Correlative photoactivated localization and scanning electron microscopy - PubMed (original) (raw)

Correlative photoactivated localization and scanning electron microscopy

Benjamin G Kopek et al. PLoS One. 2013.

Abstract

The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.

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Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflict: Harald F. Hess is a co-inventor of PALM, which was licensed to Zeiss. The full patent name and number for the PALM microscope is “Optical microscopy with phototransformable optical labels”, Patent number US 7,782,457. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1

Figure 1. Overview of correlative PALM and SEM procedure and the PALM imaging system.

(A) Flow diagram showing the steps involved in correlating PALM and SEM images. (B) PALM imaging set-up showing the 100 nm thick cryosection on an ITO coated coverslip with 80 nm Au beads that are used for image registration and alignment.

Figure 2

Figure 2. Correlated images of TFAM-mEos2 PALM data with electron micrographs.

(A) PALM image of TFAM-mEos2. (B) SEM image of mitochondria (M) in the same area imaged in A. (C) Correlated image of TFAM-mEos2 PALM data with SEM data. TFAM-mEos2 resides in the mitochondrial matrix surrounded by boundary and cristae membranes.

Figure 3

Figure 3. Correlated images of lamin B1-mEos2 PALM data with electron micrographs.

(A) Correlated image of Lamin B1-mEos2 signal with electron micrograph. White arrows point to Au beads used for alignment. (B) Higher magnification view of an area from A showing a possible nuclear pore (red arrowhead) with corresponding gap in Lamin B1-mEos2 signal. (C) SEM only image of panel B to show cellular ultrastructure. M, mitochondrion. G, Golgi apparatus.

Figure 4

Figure 4. Correlated images of peroxisome-localized mEos2-SKL PALM data with electron micrographs.

(A, C) SEM image of cells showing mitochondria (M) and other membranous organelles. Red arrowheads indicate peroxisomes as seen by overlaying PALM data of mEos2-SKL on electron micrographs (B, D).

Figure 5

Figure 5. Two-color correlative PALM and SEM data of nuclear lamina proteins.

(A) PALM image of lamin A-PS-CFP2. (B) PALM image of lamin B1-mEos2. (C) Combined lamin A-PS-CFP2 and lamin B1-mEos2 PALM data. (D) Combined two-color lamin protein PALM data correlated with electron micrograph. (E-H) Higher magnification views from boxed area in D of PALM image of lamin A-PS-CFP2 (E), PALM image of lamin B1-mEos2 (F), combined lamin A-PS-CFP2 and lamin B1-mEos2 PALM data (G), and combined two-color lamin protein PALM data correlated with electron micrograph (H). Note the cytoplasmic organelles such as mitochondria (M). White arrows in panels A-D indicate Au beads that are used to align the datasets.

Figure 6

Figure 6. Caged dye-phalloidin labeling of actin and correlated PALM/SEM.

(A) Maximum intensity projection image of confocal z-stack of a cell with actin labeled using NVOC2-carborhodamine110–PEG8–phalloidin (magenta) and the nuclear stain 4',6-diamidino-2-phenylindole (blue). (B) PALM image of caged dye labeled actin. (C) SEM of area imaged by PALM in panel B. (D) Correlated PALM and SEM of caged dye labeled actin.

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References

    1. Giepmans BNG, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312: 217–224 10.1126/science.1124618 - DOI - PubMed
    1. Morphew MK (2007) 3D immunolocalization with plastic sections. Methods Cell Biol 79: 493–513 10.1016/S0091-679X(06)79019-7 - DOI - PubMed
    1. Sjollema KA, Schnell U, Kuipers J, Kalicharan R, Giepmans BNG (2012) Correlated Light Microscopy and Electron Microscopy. Elsevier. 17 pp. doi:10.1016/B978-0-12-416026-2.00009-1. - DOI - PubMed
    1. Murphy GE, Narayan K, Lowekamp BC, Hartnell LM, Heymann JAW, et al... (2011) Correlative 3D imaging of whole mammalian cells with light and electron microscopy. J Struct Biol. doi:10.1016/j.jsb.2011.08.013. - DOI - PMC - PubMed
    1. Kukulski W, Schorb M, Welsch S, Picco A, Kaksonen M, et al. (2011) Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. The Journal of Cell Biology 192: 111–119 10.1083/jcb.201009037 - DOI - PMC - PubMed

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