Correlative Fluorescence and Electron Microscopy in 3D—Scanning Electron Microscope Perspective

Jonathan Franks1, Callen T. Wallace1, Masateru Shibata2, Mitsuo Suga3, Natasha Erdman2, Donna B. Stolz1, Simon C. Watkins1

1 Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, Pennsylvania, 2 JEOL USA Inc., Peabody, Massachusetts, 3 JEOL Ltd., Akishima, Tokyo
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.45
DOI:  10.1002/cpcy.18
Online Posting Date:  April, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The ability to correlate fluorescence microscopy (FM) and electron microscopy (EM) data obtained on biological (cell and tissue) specimens is essential to bridge the resolution gap between the data obtained by these different imaging techniques. In the past such correlations were limited to either EM navigation in two dimensions to the locations previously highlighted by fluorescence markers, or subsequent high‐resolution acquisition of tomographic information using a TEM. We present a novel approach whereby a sample previously investigated by FM is embedded and subjected to sequential mechanical polishing and backscatter imaging by scanning electron microscope. The resulting three dimensional EM tomogram of the sample can be directly correlated to the FM data. © 2017 by John Wiley & Sons, Inc.

Keywords: EM tomography; fluorescence; SEM; mechanical polishing; low vacuum

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Significance Statement
  • Introduction
  • Basic Protocol 1: Specimen Preparation for FM And EM
  • Basic Protocol 2: Sequential Mechanical Polishing and SEM Image Acquisition
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Specimen Preparation for FM And EM

  Materials
  • Cultured mammalian cells (e.g., HeLa) or fresh tissue or animal (e.g., mouse kidneys)
  • DMEM, complete (see recipe)
  • 2% (w/v) paraformaldehyde (see recipe)
  • 1× PBS
  • 30% (w/v) sucrose in PBS
  • 2‐methylbutane (Fisher Scientific, cat. no. O3551‐4)
  • Neg‐50 (Fisher Scientific, cat. no. 22‐110‐617)
  • Triton X‐100 (Sigma‐Aldrich, cat. no. 9002‐93‐ 1)
  • 5% (v/v) normal goat serum
  • 0.5% (w/v) bovine serum albumin (Sigma‐Aldrich, cat. no. A4503) in PBS (PBB)
  • Rabbit anti‐Tom20 (Santa Cruz, cat. no. sc‐11415)
  • Cy3‐labeled goat anti‐rabbit (Jackson Immunoresearch, cat. no. 111‐165‐003)
  • Alexa Fluor 488 phalloidin (Thermo Fisher Scientific, cat. no. A12379)
  • Hoechst (Sigma‐Aldrich, cat. no. B‐2883)
  • 1% (w/v) osmium tetroxide (OsO 4; see recipe)
  • 1% (w/v) tannic acid (see recipe)
  • 2% aqueous uranyl acetate (see recipe)
  • Lead aspartate en bloc stain (see recipe)
  • 30%, 50%, 70%, and 90% (v/v) ethanol in ddH 2O
  • 100% (absolute) ethanol
  • Poly/Bed 812 resin (see recipe)
  • Large gelatin capsules, 13‐mm (Electron Microscopy Sciences, cat. no. 70114)
  • 12‐ and 22‐mm glass‐bottom dish (WillCo Wells, cat. no. GWST‐3512 and GWST‐3522)
  • Liquid nitrogen
  • Cryostat
  • Nikon A1 confocal (or similar; e.g., Leica SP8), configured with 20×/0.75 NA and 60×/1.40 NA objectives, 405 nm, 488 nm, 561 nm, and 647 nm laser lines, stage automation, and montage capability
  • NIS‐Elements software (Nikon), or similar
  • Aluminum foil
  • 60°C water bath (Tapia et al., )
  • 65°C oven

Basic Protocol 2: Sequential Mechanical Polishing and SEM Image Acquisition

  Materials
  • Resin‐embedded sample in gelatin capsule on optical dish (from protocol 1)
  • 500 ml of 80°C tap water
  • Liquid nitrogen in a shallow container
  • SEM specimen holder
  • Handy Lap (JEOL Ltd.) polishing device
  • Schottky field emission SEM (JEOL JSM‐7200 F‐LV)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Bullen, A., West, T., Moores, C. A., Ashmore, J., Fleck, R. A., MacLellan‐Gibson, K., & Forge, A. (2015). Association of intracellular and synaptic organization in cochlear inner hair cells revealed by 3D electron microscopy. Journal of Cell Science, 128, 2529–2540. doi: 10.1242/jcs.170761
  Clegg, R. M. (2009). Förster resonance energy transfer—FRET: What is it, why do it, and how it's done. FRET FLIM Technology, 1, 1–57. doi: 10.1016/S0075‐7535(08)00001‐6
  Denk, W., & Horstmann, H. (2004). Serial block‐face scanning electron microscopy to reconstruct three‐dimensional tissue nanostructure. PLoS Biology, 2, e329. doi: 10.1371/journal.pbio.0020329
  Koster, A. J., & Barcena, M. (2006). Cryotomography: Low‐dose automated tomography of frozen hydrated specimens. In J. Frank (Ed.), Electron tomography: Methods for three‐dimensional visualization of structures in the cell (2nd ed.) (pp. 113–161). New York: Springer.
  Leung, B. O., & Chou, K. C. (2011). Review of super resolution fluorescence microscopy for biology. Applied Spectroscopy, 65, 967–980. doi: 10.1366/11‐06398
  Schultz, C. (2009). Fluorescent revelations. Chemistry & Biology, 16, 107–111. doi: 10.1016/j.chembiol.2009.02.001
  Simionescu, N., & Simionescu, M. (1976). Galloylglucoses of low molecular weight as mordant in electron microscopy. I. Procedure, and evidence for mordanting effect. Journal of Cell Biology, 70, 608–621. doi: 10.1083/jcb.70.3.608
  Tapia, J. C., Kasthuri, N., Hayworth, K., Schalek, R., Lichtman, J. W., Smith, S. J., & Buchanan, J. (2012). High contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy. Nature Protocols, 7, 193–206. doi: 10.1038/nprot.2011.439
  Walton, J. (1979). Lead aspartate, an en bloc contrast stain particularly useful for ultrastructural enzymology. The Journal of Histochemistry and Cytochemistry, 10, 1337–1342. doi: 10.1177/27.10.512319
  Zhang, P. (2013). Correlative cryo‐electron tomography and optical microscopy of cells. Current Opinion in Structural Biology, 23, 763‐770. doi: 10.1016/j.sbi.2013.07.017
  Zhang, X., & Liu, Z. (2008). Superlenses to overcome the diffraction limit. Nature Materials, 7, 435–441. doi: 10.1038/nmat2141
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library