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  • richardmitnick 4:09 pm on October 4, 2018 Permalink | Reply
    Tags: , Microscopy technology, Super-resolution microscopy, Super-resolution microscopy builds multicolor 3D from 2D, The researchers tested the method on human centriole complexes   

    From École Polytechnique Fédérale de Lausanne: “Super-resolution microscopy builds multicolor 3D from 2D” 

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    From École Polytechnique Fédérale de Lausanne

    04.10.18
    Nik Papageorgiou

    1
    Human centrioles labelled with antibodies against two proteins (Cep152, HsSAS-6) and imaged using super-resolution microscopy. From many individual particles showing projections of the centriole complex in various orientations (upper panel), by using a fused intermediate (yellow, lower panel), the newly developed method allows now to reconstruct a multicolor 3D model (lower panel) (credit: Christian Sieben/EPFL).

    A new technique developed by EPFL overcomes the noise and color limitations of super-resolution microscopy by creating three-dimensional reconstructions from single-color, two-dimensional images of protein complexes.

    Super-resolution microscopy is a technique that can “see” beyond the diffraction limit of light. The technique has garnered increasing interest recently, especially since its developers won the Nobel Prize in Chemistry in 2014. By exploiting fluorescence, super-resolution microscopy now allows scientists to observe cells and their interior structures and organelles in a way never before possible.

    Many of the molecular complexes inside cells are made up of multiple proteins. Since current techniques of super-resolution microscopy typically can only use one or two fluorescent colors, it is difficult to observe different proteins and decipher the complex architecture and underlying assembly mechanisms of the cell’s interior structures. An even greater challenge is to overcome the noise inherent to the super-resolution methods and fluorescent labeling, to achieve the full resolution potential.

    Scientists from the lab of Suliana Manley at EPFL have now solved both problems by developing a new method to analyze and reconstruct super-resolution images and re-align them in a way that multiple proteins can be placed within a single 3D volume. The method works with images taken with large field-of-view super-resolution microscopy, with each image containing hundreds of two-dimensional projections of a labeled structure in parallel.

    Each 2D view represents a slightly different orientation of the structure, so that with a dataset of thousands of views, the method can computationally reconstruct and align the 2D images into a 3D volume. By combining information from a large number of single images, the noise is reduced and the effective resolution of the 3D reconstruction is enhanced.

    With the help of Pierre Gönczy’s lab at EPFL, the researchers tested the method on human centriole complexes. Centrioles are pairs of cylindrical molecular assemblies that are crucial in helping the cell divide. Using the new multicolor super-resolution reconstruction method, the researchers were able to uncover the 3D architecture of four proteins critical for centriolar assembly during organelle biogenesis.

    The new approach allows for unlimited multiplexing capabilities. “With this method, if the proteins in the structure can be labeled, there is no limit to the number of colors in the 3D reconstruction,” says Suliana Manley. “Plus, the reconstruction is independent of the super-resolution method used, so we expect this analysis method and software to be of broad interest.”

    Funding
    European Research Council (ERC)
    EPFL
    Horizon 2020 (MSCA-COFUND)
    NCCR Chemical Biology

    Science paper:
    Multicolor single-particle reconstruction of protein complexes
    Nature Methods

    See the full article here .

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    EPFL is Europe’s most cosmopolitan technical university. It receives students, professors and staff from over 120 nationalities. With both a Swiss and international calling, it is therefore guided by a constant wish to open up; its missions of teaching, research and partnership impact various circles: universities and engineering schools, developing and emerging countries, secondary schools and gymnasiums, industry and economy, political circles and the general public.

     
  • richardmitnick 9:39 am on March 1, 2018 Permalink | Reply
    Tags: , , Super-resolution microscopy   

    From École Polytechnique Fédérale de Lausanne: “Super-resolution microscopy in both space and time” 

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    École Polytechnique Fédérale de Lausanne

    1
    A cell visualized with the PRISM method © T. Lasser/EPFL

    28.02.18
    Nik Papageorgiou

    2
    Workshop on Super resolution microscopy – Copyright: Shem Johnson
    3

    In a breakthrough for biological imaging, EPFL scientists have developed the first microscope platform that can perform super-resolution spatial and temporal imaging, capturing unprecedented views inside living cells. The landmark paper is published in Nature Photonics.

    Super-resolution microscopy is a technique that can “see” beyond the diffraction of light, providing unprecedented views of cells and their interior structures and organelles. The technique has garnered increasing interest recently, especially since its developers won the Nobel Prize in Chemistry in 2014.

    But super-resolution microscopy comes with a big limitation: it only offers spatial resolution. That might suffice for static samples, like solid materials or fixed cells, but when it comes to biology, things become more complicated. Living cells are highly dynamic and depend on a complex set of biological processes that occur across sub- second timescales, constantly changing. So if we are to visualize and understand how living cells function in health and disease, we need a high time (or “temporal”) resolution as well.

