scholarly journals Revealing Nanoscale Morphology of the Primary Cilium Using Super-Resolution Fluorescence Microscopy

2019 ◽  
Vol 116 (2) ◽  
pp. 319-329 ◽  
Author(s):  
Joshua Yoon ◽  
Colin J. Comerci ◽  
Lucien E. Weiss ◽  
Ljiljana Milenkovic ◽  
Tim Stearns ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Primary Cilium ◽  
Super Resolution ◽  
Nanoscale Morphology

scholarly journals Revealing the nanoscale morphology of the primary cilium using super-resolution fluorescence microscopy

2018 ◽  
Author(s):  
Joshua Yoon ◽  
Colin J. Comerci ◽  
Lucien E. Weiss ◽  
Ljiljana Milenkovic ◽  
Tim Stearns ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Primary Cilium ◽  
Mammalian Cells ◽  
Morphological Changes ◽  
Three Dimensional ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Underlying Structure ◽  
Nanoscale Morphology

ABSTRACTSuper-resolution (SR) microscopy has been used to observe structural details beyond the diffraction limit of ~250 nm in a variety of biological and materials systems. By combining this imaging technique with both computer-vision algorithms and topological methods, we reveal and quantify the nanoscale morphology of the primary cilium, a tiny tubular cellular structure (~2-6 μm long and 200-300 nm diameter). The cilium in mammalian cells protrudes out of the plasma membrane and is important in many signaling processes related to cellular differentiation and disease. After tagging individual ciliary transmembrane proteins, specifically Smoothened (SMO), with single fluorescent labels in fixed cells, we use three-dimensional (3D) single-molecule SR microscopy to determine their positions with a precision of 10-25 nm. We gain a dense, pointillistic reconstruction of the surfaces of many cilia, revealing large heterogeneity in membrane shape. A Poisson surface reconstruction (PSR) algorithm generates a fine surface mesh, allowing us to characterize the presence of deformations by quantifying the surface curvature. Upon impairment of intracellular cargo transport machinery by genetic knockout or small-molecule treatment of cells, our quantitative curvature analysis shows significant morphological differences not visible by conventional fluorescence microscopy techniques. Furthermore, using a complementary SR technique, 2-color, 2D STimulated Emission Depletion (STED) microscopy, we find that the cytoskeleton in the cilium, the axoneme, also exhibits abnormal morphology in the mutant cells, similar to our 3D results on the SMO-measured ciliary surface. Our work combines 3D SR microscopy and computational tools to quantitatively characterize morphological changes of the primary cilium under different treatments and uses STED to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest.

scholarly journals Quantifying Nanoscale Morphological Features of the Primary Cilium Membrane using Super-Resolution Fluorescence Microscopy

2018 ◽  
Vol 114 (3) ◽  
pp. 268a
Author(s):  
Joshua Yoon ◽  
Lucien Weiss ◽  
Ljiljana Milenkovic ◽  
Tim Stearns ◽  
W.E. Moerner
Keyword(s):  
Fluorescence Microscopy ◽  
Primary Cilium ◽  
Super Resolution ◽  
Morphological Features

scholarly journals Advanced Static and Dynamic Fluorescence Microscopy Techniques to Investigate Drug Delivery Systems

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 861
Author(s):  
Jacopo Cardellini ◽  
Arianna Balestri ◽  
Costanza Montis ◽  
Debora Berti
Keyword(s):  
Drug Delivery ◽  
Fluorescence Microscopy ◽  
Drug Delivery Systems ◽  
Laser Scanning ◽  
Super Resolution ◽  
Laser Scanning Confocal Microscopy ◽  
Delivery Systems ◽  
Scanning Confocal Microscopy ◽  
Biological Phenomena

In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments.

scholarly journals Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

The Analyst ◽  
2014 ◽  
Vol 139 (12) ◽  
pp. 3174-3178 ◽  
Author(s):  
Ian L. Gunsolus ◽  
Dehong Hu ◽  
Cosmin Mihai ◽  
Samuel E. Lohse ◽  
Chang-soo Lee ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Cell Surface ◽  
Bacterial Cell ◽  
Super Resolution ◽  
Bacterial Cell Surface

Super-resolution FRET measurements

Nanoscale ◽  
2021 ◽  
Author(s):  
Fernando D Stefani ◽  
Alan M. M. Szalai ◽  
Cecilia Zaza
Keyword(s):  
Energy Transfer ◽  
Fluorescence Microscopy ◽  
Biological Systems ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Dynamic Architecture ◽  
Förster Resonance

Super-resolution fluorescence microscopy and Förster Resonance Energy Transfer (FRET) form a well-established family of techniques that has provided unique tools to study the dynamic architecture and functionality of biological systems,...

scholarly journals Motion Analysis of Live Objects by Super-Resolution Fluorescence Microscopy

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Chunyan Yao ◽  
Jianwei Zhang ◽  
Guang Wu ◽  
Houxiang Zhang
Keyword(s):  
Fluorescence Microscopy ◽  
Motion Analysis ◽  
Cell Cancer ◽  
Super Resolution ◽  
Biological Engineering ◽  
Related Field ◽  
Cellular Analysis ◽  
Detection And Tracking ◽  
Motion Behavior ◽  
Analysis Of Motion

Motion analysis plays an important role in studing activities or behaviors of live objects in medicine, biotechnology, chemistry, physics, spectroscopy, nanotechnology, enzymology, and biological engineering. This paper briefly reviews the developments in this area mostly in the recent three years, especially for cellular analysis in fluorescence microscopy. The topic has received much attention with the increasing demands in biomedical applications. The tasks of motion analysis include detection and tracking of objects, as well as analysis of motion behavior, living activity, events, motion statistics, and so forth. In the last decades, hundreds of papers have been published in this research topic. They cover a wide area, such as investigation of cell, cancer, virus, sperm, microbe, karyogram, and so forth. These contributions are summarized in this review. Developed methods and practical examples are also introduced. The review is useful to people in the related field for easy referral of the state of the art.

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