We thank Harald Hess and Eric Betzig for access to the PALM microscope for proof-of-principle experiments, Richard Fetter for sharing fixation protocols, reagents and encouragement. We thank Michael Davidson, Geraldine Seydoux, Stefan Eimer, Rudolf Leube, Keith Nehrke, Christian Fr&oslash;kjr-Jensen, Aude Ada-Nguema and Marc Hammarlund for DNA constructs. We also thank Carl Zeiss Inc. for providing access to the Zeiss PAL-M, a beta version of the Zeiss Elyra P.1 PALM microscope.

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Production and Free Access to this article is sponsored by Carl Zeiss, Inc.

Here we describe how to preserve fluorescent proteins in plastic, localize the fluorescent proteins in sections, and image the ultrastructure using electron microscopy. Proteins were localized below the diffraction limit using PALM microscopy to nanometer resolution. To adapt this protocol to particular specimens, the following parameters should be considered: fluorophore, quantification, and alignment. 
The choice of fluorescent protein or organic fluorophore depends on the application and the model system. We have tested a variety of fluorescent proteins, including EGFP, YFP, Citrine, mEosFP, mEos2, tdEos, mOrange, PA-mCherry, and Dendra12. The preservation of fluorescence from each fluorophore was similar, suggesting that all fluorescent proteins can be preserved using the described method. We chose tdEos because it expressed well in C. elegans, proteins remained functional when fused to tdEos, and because its photo-activation characteristics were optimal for PALM microscopy. However, aggregation or failed expression of tdEos has been occasionally observed12. 
Depending on the application, a different fluorophore may be better suited. In many cases, it is not necessary to use a photo-activated fluorescent protein. Simple correlative fluorescence electron microscopy does not require photo-activated fluorescent protein. GFP or organic dyes can be used to image fluorescence from tagged proteins in sections above the diffraction limit. For example, one can image an axon in a neuropil using fluorescence microscopy and correlate the fluorescence signal with a particular axon in an electron micrograph by imaging the fluorescence on a fluorescence microscope. Other super-resolution techniques, such as stimulated emission depletion microscopy (STED)12, ground state depletion microscopy followed by individual molecule return (GSDIM)13, and structured illumination microscopy (SIM)14, do not require photo-activated fluorescent proteins. Moreover, super-resolution imaging techniques that use organic dyes9,15,16 or the intrinsic property of fluorescent probes17 are readily applicable. 
In PALM, the number of molecules can be quantified because fluorescence of each molecule is separated spatially and temporally. However, quantification may be misleading for four reasons: oxidation, undercounting, overcounting, and overexpression. First, a fraction of the fluorescent proteins can be denatured or oxidized during sample processing5,12. Although ~90% of the fluorescence signal was preserved through fixation and embedding in our protocol, oxidation of the fluorescent protein may occur after the specimen has been sectioned and the surface exposed to oxygen. Second, the activation of photo-activatable proteins is stochastic, and thus multiple molecules can be activated in a given diffraction limited spot8. Fluorescence from the multiple molecules will appear as one spot, and thus the total number of proteins will be undercounted. Third, a similar problem can lead to overcounting. In PALM, each fluorescent protein is localized and then "erased" by bleaching. However, fluorescent proteins can return from the dark state without being permanently bleached18. Such molecules will then be counted multiple times. Fourth, tagged proteins are expressed as transgenes and are often present in multiple copies, which can lead to overexpression. Therefore, quantification from PALM can be used to estimate but not precisely determine the number of molecules in a given location. 
The alignment of a PALM image with an electron micrograph can also be challenging because of the resolution difference in light and electron microscopy and distortion caused by the electron beam. Gold particles serve as tightly localized fiducial markers in electron micrographs. However, fluoresecence from gold particles is not photo-activated, and appears as a large diffraction-limited spot. Thus, the placement of a fluorescence image over an electron micrograph is an estimate. Distortions can also arise from interactions of electrons with the plastic section. Acrylic resins such as GMA are less stable under the electron beam, and the dimensions of the plastic can be altered. Under these circumstances, aligning the fluorescence with ultrastructure may require non-linear transformation of the fiducial markers.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent or equipment</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>High-pressure freezer</td>
 <td>ABRA</td>
 <td>HPM 010</td>
 <td>EMPact and HPM 100 from Leica or hpf-01 from Wohlwend can also 			be used.</td>
 </tr>
 <tr>
 <td>Automated freeze substitution unit</td>
 <td>Leica</td>
 <td>AFS 2</td>
 <td>AFS 1 can also be used.</td>
 </tr>
 <tr>
 <td>Zeiss PALM</td>
 <td>Zeiss</td>
 <td>ELYRA P.1</td>
 <td>Nikon and Vutara also sell commercial PALM microscopes. </td>
 </tr>
 <tr>
 <td>Scanning electron microscope</td>
 <td>FEI</td>
 <td>Nova nano</td>
 <td>Other high-resolution SEM microscopes can be used.</td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>EMS</td>
 <td>RT10016</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethanol </td>
 <td>Sigma-aldrich</td>
 <td>459844-1L</td>
 <td></td>
 </tr>
 <tr>
 <td>Osmium tetroxide</td>
 <td>EMS</td>
 <td>RT19134</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium permanganate</td>
 <td>EMS</td>
 <td>RT20200</td>
 <td></td>
 </tr>
 <tr>
 <td>Albumin from bovine serum</td>
 <td>Sigma-aldrich</td>
 <td>A3059-50G</td>
 <td></td>
 </tr>
 <tr>
 <td>Uranyl acetate</td>
 <td>Polysciences</td>
 <td>21447-25</td>
 <td>pH of uranyl acetate from this company is slightly higher.</td>
 </tr>
 <tr>
 <td>Glycol methacrylate (GMA)</td>
 <td>SPI</td>
 <td>02630-AA</td>
 <td>Low acid, TEM grade.</td>
 </tr>
 <tr>
 <td>N,N-Dimethyl-p-toluidine</td>
 <td>Sigma-aldrich</td>
 <td>D9912</td>
 <td></td>
 </tr>
 <tr>
 <td>Cryo vials</td>
 <td>Nalgene</td>
 <td>5000-0020</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass vials</td>
 <td>EMS</td>
 <td>72632</td>
 <td></td>
 </tr>
 <tr>
 <td>Aclar film</td>
 <td>EMS</td>
 <td>50425-10</td>
 <td></td>
 </tr>
 <tr>
 <td>BEEM capsule</td>
 <td>EBSciences</td>
 <td>TC</td>
 <td>Polypropylene</td>
 </tr>
 <tr>
 <td>3/8" DISC punches</td>
 <td>Ted Pella</td>
 <td>54741</td>
 <td></td>
 </tr>
 <tr>
 <td>Gold nanoparticles</td>
 <td>microspheres-nanospheres.com</td>
 <td>790122-010 </td>
 <td>Request 2x concentrated solution</td>
 </tr>
 </tbody>
</table>

nano-fem: protein localization using photo-activated localization microscopy and electron microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. 
Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. 
Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. 
We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

Watch this Scientific Journal Video about Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy at JoVE.com

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

nano-fem-protein-localization-using-photo-activated-localization

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Biology

15.9K vistas.  University of Utah. Se describe un método para localizar proteínas etiquetadas fluorescentemente en micrografías electrónicas. La fluorescencia se localiza primero mediante foto-activado microscopía de localización en secciones ultrafinas. Estas imágenes se alinean entonces a micrografías electrónicas de la misma sección.