    A team led by Professor Theo Lasser, the head of the Laboratory of Biomedical Optics (LOB) at EPFL has now made strides to address the issue by developing a technique that can perform both 3D super-resolution microscopy and fast 3D phase imaging in a single instrument. Phase imaging is a technique that translates the changes in the phase of light caused by cells and their organelles into refractive index maps of the cells themselves.

    The unique platform, which is referred as a “4D microscope”, combines the sensitivity and high time-resolution of phase imaging with the specificity and high spatial resolution of fluorescence microscopy. The researchers developed a novel algorithm that can recover the phase information from a stack of bright-field images taken by a classical microscope.

    “With this algorithm, we present a new way to achieve 3D quantitative phase microscopy using a conventional bright-field microscope,” says Adrien Descloux, one of the lead authors of the paper. “This allows direct visualization and analysis of subcellular structures in living cells without labeling.”

    To achieve fast 3D imaging, the scientists custom-designed an image-splitting prism, which allows the simultaneous recording of a stack of eight z-displaced images. This means that the microscope can perform high-speed 3D phase imaging across a volume of 2.5μm x 50μm x 50μm. The microscope’s speed is basically limited by the speed of its camera; for this demonstration, the team was able to image intracellular dynamics at up to 200 Hz. “With the prism as an add-on, you can turn a classical microscope into an ultra-fast 3D imager,” says Kristin Grussmayer, another one of the paper’s lead authors.

    The prism is also suited for 3D fluorescence imaging, which the scientists tested using super-resolution optical fluctuation imaging (SOFI). This method exploits the blinking of fluorescent dyes to improve 3D resolution through correlation analysis of the signal. Using this, the researchers performed 3D super-resolution imaging of stained structures in the cells, and combined it with 3D label-free phase imaging. The two techniques complemented each other very well, revealing fascinating images of the inner architecture, cytoskeleton, and organelles also in living cells across different time points.

    “We are thrilled by these results and the possibilities offered by this technique,” says Professor Hilal Lashuel, whose lab at EPFL teamed up with Professor Lasser’s in using the new technique to study the mechanisms by which protein aggregation contributes to the development and progression of neurodegenerative diseases, such as Parkinson’s and Alzheimer’s. “The technical advances enabled high-resolution visualization of the formation of pathological alpha synuclein aggregates in hippocampal neurons.”

    The team has named the new microscopy platform PRISM, for Phase Retrieval Instrument with Super-resolution Microscopy. “We offer PRISM as a new microscopy tool and anticipate that it will be rapidly used in the life science community to expand the scope for 3D high-speed imaging for biological investigations,” says Theo Lasser. “We hope that it will become a regular workhorse for neuroscience and biology.”

    Funding
    European Union (Horizon 2020, Marie Skłodowska-Curie Grant Agreement and AD-gut European consortium), Swiss National Science Foundation (SNSF

    See the full article here .

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    EPFL is Europe’s most cosmopolitan technical university. It receives students, professors and staff from over 120 nationalities. With both a Swiss and international calling, it is therefore guided by a constant wish to open up; its missions of teaching, research and partnership impact various circles: universities and engineering schools, developing and emerging countries, secondary schools and gymnasiums, industry and economy, political circles and the general public.

     
  • richardmitnick 7:06 am on February 1, 2018 Permalink | Reply
    Tags: , STORM-stochastic optical reconstruction microscopy, Super-resolution microscopy,   

    From UC Berkeley: “Super-resolution microscopy reveals fine detail of cellular mesh” 

    UC Berkeley

    UC Berkeley

    January 30, 2018
    Robert Sanders
    rlsanders@berkeley.edu

    One of today’s sharpest imaging tools, super-resolution microscopy, produces sparkling images of what until now has been the blurry interior of cells, detailing not only the cell’s internal organs and skeleton, but also providing insights into cells’ amazing flexibility.

    1
    Super-resolution microscopy reveals the two-dimensional triangular protein meshwork underlying the membrane of the red blood cell. Ke Xu image.

    In the current issue of the journal Cell Reports, Ke Xu and his colleagues at UC Berkeley use the technique to provide a sharp view of the geodesic mesh that supports the outer membrane of a red blood cell, revealing why such cells are sturdy yet flexible enough to squeeze through narrow capillaries as they carry oxygen to our tissues.

    The discovery could eventually help uncover how the malaria parasite hijacks this mesh, called the sub-membrane cytoskeleton, when it invades and eventually destroys red blood cells.

