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

2022 ◽  
Vol 11 (1) ◽  
Author(s):  
Meiling Guan ◽  
Miaoyan Wang ◽  
Karl Zhanghao ◽  
Xu Zhang ◽  
Meiqi Li ◽  
...  
Keyword(s):  
Fluorescence Anisotropy ◽  
Structural Changes ◽  
Super Resolution ◽  
Live Cells ◽  
Polarization Modulation ◽  
Polarization Microscopy ◽  
Dipole Orientation ◽  
Subcellular Organelles ◽  
Lock In

AbstractThe 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.

scholarly journals Super-resolution fluorescence polarization microscopy

2017 ◽  
Vol 11 (01) ◽  
pp. 1730002 ◽  
Author(s):  
Karl Zhanghao ◽  
Juntao Gao ◽  
Dayong Jin ◽  
Xuedian Zhang ◽  
Peng Xi
Keyword(s):  
Fluorescence Polarization ◽  
Super Resolution ◽  
Biological Imaging ◽  
Biological Macromolecules ◽  
Polarization Microscopy ◽  
Dipole Orientation ◽  
Fluorescence Polarization Microscopy ◽  
Microscopy Techniques ◽  
Cellular Organelles

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.

High spatiotemporal resolution and low photo-toxicity fluorescence imaging in live cells and in vivo

2019 ◽  
Vol 47 (6) ◽  
pp. 1635-1650 ◽  
Author(s):  
Xiaohong Peng ◽  
Xiaoshuai Huang ◽  
Ke Du ◽  
Huisheng Liu ◽  
Liangyi Chen
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
Cell Biology ◽  
Critical Role ◽  
Super Resolution ◽  
Light Sheet ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
And Function

Taking advantage of high contrast and molecular specificity, fluorescence microscopy has played a critical role in the visualization of subcellular structures and function, enabling unprecedented exploration from cell biology to neuroscience in living animals. To record and quantitatively analyse complex and dynamic biological processes in real time, fluorescence microscopes must be capable of rapid, targeted access deep within samples at high spatial resolutions, using techniques including super-resolution fluorescence microscopy, light sheet fluorescence microscopy, and multiple photon microscopy. In recent years, tremendous breakthroughs have improved the performance of these fluorescence microscopies in spatial resolution, imaging speed, and penetration. Here, we will review recent advancements of these microscopies in terms of the trade-off among spatial resolution, sampling speed and penetration depth and provide a view of their possible applications.

scholarly journals Probing DNA interactions with proteins using a single-molecule toolbox: inside the cell, in a test tube and in a computer

2015 ◽  
Vol 43 (2) ◽  
pp. 139-145 ◽  
Author(s):  
Adam J. M. Wollman ◽  
Helen Miller ◽  
Zhaokun Zhou ◽  
Mark C. Leake
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Super Resolution ◽  
Magnetic Tweezers ◽  
In Vivo Studies ◽  
Live Cells ◽  
Molecular Heterogeneity ◽  
Molecular Signatures

DNA-interacting proteins have roles in multiple processes, many operating as molecular machines which undergo dynamic meta-stable transitions to bring about their biological function. To fully understand this molecular heterogeneity, DNA and the proteins that bind to it must ideally be interrogated at a single molecule level in their native in vivo environments, in a time-resolved manner, fast enough to sample the molecular transitions across the free-energy landscape. Progress has been made over the past decade in utilizing cutting-edge tools of the physical sciences to address challenging biological questions concerning the function and modes of action of several different proteins which bind to DNA. These physiologically relevant assays are technically challenging but can be complemented by powerful and often more tractable in vitro experiments which confer advantages of the chemical environment with enhanced detection signal-to-noise of molecular signatures and transition events. In the present paper, we discuss a range of techniques we have developed to monitor DNA–protein interactions in vivo, in vitro and in silico. These include bespoke single-molecule fluorescence microscopy techniques to elucidate the architecture and dynamics of the bacterial replisome and the structural maintenance of bacterial chromosomes, as well as new computational tools to extract single-molecule molecular signatures from live cells to monitor stoichiometry, spatial localization and mobility in living cells. We also discuss recent developments from our laboratory made in vitro, complementing these in vivo studies, which combine optical and magnetic tweezers to manipulate and image single molecules of DNA, with and without bound protein, in a new super-resolution fluorescence microscope.

