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

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.

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

Biophysical Journal ◽

10.1016/j.bpj.2018.11.3136 ◽

2019 ◽

Vol 116 (2) ◽

pp. 319-329 ◽

Cited By ~ 8

Author(s):

Joshua Yoon ◽

Colin J. Comerci ◽

Lucien E. Weiss ◽

Ljiljana Milenkovic ◽

Tim Stearns ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Primary Cilium ◽

Super Resolution ◽

Nanoscale Morphology

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3D Multicolor Nanoscopy at 10,000 Cells a Day

10.1101/606954 ◽

2019 ◽

Cited By ~ 5

Author(s):

Andrew E S Barentine ◽

Yu Lin ◽

Miao Liu ◽

Phylicia Kidd ◽

Leonhard Balduf ◽

...

Keyword(s):

Single Molecule ◽

Mammalian Cells ◽

High Speed ◽

Three Dimensional ◽

Super Resolution ◽

High Volume ◽

Volume Data ◽

Cell Nuclei ◽

Fully Integrated ◽

Resolution Imaging

ABSTRACTDiffraction-unlimited single-molecule switching (SMS) nanoscopy techniques like STORM /(F)PALM enable three-dimensional (3D) fluorescence imaging at 20-80 nm resolution and are invaluable to investigate sub-cellular organization. They suffer, however, from low throughput, limiting the output of a days worth of imaging to typically a few tens of mammalian cells. Here we develop an SMS imaging platform that combines high-speed 3D single-molecule data acquisition with an automated, fully integrated, high-volume data processing pipeline. We demonstrate 2-color 3D super-resolution imaging of over 10,000 mammalian cell nuclei in about 26 hours, connecting the traditionally low-throughput super-resolution community to the world of omics approaches.

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Light Sheet Illumination for 3D Single-Molecule Super-Resolution Imaging of Neuronal Synapses

Frontiers in Synaptic Neuroscience ◽

10.3389/fnsyn.2021.761530 ◽

2021 ◽

Vol 13 ◽

Author(s):

Gabriella Gagliano ◽

Tyler Nelson ◽

Nahima Saliba ◽

Sofía Vargas-Hernández ◽

Anna-Karin Gustavsson

Keyword(s):

Single Molecule ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Three Dimensional ◽

Super Resolution ◽

Light Sheet ◽

Three Dimensions ◽

Single Molecule Imaging ◽

Single Molecule Tracking ◽

Resolution Imaging

The function of the neuronal synapse depends on the dynamics and interactions of individual molecules at the nanoscale. With the development of single-molecule super-resolution microscopy over the last decades, researchers now have a powerful and versatile imaging tool for mapping the molecular mechanisms behind the biological function. However, imaging of thicker samples, such as mammalian cells and tissue, in all three dimensions is still challenging due to increased fluorescence background and imaging volumes. The combination of single-molecule imaging with light sheet illumination is an emerging approach that allows for imaging of biological samples with reduced fluorescence background, photobleaching, and photodamage. In this review, we first present a brief overview of light sheet illumination and previous super-resolution techniques used for imaging of neurons and synapses. We then provide an in-depth technical review of the fundamental concepts and the current state of the art in the fields of three-dimensional single-molecule tracking and super-resolution imaging with light sheet illumination. We review how light sheet illumination can improve single-molecule tracking and super-resolution imaging in individual neurons and synapses, and we discuss emerging perspectives and new innovations that have the potential to enable and improve single-molecule imaging in brain tissue.

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

10.1242/prelights.5216 ◽

2018 ◽

Author(s):

Gautam Dey

Keyword(s):

Fluorescence Microscopy ◽

Primary Cilium ◽

Super Resolution ◽

Nanoscale Morphology

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Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

International Journal of Molecular Sciences ◽

10.3390/ijms22041903 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1903

Author(s):

Ivona Kubalová ◽

Alžběta Němečková ◽

Klaus Weisshart ◽

Eva Hřibová ◽

Veit Schubert

Keyword(s):

Single Molecule ◽

Imaging Techniques ◽

Chromatin Condensation ◽

Super Resolution ◽

Stimulated Emission ◽

Wide Field ◽

Structured Illumination Microscopy ◽

Metaphase Chromosomes ◽

Localization Microscopy ◽

Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

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Three-dimensional residual channel attention networks denoise and sharpen fluorescence microscopy image volumes

10.21203/rs.3.rs-68002/v1 ◽

2020 ◽

Author(s):

Jiji Chen ◽

Hideki Sasaki ◽

Hoyin Lai ◽

Yijun Su ◽

Jiamin Liu ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Network Performance ◽

Resolution Enhancement ◽

Three Dimensional ◽

Ground Truth ◽

Time Lapse ◽

Stimulated Emission ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Attention Networks

Abstract We demonstrate residual channel attention networks (RCAN) for restoring and enhancing volumetric time-lapse (4D) fluorescence microscopy data. First, we modify RCAN to handle image volumes, showing that our network enables denoising competitive with three other state-of-the-art neural networks. We use RCAN to restore noisy 4D super-resolution data, enabling image capture over tens of thousands of images (thousands of volumes) without apparent photobleaching. Second, using simulations we show that RCAN enables class-leading resolution enhancement, superior to other networks. Third, we exploit RCAN for denoising and resolution improvement in confocal microscopy, enabling ~2.5-fold lateral resolution enhancement using stimulated emission depletion (STED) microscopy ground truth. Fourth, we develop methods to improve spatial resolution in structured illumination microscopy using expansion microscopy ground truth, achieving improvements of ~1.4-fold laterally and ~3.4-fold axially. Finally, we characterize the limits of denoising and resolution enhancement, suggesting practical benchmarks for evaluating and further enhancing network performance.

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3D tracking of extracellular vesicles by holographic fluorescence imaging

Science Advances ◽

10.1126/sciadv.abc2508 ◽

2020 ◽

Vol 6 (45) ◽

pp. eabc2508

Author(s):

Matz Liebel ◽

Jaime Ortega Arroyo ◽

Vanesa Sanz Beltrán ◽

Johann Osmond ◽

Ala Jo ◽

...

Keyword(s):

Extracellular Vesicles ◽

Single Molecule ◽

Three Dimensional ◽

Super Resolution ◽

Fluorescent Detection ◽

Live Cells ◽

Fluorescent Light ◽

3D Tracking ◽

Anisotropic Motion ◽

Lag Times

Fluorescence microscopy is the method of choice in biology for its molecular specificity and super-resolution capabilities. However, it is limited to a narrow z range around one observation plane. Here, we report an imaging approach that recovers the full electric field of fluorescent light with single-molecule sensitivity. We expand the principle of digital holography to fast fluorescent detection by eliminating the need for phase cycling and enable three-dimensional (3D) tracking of individual nanoparticles with an in-plane resolution of 15 nm and a z-range of 8 mm. As a proof-of-concept biological application, we image the 3D motion of extracellular vesicles (EVs) inside live cells. At short time scales (<4 s), we resolve near-isotropic 3D diffusion and directional transport. For longer lag times, we observe a transition toward anisotropic motion with the EVs being transported over long distances in the axial plane while being confined in the horizontal dimension.

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