Development of correlative light and electron microscopy to observe green fluorescent protein-labeled organelles embedded in resin using field-emission electron scanning microscope

Abstract Correlative light and electron microscopy (CLEM) enables ultrastructural-level analysis of fluorescence-labeled proteins by combining images obtained from both fluorescence and electron microscopies. A technical challenge with the CLEM method is the effective detection of fluorescence from samples embedded in resins, which generally cause fluorescence decay. To overcome this issue, we developed a method for fluorescence recovery of green fluorescent protein (GFP) in resin-embedded semi-thin sections using commercially available antifade reagents. By applying this method, we successfully obtained CLEM images using field-emission scanning electron microscopy with moderately enhanced GFP signals, demonstrating the efficacy of this simple fluorescence recovery method.

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Out with the Old, In with the New

Microscopy Today ◽

10.1017/s1551929500052251 ◽

2003 ◽

Vol 11 (1) ◽

pp. 3-4

Author(s):

Stephen W. Carmichael

Keyword(s):

Electron Microscopy ◽

Green Fluorescent Protein ◽

Temporal Resolution ◽

Fluorescent Protein ◽

Fluorescence And Electron Microscopy ◽

A Cell ◽

Green Fluorescent ◽

Microscopic Techniques ◽

Biologic System ◽

Over Time

Temporal resolution has long been a challenge to microscopists. Certainly, spatial resolution has occupied center stage, but we're all concerned about what happens over time in a biologic system, for example, a cell. Tags such as green fluorescent protein (GFP) have been used with confocal microscopy and other light microscopic techniques to achieve outstanding temporal resolution, but good spatial and temporal resolution have proven to be difficult to achieve simultaneously. This has been accomplished in a remarkable study by Guido Gaietta, Thomas Deerinck, Stephen Adams, James Bouwer, Oded Tour, Dale Laird, Gina Sosinsky, Roger Tsien, and Mark Ellisman, who demonstrated a pulse-chase technique that correlates with both fluorescence and electron microscopy.

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Localization of Green Fluorescent Protein Absorbance by Energy Filtered Transmission Electron Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927600034115 ◽

2000 ◽

Vol 6 (S2) ◽

pp. 324-325

Author(s):

J. A. Davis ◽

R. G. Garces ◽

J.-Y. Diao ◽

F. P. Ottensmeyer

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Green Fluorescent Protein ◽

Energy Loss ◽

Energy Resolution ◽

Fluorescent Protein ◽

High Energy ◽

High Energy Resolution ◽

Transmission Electron ◽

Green Fluorescent

Energy filtered transmission electron microscopy has the potential to provide high resolution, spatially resolved, atomic and chemical information. However, aberrations generated by the electron spectrometer blur the energy resolution and limit the atomic or molecular distributions that can be studied. Energy absorptions corresponding to the visible light range fall below an energy loss of 5 eV. The selection of electrons that have lost an amount of energy corresponding to chromophore absorption by the sample thus requires a spectrometer with a high energy resolution over the full image plane. A corrected prism-mirror-prism filter that has a resolution of 1.1 eV, sufficient to select these low energy loss electrons, was developed and installed by us in a Zeiss EM902. Its imaging capability was verified for a number of different chromophores. The chromophore currently under study is that of the green fluorescent protein (GFP).

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Visualizing Green Fluorescent Protein and Fluorescence Associated with a Gold Conjugate in Thin Sections with Correlative Confocal and Electron Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927604886781 ◽

2004 ◽

Vol 10 (S02) ◽

pp. 156-157 ◽

Cited By ~ 2

Author(s):

Paul. A Sims ◽

Jeff Hardin

Keyword(s):

Electron Microscopy ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Thin Sections ◽

Gold Conjugate ◽

Green Fluorescent ◽

Confocal And Electron Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

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Better Protein Localization

Microscopy Today ◽

10.1017/s1551929511000861 ◽

2011 ◽

Vol 19 (5) ◽

pp. 8-10

Author(s):

Stephen W. Carmichael ◽

Philip Oshel

Keyword(s):

Electron Microscopy ◽

Green Fluorescent Protein ◽

Cell Membrane ◽

Fluorescence Microscopy ◽

Light Microscopy ◽

Fluorescent Protein ◽

Protein Localization ◽

On Line ◽

Specific Proteins ◽

Green Fluorescent

Localizing specific proteins within cells, tissues, and organisms has been a goal of microscopists for generations. In the early 1990s, a breakthrough was made when a molecule originally derived from a jellyfish was introduced as a probe for fluorescence microscopy. This molecule is green fluorescent protein (GFP), and it has become well known for its usefulness in localizing proteins at the level of the light microscope. It is also well known that electron microscopy (EM) offers far superior spatial resolution over light microscopy, but the application of probes to localize specific proteins has required antibodies conjugated with colloidal metals (such as gold). Delivery of antibodies into the cell commonly requires detergents to permeabilize the cell membrane, which compromises the ultrastructural detail. Another breakthrough was recently published on-line by Xiaokun Shu, Varda Lev-Ram, Thomas Deerinck, Yingchuan Qi, Ericka Ramko, Michael Davidson, Yishi Jin, Mark Ellisman, and Roger Tsien: they have developed a method similar to using GFP for light microscopy, but for specifically tagging proteins at the EM level.

