Publications
Fourier Channel Attention Powered Lightweight Network for Image Segmentation
Authors: F. Zhou, Y.H. Liu, Z. Chen, K. Zhanghao*, D.Y. Jin*
IEEE Journal of Translational Engineering in Health and Medicine • 2023
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.
Laterally swept light-sheet microscopy enhanced by pixel reassignment for photon-efficient volumetric imaging
Authors: L.Qiao#, H.J.Li#, S.Y.Zhong#, X.Z.Xu, F.Su, P.Xi, D.Y. Jin*, K.Zhanghao*
Advanced Photonics Nexus • 2022
In light-sheet fluorescence microscopy, the axial resolution and field of view are mutually constrained. Axially swept light-sheet microscopy (ASLM) can decouple the trade-off, but the confocal detection scheme using a rolling shutter also rejects fluorescence signals from the specimen in the field of interest, which sacrifices the photon efficiency. Here, we report a laterally swept light-sheet microscopy (LSLM) scheme in which the focused beam is first scanned along the axial direction and subsequently laterally swept with the rolling shutter. We show that LSLM can obtain a higher photon efficiency when similar axial resolution and field of view can be achieved. Moreover, based on the principle of image scanning microscopy, applying the pixel reassignment to the LSLM images, hereby named iLSLM, improves the optical sectioning. Both simulation and experimental results demonstrate the higher photon efficiency with similar axial resolution and optical sectioning. Our proposed scheme is suitable for volumetric imaging of specimens that are susceptible to photobleaching or phototoxicity.
Polarization Modulation with Optical Lock-in Detection Reveals Universal Fluorescence Anisotropy of Subcellular Structures in Live Cells
Authors: M.L. Guan#, M.Y. Wang#, K Zhanghao#, X. Zhang, M.Q. Li, W.H. Liu, J. Niu, X.S. Yang, L. Chen, Z.L. Jing, M.Q. Zhang, D.Y. Jin, P. Xi, J.T. Gao†
Light: Science & Applications • 2022
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.
Axially Overlapped Multi-Focus Light Sheet with Enlarged Field of View
Authors: H.J. Li, Z.H. Wu, Z.C. Yang, K. Zhanghao*, P. Xi, D.Y. Jin
Applied Physics Letters • 2021
Light sheet fluorescence microscopy provides optical sectioning and is widely used in volumetric imaging of large specimens. However, the axial resolution and the lateral Field of View (FoV) of the system, defined by the light sheet, typically limit each other due to the spatial band product of the excitation objective. Here, we develop a simple multi-focus scheme to extend the FoV, where a Gaussian light sheet can be focused at three or more consecutive positions. Axially overlapped multiple light sheets significantly enlarge the FoV with improved uniformity and negligible loss in axial resolution. By measuring the point spread function of fluorescent beads, we demonstrated that the obtained light sheet has a FoV of 450 μm and a maximum axial FWHM of 7.5 μm. Compared with the conventional single-focus one, the multi-focus Gaussian light sheet displays a significantly improved optical sectioning ability over the full FoV when imaging cells and zebrafish.
High-dimensional Super-Resolution Imaging Reveals Heterogeneity and Dynamics of Subcellular Lipid Membranes
Authors: K. Zhanghao#, *, W.H. Liu#, M.Q. Li#, Z.H. Wu, X.S. Wang, X.Y. Chen, C.Y. Shan, H.Q. Wang, X.W. Chen, Q.H. Dai, D.Y. Jin*, P. Xi*
Nature Communications • 2020
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.
Structured Illumination Microscopy using Digital Micro-mirror Device and Coherent Light Source
Authors: M.Q. Li#, Y.N. Li#, W.H. Liu, A. Lai, S. Jiang, D.Y. Jin, H.P. Yang, S. Wang, K. Zhanghao*, P. Xi.*
Applied Physics Letters • 2020
Structured illumination microscopy (SIM) achieves doubled spatial resolution through exciting the specimen with high-contrast, high-frequency sinusoidal patterns. Such an illumination pattern can be generated by laser interference or incoherent structured patterns. Opto-electronic devices, such as a Spatial Light Modulator (SLM) or a Digital Micro-mirror Device (DMD), can provide rapid switch of illumination patterns for SIM. Although the DMD is much more cost-effective than the SLM, it was previously restricted in association with incoherent light sources, as its diffractive orders are related to the incident angle and the wavelength of coherent incidence. To extend its application with coherent illumination, here, we model the DMD as a blazed grating and simulate the effect with DMD pattern changes in the SIM. With careful analysis of the illumination contrast along different angles and phases, we report a fast, high-resolution, and cost-efficient SIM with DMD modulation. Our home-built laser interference-based DMD-SIM (LiDMD-SIM) reveals the nuclear pore complex and microtubule in mammalian cells with doubled spatial resolution. We further proposed the multi-color LiDMD-SIM concept by jointly employing the DMD ON/OFF states with different incident angles for different wavelengths, with high contrast and maximum resolution enhancement.
Enhanced Reconstruction of Structured Illumination Microscopy on Polarized Specimen
Authors: X. Chen#, K. Zhanghao#, M.Q. Li, W.H. Liu, P. Xi*, Q.H. Dai.*
Optics Express • 2020
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
Authors: K. Zhanghao#, *, X. Chen#, W. Liu, M.Q. Li, Y.Q. Liu, Y.M. Wang, S. Luo, X. Wang, C.Y. Shan, H. Xie, J.T. Gao, X.W. Chen, D.Y. Jin, X.D. Li, Y. Zhang, Q. Dai*, P. Xi*
Nature Communications • 2019
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.
Super-resolution Fluorescence Polarization Microscopy
Authors: K. Zhanghao, J.T. Gao, D.Y. Jin, X.D. Zhang*, P. Xi*
Journal of Innovative Optical Health Sciences • 2017
Fluorescence polarization is related to the dipole orientation of chromophores, making fluorescence polarization microscopy possible to reveal structures and functions of tagged cellular organelles and biological macromolecules. Several recent super resolution techniques have been applied to fluorescence polarization microscopy, achieving dipole measurement at nanoscale. In this review, we summarize both diffraction limited and super resolution fluorescence polarization microscopy techniques, as well as their applications in biological imaging.
Super-resolution Dipole Orientation Mapping via Polarization Demodulation
Authors: K. Zhanghao#, L. Chen#, X.S. Yang, M.Y. Wang, Z.L. Jing, H.B. Han, M. Q. Zhang, D.Y. Jin*, J.T. Gao*, P. Xi*
Light: Science & Applications • 2016
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.