scholarly journals Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals

2011 ◽  
Vol 193 (6) ◽  
pp. 1065-1081 ◽  
Author(s):  
Bradley S. DeMay ◽  
Xiaobo Bai ◽  
Louisa Howard ◽  
Patricia Occhipinti ◽  
Rebecca A. Meseroll ◽  
...  
Keyword(s):  
Fluorescence Anisotropy ◽  
Dipole Moments ◽  
Live Cells ◽  
Epithelial Cell Line ◽  
Polarized Fluorescence ◽  
Additional Support ◽  
Membrane Compartmentalization ◽  
Order Structures

The septins are conserved, GTP-binding proteins important for cytokinesis, membrane compartmentalization, and exocytosis. However, it is unknown how septins are arranged within higher-order structures in cells. To determine the organization of septins in live cells, we developed a polarized fluorescence microscopy system to monitor the orientation of GFP dipole moments with high spatial and temporal resolution. When GFP was fused to septins, the arrangement of GFP dipoles reflected the underlying septin organization. We demonstrated in a filamentous fungus, a budding yeast, and a mammalian epithelial cell line that septin proteins were organized in an identical highly ordered fashion. Fluorescence anisotropy measurements indicated that septin filaments organized into pairs within live cells, just as has been observed in vitro. Additional support for the formation of pairs came from the observation of paired filaments at the cortex of cells using electron microscopy. Furthermore, we found that highly ordered septin structures exchanged subunits and rapidly rearranged. We conclude that septins assemble into dynamic, paired filaments in vivo and that this organization is conserved from yeast to mammals.

Fine Structural Changes in the Microvasculature of the Mouse Pinna: Aging and Radiation Injury

1974 ◽  
Vol 32 ◽  
pp. 54-55
Author(s):  
S. Phyllis Steamer ◽  
Rosemarie L. Devine
Keyword(s):  
Structural Changes ◽  
Radiation Treatment ◽  
Arterial Stenosis ◽  
Ultrastructural Changes ◽  
Vascular Effects ◽  
Microvascular Changes ◽  
Surrounding Connective Tissue ◽  
Fission Neutrons ◽  
Total Body

The importance of radiation damage to the skin and its vasculature was recognized by the early radiologists. In more recent studies, vascular effects were shown to involve the endothelium as well as the surrounding connective tissue. Microvascular changes in the mouse pinna were studied in vivo and recorded photographically over a period of 12-18 months. Radiation treatment at 110 days of age was total body exposure to either 240 rad fission neutrons or 855 rad 60Co gamma rays. After in vivo observations in control and irradiated mice, animals were sacrificed for examination of changes in vascular fine structure. Vessels were selected from regions of specific interest that had been identified on photomicrographs. Prominent ultrastructural changes can be attributed to aging as well as to radiation treatment. Of principal concern were determinations of ultrastructural changes associated with venous dilatations, segmental arterial stenosis and tortuosities of both veins and arteries, effects that had been identified on the basis of light microscopic observations. Tortuosities and irregularly dilated vein segments were related to both aging and radiation changes but arterial stenosis was observed only in irradiated animals.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

scholarly journals 3D super-resolved imaging in live cells using sub-diffractive plasmonic localization of hybrid nanopillar arrays

Nanophotonics ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2847-2859
Author(s):  
Soojung Kim ◽  
Hyerin Song ◽  
Heesang Ahn ◽  
Seung Won Jun ◽  
Seungchul Kim ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Imaging Techniques ◽  
Optical Loss ◽  
Super Resolution ◽  
Living Cells ◽  
Live Cells ◽  
Complex Biological System ◽  
Observation Volume ◽  
Resolution Imaging ◽  
Nanopillar Arrays

AbstractAnalysing dynamics of a single biomolecule using high-resolution imaging techniques has been had significant attentions to understand complex biological system. Among the many approaches, vertical nanopillar arrays in contact with the inside of cells have been reported as a one of useful imaging applications since an observation volume can be confined down to few-tens nanometre theoretically. However, the nanopillars experimentally are not able to obtain super-resolution imaging because their evanescent waves generate a high optical loss and a low signal-to-noise ratio. Also, conventional nanopillars have a limitation to yield 3D information because they do not concern field localization in z-axis. Here, we developed novel hybrid nanopillar arrays (HNPs) that consist of SiO2 nanopillars terminated with gold nanodisks, allowing extreme light localization. The electromagnetic field profiles of HNPs are obtained through simulations and imaging resolution of cell membrane and biomolecules in living cells are tested using one-photon and 3D multiphoton fluorescence microscopy, respectively. Consequently, HNPs present approximately 25 times enhanced intensity compared to controls and obtained an axial and lateral resolution of 110 and 210 nm of the intensities of fluorophores conjugated with biomolecules transported in living cells. These structures can be a great platform to analyse complex intracellular environment.

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