    “People know that the parasite interacts with the cytoskeleton, but how it does it is unclear because there has been no good way to look at the structure,” said Xu, an assistant professor of chemistry. “Now that we have resolved what is really going on in a normal healthy cell, we can ask what changes under infection with parasites and how drugs affect the interaction.”

    Typical human cells have a two-dimensional skeleton that supports the outer membrane and a three-dimensional interior skeleton that supports all the organelles inside and serves as a transportation system throughout the cell.

    Red blood cells, however, have only the membrane supports and no internal scaffolding, so they’re basically a balloon filled with molecules of oxygen-carrying hemoglobin. Because of their simpler structure, red blood cells are ideal for studying the skeleton that supports the membrane in all cells.

    Electron microscope images earlier showed that the sub-membrane cytoskeleton in red blood cells is a triangular mesh of proteins, reminiscent of a geodesic dome. But measurements of the size of the triangular subunits were made by flattening out the domed membrane of a dead and dried-out cell, which distorts the structure.

    STORMing the cytoskeleton

    Xu was a postdoctoral fellow in the Harvard University lab of one of the inventors of super-resolution microscopy, Xiaowei Zhuang, and is an expert on the version called STORM (stochastic optical reconstruction microscopy). Super-resolution microscopy gives about 10 times better resolution than standard light microscopy and works well with wet and live cells.

    2
    Labeling one end of the spectrin molecule with a dye reveals where it connects with the actin protein at the vertices of the triangular mesh. Super-resolution microscopy revealed a 80-nanometer distance between vertices, as well as unsuspected gaps in the mesh – weak points that may allow the red blood cell to reshape itself without breaking.

    Using STORM, Xu, former Berkeley postdoc Leiting Pan and graduate student Rui Yan were able to image the full sub-membrane cytoskeleton of fresh red blood cells and discovered that the triangles of the mesh are about half the size of found in earlier measurements done with electron microscopy: each side is 80 nanometers long, instead of 190 nanometers.

    The distinction is critical: The building blocks of the mesh are a protein called spectrin, which can be stretched to a maximum of about 190 nanometers in length. If the mesh were made of stretched spectrin, it would be rigid, Xu said. But since its normal length is a relaxed 80 nanometers, it acts like a spring.
    “It is more like a spring in its relaxed state, where it has much flexibility under compression or stretching, so that gives red blood cells a lot of elasticity under different physiological conditions, such as squeezing through a narrow capillary,” Yan said.

    At the vertices of the mesh, where five to six spectrin proteins come together, is a different protein: actin. Actin is a standard part of the sub-membrane cytoskeleton and one of the main structural components of the cell.

    Tears in the mesh

    Interestingly, STORM revealed never-before-seen holes in the cytoskeletal mesh that may also be critical to its flexibility.

    “This is a defect in the network, but there might be a reason for it,” said Xu, who is also a Chan Zuckerberg Biohub Investigator. “The cell would want to change structure rapidly as it goes through the capillaries, and having those defects is helpful in reorganizing the shape without breaking the mesh. It can act as a weak point as they try to squeeze through things, they can start to bend around those points.”

    3
    Labeling of the spectrin molecule in the axon of a neuron, showing that they are stretched to their full length of 190 nanometers.

    Xu actually discovered the key structural role of spectrin. While still at Harvard, he used STORM to look at the skeletal structure of neurons, and discovered that actin proteins form precisely spaced rings along the entire length of the axon – which can be as much as a foot long – much like the ribs of a snake. They are separated by exactly 190 nanometers, and when he looked through textbooks for proteins with that length, he came across spectrin. He subsequently used STORM to confirm that in its stretched state, spectrin proteins are the spacers between the rings, keeping them precisely separated.

    “The ringed skeleton makes the axon a very stable but bendable structure,” Xu said, whereas the regular spacing may be key to its electrical conductivity.

    Super-resolution microscopy employs a trick to overcome the diffraction limit of light microscopy, which prevents conventional light microscopes from resolving things smaller than half the size of the wavelength of the light, which for visible light is about 300 nanometers.

    4
    STORM can provide clear images of the interior skeleton of a cell, such as this epithelial cell.

    STORM involves attaching a blinking light source to individual molecules and then isolating each light’s position independently of the others, building up a complete image much like the 1880s artists who developed pointillism, producing images from individual dots of paint.

    Typically chemists attach these flashing sources to all molecules of the same type in a cell, such as all actin molecules, but since only a small percentage of the sources blink on at any one time, it’s possible to pinpoint the exact location of each. Today’s best resolution is about 10 nanometers, Xu said, which is about the size of a single protein or molecule.

    The work was supported by the National Natural Science Foundation of China, a Pew Biomedical Scholars Award and a Packard Fellowship for Science and Engineering. Coauthor and postdoc Wan Li contributed to experimental design and data analysis.

    See the full article here .

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