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Gold labelling of Green Fluorescent Protein (GFP)-tag inside cells using recombinant nanobodies conjugated to 2.4 nm thiolate-coated gold nanoparticles

Nanoscale Advances ◽

10.1039/d1na00256b ◽

2021 ◽

Author(s):

Nadja Groysbeck ◽

Mariel Donzeau ◽

Audrey Stoessel ◽

Anne Marie Haeberlé ◽

Stéphane Ory ◽

...

Keyword(s):

Electron Microscopy ◽

Gold Nanoparticles ◽

Green Fluorescent Protein ◽

High Resolution ◽

Fluorescent Protein ◽

High Resolution Electron Microscopy ◽

Resolution Electron ◽

Gold Labelling ◽

Green Fluorescent ◽

Gfp Tag

The advances in the microscopy technology have prompted efforts to improve the reagents required to recognize specific molecules within the intracellular environment. For high-resolution electron microscopy, conjugation of selective binders...

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Fusion of DARPin to aldolase enables visualization of small protein by cryoEM

10.1101/455063 ◽

2018 ◽

Cited By ~ 3

Author(s):

Qing Yao ◽

Sara J. Weaver ◽

Jee-Young Mock ◽

Grant J. Jensen

Keyword(s):

Electron Microscopy ◽

Green Fluorescent Protein ◽

Phage Display ◽

Single Particle ◽

Fluorescent Protein ◽

Ankyrin Repeat ◽

Rabbit Muscle ◽

Small Protein ◽

Ankyrin Repeat Protein ◽

Green Fluorescent

AbstractIn recent years, solving protein structures by single particle cryogenic electron microscopy (cryoEM) has become a crucial tool in structural biology. While exciting progress is being made towards the visualization of smaller and smaller macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of single particle cryoEM. To overcome this problem, we implemented a platform strategy in which a small protein target was rigidly attached to a large, symmetric base via a selectable adapter. Seven designs were tested. In the best construct, a designed ankyrin repeat protein (DARPin) was rigidly fused to tetrameric rabbit muscle aldolase through a helical linker. The DARPin retained its ability to bind its target, the 27 kDa green fluorescent protein (GFP). We solved the structure of this complex to 3.0 Å resolution overall, with 5 to 8 Å resolution in the GFP region. As flexibility in the DARPin limited the overall resolution of the target, we describe strategies to rigidify this element.Author summarySingle particle cryogenic electron microscopy (cryoEM) is a technique that uses images of purified proteins to determine their atomic structure. Unfortunately, the majority of proteins in the human and bacterial proteomes are too small to be analyzed by cryoEM. Over the years, several groups have suggested the use of a platform to increase the size of small protein targets. The platform is composed of a large protein base and a selectable adapter that binds the target protein. Here we report a platform based on tetrameric rabbit muscle aldolase that is fused to a Designed Ankyrin Repeat Protein (DARPin). Phage display libraries can be used to generate DARPins against target proteins. The residues mutated in a phage display library to generate a DARPin against a new target do not overlap with the DARPin-base fusion in the platform, thus changing the DARPin identity will not disrupt the platform design. The DARPin adapter used here is capable of binding Green Fluorescent Protein (GFP). We report the structure of GFP to 5 to 8 Å local resolution by single particle cryoEM. Our analysis demonstrates that flexibility in the DARPin-aldolase platform prevents us from achieving higher resolution in the GFP region. We suggest changes to the DARPin design to rigidify the DARPin-aldolase platform. This work expands on current platforms and paves a generally applicable way toward structure determination of small proteins by cryoEM.

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Streamlined Embedding of Cell Monolayers on Gridded Glass-Bottom Imaging Dishes for Correlative Light and Electron Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927610094092 ◽

2010 ◽

Vol 16 (6) ◽

pp. 747-754 ◽

Cited By ~ 22

Author(s):

Hugo H. Hanson ◽

James E. Reilly ◽

Rebecca Lee ◽

William G. Janssen ◽

Greg R. Phillips

Keyword(s):

Electron Microscopy ◽

Laser Scanning ◽

Fluorescent Protein ◽

Light And Electron Microscopy ◽

Laser Scanning Confocal Microscopy ◽

Step Method ◽

Cell Monolayers ◽

Scanning Confocal Microscopy ◽

Transmission Electron ◽

Green Fluorescent

AbstractCorrelative light and electron microscopy (CLEM) has facilitated study of intracellular trafficking. Routine application of CLEM would be advantageous for many laboratories but previously described techniques are particularly demanding, even for those with access to laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). We describe streamlined methods for TEM of green fluorescent protein (GFP)-labeled organelles after imaging by LSCM using gridded glass bottom imaging dishes. GFP-MAP 1A/1B LC3 (GFP-LC3) transfected cells were treated with rapamycin, fixed and imaged by LSCM. Confocal image stacks were acquired enabling full visualization of each GFP-LC3 labeled organelle. After LSCM, cells were embedded for TEM using a simplified two step method that stabilizes the glass bottom such that the block can be separated from the glass by mild heating. All imaging and TEM processing are performed in the same dish. The LSCM imaged cells were relocated on the block and serial sectioned. Correlation of LSCM, DIC, and TEM images was facilitated by cellular landmarks. All GFP labeled structures were successfully reidentified and imaged by serial section TEM. This method could make CLEM more accessible to nonspecialized laboratories with basic electron microscopy expertise and could be used routinely to confirm organelle localization of fluorescent puncta.

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