SDOM was employed to explore the universal anisotropy of a large variety of GFP-tagged subcellular organelles, including mitochondria, lysosome, Golgi, endosome, etc. We found that OUF (Orientation Uniformity Factor) of OLID-SDOM can be specific for different subcellular organelles, indicating that the anisotropy was related to the function of the organelles, and OUF can potentially be an indicator to distinguish normal and abnormal cells (even cancer cells). Furthermore, dual-color super-resolution OLID-SDOM imaging of lysosomes and actins demonstrates its potential in studying dynamic molecular interactions. The subtle anisotropy changes of expanding and shrinking dendritic spines in live neurons were observed with real-time OLID-SDOM. Revealing previously unobservable fluorescence anisotropy in various samples and indicating their underlying dynamic molecular structural changes, OLID-SDOM expands the toolkit for live cell research.

The largest isoform of Ankyrin-G is required for lattice structure of the axon initial segment.

Alzheimer's disease (AD) is the most frequent neurodegenerative disease and a common dementia in elderly individuals. Previous studies found a strong correlation between axon initial segment (AIS) defects and AD, but it remains unclear whether AD itself changes the arrangement of AIS components, and the mechanisms by which adaptor proteins and ion channels in the AIS are disturbed in AD are not well understood. With super-resolution structured illumination microscopy (SIM) revealing axonal structures, here we imaged the lattice structure of completely assembled AIS in APP/PS1 neurons. By analyzing the images with Gaussian fitting and 1D mean autocorrelation, we found dual spacings (∼200 nm and ∼370 nm) of Ankyrin-G (AnkG), Nav1.2 and ßIV-spectrin in AD model APP/PS1 mice due to the low-expressed 480-kDa AnkG. To identify the roles of each AnkG isoform, two isoforms were separately expressed in neurons from AnkG conditional knockout mice. Mice rescued with 270-kDa AnkG displayed dual spacings of AnkG components in cultured neurons and impaired in spatial memory, while transgenic mice expressing 480-kDa AnkG showed a normal molecular distribution in the AIS and normal cognitive performance. Our findings provide new insight into the mechanisms underlying impaired cognition associated with neurodegenerative diseases such as AD.

Iterative tomography with digital adaptive optics permits hour-long intravital observation of 3D subcellular dynamics at millisecond scale.

Long-term subcellular intravital imaging in mammals is vital to study diverse intercellular behaviors and organelle functions during native physiological processes. However, optical heterogeneity, tissue opacity, and phototoxicity pose great challenges. Here, we propose a computational imaging framework, termed digital adaptive optics scanning light-field mutual iterative tomography (DAOSLIMIT), featuring high-speed, high-resolution 3D imaging, tiled wavefront correction, and low phototoxicity with a compact system. By tomographic imaging of the entire volume simultaneously, we obtained volumetric imaging across 225 × 225 × 16 µm3, with a resolution of up to 220 nm laterally and 400 nm axially, at the millisecond scale, over hundreds of thousands of time points. To establish the capabilities, we investigated large-scale cell migration and neural activities in different species and observed various subcellular dynamics in mammals during neutrophil migration and tumor cell circulation.

High-dimensional super-resolution imaging reveals heterogeneity and dynamics of subcellular lipid membranes.

Lipid membranes are found in most intracellular organelles, and their heterogeneities play an essential role in regulating the organelles' biochemical functionalities. Here we report a Spectrum and Polarization Optical Tomography (SPOT) technique to study the subcellular lipidomics in live cells. Simply using one dye that universally stains the lipid membranes, SPOT can simultaneously resolve the membrane morphology, polarity, and phase from the three optical-dimensions of intensity, spectrum, and polarization, respectively. These high-throughput optical properties reveal lipid heterogeneities of ten subcellular compartments, at different developmental stages, and even within the same organelle. Furthermore, we obtain real-time monitoring of the multi-organelle interactive activities of cell division and successfully reveal their sophisticated lipid dynamics during the plasma membrane separation, tunneling nanotubules formation, and mitochondrial cristae dissociation. This work suggests research frontiers in correlating single-cell super-resolution lipidomics with multiplexed imaging of organelle interactome.

Enhanced reconstruction of structured illumination microscopy on a polarized specimen.

Structured illumination microscopy (SIM) requires polarization control to guarantee the high-contrast illumination pattern. However, this modulated polarization will induce artifacts in SIM when imaging fluorescent dipoles. Here we proposed the polarization weighted recombination of frequency components to reconstruct SIM data with suppressed artifacts and better resolving power. Both the simulation results and experimental data demonstrate that our algorithm can obtain isotropic resolution on dipoles and resolve a clearer structure in high-density sections compared to the conventional algorithm. Our work reinforces the SIM theory and paves the avenue for the application of SIM on a polarized specimen.

Super-resolution imaging of fluorescent dipoles via polarized structured illumination microscopy.

Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of bio-complexes. However, current techniques remain difficult to resolve the dipole assemblies on subcellular structures and their dynamics in living cells at super-resolution level. Here we report polarized structured illumination microscopy (pSIM), which achieves super-resolution imaging of dipoles by interpreting the dipoles in spatio-angular hyperspace. We demonstrate the application of pSIM on a series of biological filamentous systems, such as cytoskeleton networks and λ-DNA, and report the dynamics of short actin sliding across a myosin-coated surface. Further, pSIM reveals the side-by-side organization of the actin ring structures in the membrane-associated periodic skeleton of hippocampal neurons and images the dipole dynamics of green fluorescent protein-labeled microtubules in live U2OS cells. pSIM applies directly to a large variety of commercial and home-built SIM systems with various imaging modality.

Polarization-based super-resolution imaging of surface-enhanced Raman scattering nanoparticles with orientational information.

Raman scattering provides key information of the biological environment through light-molecule interaction; yet, it is generally very weak to detect. Surface-enhanced Raman scattering (SERS) can boost the Raman signal by several orders-of-magnitude, and thus is highly attractive for biochemical sensing. However, conventional super-resolution imaging of SERS is challenging as the Raman signal is generated from the virtual state which cannot be easily modulated as fluorescence. Here, we demonstrate super-resolution microscopy with a surface-enhanced Raman scattering (SERS) signal, with a resolution of approximately 50 nm. By modulating the polarization angle of the excitation laser, the SERS nanorods display a dramatic anisotropy effect, allowing nanoscale orientation determination of multiple dipoles with dense concentration. Furthermore, a well-established defocused analysis was performed to reconfirm the orientation accuracy of super-resolved SERS nanorods. Sub-diffraction resolution was achieved in the imaging of SERS nanorod labeled vesicles in fixed macrophages. Finally, we demonstrate dynamic SERS nanorod tracking in living macrophages, which provides not only the particle trajectory with high spatial resolution but also the rotational changes at the nanometer scale. This pioneering study paves a new way for subcellular super-resolution imaging with the SERS effect, shedding light on wider biological applications.

Developing novel methods to image and visualize 3D genomes.

To investigate three-dimensional (3D) genome organization in prokaryotic and eukaryotic cells, three main strategies are employed, namely nuclear proximity ligation-based methods, imaging tools (such as fluorescence in situ hybridization (FISH) and its derivatives), and computational/visualization methods. Proximity ligation-based methods are based on digestion and re-ligation of physically proximal cross-linked chromatin fragments accompanied by massively parallel DNA sequencing to measure the relative spatial proximity between genomic loci. Imaging tools enable direct visualization and quantification of spatial distances between genomic loci, and advanced implementation of (super-resolution) microscopy helps to significantly improve the resolution of images. Computational methods are used to map global 3D genome structures at various scales driven by experimental data, and visualization methods are used to visualize genome 3D structures in virtual 3D space-based on algorithms. In this review, we focus on the introduction of novel imaging and visualization methods to study 3D genomes. First, we introduce the progress made recently in 3D genome imaging in both fixed cell and live cells based on long-probe labeling, short-probe labeling, RNA FISH, and the CRISPR system. As the fluorescence-capturing capability of a particular microscope is very important for the sensitivity of bioimaging experiments, we also introduce two novel super-resolution microscopy methods, SDOM and low-power super-resolution STED, which have potential for time-lapse super-resolution live-cell imaging of chromatin. Finally, we review some software tools developed recently to visualize proximity ligation-based data. The imaging and visualization methods are complementary to each other, and all three strategies are not mutually exclusive. These methods provide powerful tools to explore the mechanisms of gene regulation and transcription in cell nuclei.

Super-resolution dipole orientation mapping via polarization demodulation.

Fluorescence polarization microscopy (FPM) aims to detect the dipole orientation of fluorophores and to resolve structural information for labeled organelles via wide-field or confocal microscopy. Conventional FPM often suffers from the presence of a large number of molecules within the diffraction-limited volume, with averaged fluorescence polarization collected from a group of dipoles with different orientations. Here, we apply sparse deconvolution and least-squares estimation to fluorescence polarization modulation data and demonstrate a super-resolution dipole orientation mapping (SDOM) method that resolves the effective dipole orientation from a much smaller number of fluorescent molecules within a sub-diffraction focal area. We further apply this method to resolve structural details in both fixed and live cells. For the first time, we show that different borders of a dendritic spine neck exhibit a heterogeneous distribution of dipole orientation. Furthermore, we illustrate that the dipole is always perpendicular to the direction of actin filaments in mammalian kidney cells and radially distributed in the hourglass structure of the septin protein under specific labelling. The accuracy of the dipole orientation can be further mapped using the orientation uniform factor, which shows the superiority of SDOM compared with its wide-field counterpart as the number of molecules is decreased within the smaller focal area. Using the inherent feature of the orientation dipole, the SDOM technique, with its fast imaging speed (at sub-second scale), can be applied to a broad range of fluorescently labeled biological systems to simultaneously resolve the valuable dipole orientation information with super-resolution imaging.