Department of Biology, Howard Hughes Medical Institute, University of Utah
Watanabe, S., Richards, J., Hollopeter, G., Hobson, R. J., Davis, W. M., Jorgensen, E. M. Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy. J. Vis. Exp. (70), e3995, doi:10.3791/3995 (2012).
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.
1. High-pressure Freezing
3. Infiltration and Polymerization
Steps 3.1-3.3 are carried out in the same cryovials used for freeze-substitution. Infiltration and polymerization are carried out at -30 °C in the AFS to preserve the fluorescent protein.
5. PALM Imaging
6. SEM Imaging
7. Aligning PALM and EM Images
8. Representative Results
Histone tagged with tdEos can be stably expressed in the nematode C. elegans, and the transgenic animals were processed using the protocol described above. The PALM and electron micrographs were acquired from the same section (Figure 1). To align the images, the sum TIRF image, which sums the fluorescence over the entire time course, is overlaid on the electron micrograph. The gold nanoparticles appear in both the fluorescence and electron micrographs and can be used to align the two images using the 'transform' function in Photoshop (Figure 1A and B). Then, the same 'transform' value was applied to the PALM image (Figure 1C). At this magnification, structural detail cannot be distinguished, so we zoomed into a region near the top end of the micrograph (Figure 2). In the high magnification image, subcellular details such as a nucleus, a nucleolus, nuclear pores, and endoplasmic reticulum could be observed. Moreover, the tagged Histone molecules are exclusively localized to the nucleus but not to the nucleolus, as expected. The correlative PALM and electron microscopy thus allows for protein localization at the highest resolution.
Five problems can compromise the quality of the images. First, ice crystal damage can distort ultrastructure (Figure 3A and B). Placing specimens in a cryo-protectant such as bacteria, which reduces the propagation of ice crystals, can reduce this damage. Nevertheless, one must still screen specimens by electron microscopy and discard those with freezing artifacts. Second, GMA does not cross-link to tissues like epoxy resins, and thus specimens often break loose from the surrounding plastic and stretch, shrink or even fall out of the section (Figure 3C and D). Dissection of the sample away from the bacteria or other cryo-protectant before embedding provides for greater adhesion of the plastic to the specimen (Figure 3C). Similarly, structures such as lipid droplets in the gut often dissociate from the tissues due to the absence of cross-linking (Figure 3E). Third, the incomplete polymerization of plastic causes stretching or folding of tissues (Figure 3F); the presence of oxygen in the sample also impedes the polymerization of GMA. Fourth, the poor sectioning quality of GMA often results in an inconsistent morphology (Figure 3G and H). GMA sections should be cut at 70 nm or thicker and at a speed of around 1.6 mm/s to minimize sectioning artifacts. Fifth, background autofluorescence from dust on the coverslip or section is inevitable. Autofluorescence from dust can be minimized by using pre-cleaned coverslips and by avoiding dust contamination from kimwipes and filter paper as described in the protocol. The PALM analysis program can edit out signals from the specimen or plastic that fluoresce longer than typical signals from fluorescent proteins (Figure 2 A and B). The final image will therefore be free of such artifacts.
Figure 1. Aligning fluorescence and electron micrographs using gold nanoparticles. (A) A low magnification electron micrograph from a cross section of C. elegans expressing the tagged histone tdEos::HIS-11. White arrows indicate 100 nm electron-dense gold nanoparticles applied prior to PALM imaging that serve as fiducial marks. (B) The gold beads fluoresce upon exposure to ~580 nm light and create fiducial marks in the fluorescence image. The sum TIRF image is aligned onto an electron micrograph based on the location of the fiducial marks. The sum TIRF image represents all the photons detected by the camera during the experimental time course. Note that the bright spots on the upper left (white arrow) arise from the clusters of gold particles. (C) A PALM image is then added to the electron micrograph and rotated and translated based on the values determined from the alignment of the sum TIRF image in (B). Click here to view larger figure.
Figure 2. Correlative nano-fEM using Histone fusion proteins. (A) Sum TIRF image of tdEos::HIS-11 acquired from a thin section (70nm). (B) Corresponding PALM image of tdEos::HIS-11. Autofluorescence (white arrow) lasting longer than 500 msec was filtered out by the PALM program. (C) Electron micrograph of a nucleus acquired from the same section. (D) Correlative PALM image and electron micrograph of tdEos::HIS-11. Fluorescence is tightly localized to the chromatin in the nucleus.
Figure 3. Problems associated with nano fEM. (A) Electron micrograph of a C. elegans body muscle without ice crystal damage. (B) Electron micrograph of a body muscle with ice crystal damage. Instead of discrete cross-sections, actin and myosin filaments are collapsed into aggregates due to the formation of ice crystals. (C, D) Low magnification electron micrographs, showing the dissociation of worms from the surrounding media. The section is more distorted in a specimen that is surrounded by the cryo-protecting bacteria (D) than when the specimen is surrounded by plastic (C). The bacterial cryo-protectant in the gallette should be dissected away from the fixed sample before plastic embedding. Note that the animal on the right in (D) was sectioned obliquely, and thus the shape is not due to the distortion of tissues. (E) Electron micrograph of intestine, showing dropouts of tissues (black arrows). (F) Electron micrograph of nerve ring, showing folding of sections due to the incomplete polymerization of plastic (black arrows). (G, H) Electron micrographs of neurons from the same specimen, sectioned on different dates. The preservation of tissues is superb on one day (G), but such morphology is obscured by the inconsistent sectioning quality (H). Click here to view larger figure.
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.
Production and Free Access to this article is sponsored by Carl Zeiss, Inc.
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 Frøkjr-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.
|High-pressure freezer||ABRA||HPM 010||EMPact and HPM 100 from Leica or hpf-01 from Wohlwend can also be used.|
|Automated freeze substitution unit||Leica Microsystems||AFS 2||AFS 1 can also be used.|
|Zeiss PALM||Carl Zeiss, Inc.||ELYRA P.1||Nikon and Vutara also sell commercial PALM microscopes.|
|Scanning electron microscope||FEI||Nova nano||Other high-resolution SEM microscopes can be used.|
|Albumin from bovine serum||Sigma-Aldrich||A3059-50G|
|Uranyl acetate||Polysciences, Inc.||21447-25||pH of uranyl acetate from this company is slightly higher.|
|Glycol methacrylate (GMA)||SPI Supplies||02630-AA||Low acid, TEM grade.|
|Cryo vials||Nalge Nunc international||5000-0020|
|3/8" DISC punches||Ted Pella, Inc.||54741|
|Gold nanoparticles||microspheres-nanospheres.com||790122-010||Request 2x concentrated solution|