Serial Block Face‐Scanning Electron Microscopy: A Method to Study Retinal Degenerative Phenotypes

Debarshi Mustafi1, Sandra Kikano1, Krzysztof Palczewski1

1 School of Medicine, Case Western Reserve University, Cleveland, Ohio
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo140169
Online Posting Date:  December, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Retinal degenerative conditions can vary in their clinical features and often present with subtle phenotypic features before the onset of clinically overt disease. To capture these isolated events that precipitate disease, large representative areas of the retina must be imaged at high resolution. Compared to light microscopic methods, traditional electron microscopy can provide images at sufficient resolution to detect subtle pathologic changes in the retina, but are limited to the area being surveyed. The advent of serial block face‐scanning electron microscopy (SBF‐SEM) provides the resolution needed with the unprecedented advantage of imaging large volumes of retinal tissue. Furthermore, automation of SBF‐SEM bypasses errors from manual sectioning and can produce reliable serial sections as thin as 25 nanometers. Moreover, the three‐dimensional structures generated can highlight cellular connectivity and interactions in the retina and reveal pathological changes. Using SBF‐SEM, we have identified subtle phenotypic features in mouse models of various human retinal dystrophies. This method will allow researchers to identify and monitor the time course of these pathologies. This article provides details on SBF‐SEM methodology and its application to mouse models of retinal degeneration. © 2014 by John Wiley & Sons, Inc.

Keywords: retina; retinal degeneration; scanning electron microscopy; serial block face; vision; photoreceptor(s)

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Retinal Tissue for Serial Block Face Imaging
  • Basic Protocol 2: Preparation of Retinal Tissue for Serial Block Face Imaging
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of Retinal Tissue for Serial Block Face Imaging

  Materials
  • Mice, e.g., C57BL/6J anim als (Jackson Laboratory)
  • Paraformaldehyde solution (4% in PBS by volume from stock solution; see recipe for PBS)
  • Osmium tetroxide (OsO 4) solution (2% in PBS by volume from stock solution; see recipe for PBS)
  • Potassium ferrocyanide (3% in PBS by volume from stock solution; see recipe for PBS)
  • Filtered water
  • Uranyl acetate solution (0.25% in PBS by volume from stock solution; see recipe for PBS)
  • Ethanol
  • Propylene oxide
  • Poly/Bed 812 Embedding kit (Poly/Bed, DDSA, NMA), with DMP‐30 (Ted Pella)
  • Basic dissection kit including:
    • Forceps
    • Scissors
    • Scalpel
    • Micro‐dissection scissors
  • Surgical dissecting microscope
  • 18½ ‐G needle
  • Shaker set to 37°C
  • 96‐well plates
  • Aluminum foil
  • Small vials with caps
  • Tissue culture rotator (Ted Pella)
  • Desiccator with vacuum
  • Incubator set to 73°C
  • Block mold
  • Additional reagents and equipment for cervical dislocation (see Donovan and Brown, )

Basic Protocol 2: Preparation of Retinal Tissue for Serial Block Face Imaging

  Materials
  • Block‐embedded retinal tissue (from Basic Protocol 1)
  • Uranyl acetate
  • Diamond knife (Diatome)
  • Carbon grids (200 mesh from Ted Pella)
  • Transmission electron microscope (Tecnai T12 electron microscope, FEI)
  • Ultramicrotome (Leica UCT)
  • Aluminum SEM stubs (1/2-in. slotted head, 1/8-in. pin; Ted Pella)
  • Carbon cement (Ted Pella)
  • Serial block face‐scanning electron microscope (Quanta 200 FEG ESEM; FEI)
  • Microtome (3View, Gatan)
  • Digital microscope software (e.g., Digital Micrograph, Gatan)
  • Fiji software (http://fiji.sc/Fiji)
  • Reconstruct program (see Fiala, )
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Chen, Y., Palczewska, G., Mustafi, D., Golczak, M., Dong, Z., Sawada, O., Maeda, T., Maeda, A., and Palczewski, K. 2013. Systems pharmacology identifies drug targets for Stargardt disease‐associated retinal degeneration. J. Clin. Invest. 123:5119‐5134.
  Denk, W. and Horstmann, H. 2004. Serial block‐face scanning electron microscopy to reconstruct three‐dimensional tissue nanostructure. PLoS Biol. 2:e329.
  Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
  Du, Y., Veenstra, A., Palczewski, K., and Kern, T.S. 2013. Photoreceptor cells are major contributors to diabetes‐induced oxidative stress and local inflammation in the retina. Proc. Natl. Acad. Sci. U.S.A. 110:16586‐16591.
  Fiala, J.C. 2005. Reconstruct: A free editor for serial section microscopy. J. Microsc. 218:52‐61.
  Livet, J., Weissman, T.A., Kang, H., Draft, R.W., Lu, J., Bennis, R.A., Sanes, J.R., and Lichtman, J.W. 2007. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450:56‐62.
  Mustafi, D., Kevany, B.M., Genoud, C., Okano, K., Cideciyan, A.V., Sumaroka, A., Roman, A.J., Jacobson, S.G., Engel, A., Adams, M.D., and Palczewski, K. 2011. Defective photoreceptor phagocytosis in a mouse model of enhanced S‐cone syndrome causes progressive retinal degeneration. FASEB J. 25:3157‐3176.
  Mustafi, D., Maeda, T., Kohno, H., Nadeau, J.H., and Palczewski, K. 2012. Inflammatory priming predisposes mice to age‐related retinal degeneration. J. Clin. Invest. 122:2989‐3001.
  Mustafi, D., Kevany, B.M., Genoud, C., Bai, X., and Palczewski, K. 2013. Photoreceptor phagocytosis is mediated by phosphoinositide signaling. FASEB J. 27:4585‐4595.
  Nickell, S., Park, P.S., Baumeister, W., and Palczewski, K. 2007. Three‐dimensional architecture of murine rod outer segments determined by cryoelectron tomography. J. Cell Biol. 177:917‐925.
  Palczewski, K. 2012. Chemistry and biology of vision. J. Biol. Chem. 287:1612‐1619.
  Zhang, N, Kolesnikov, AV, Jastrzebska, B, Mustafi, D, Sawada, O, Maeda, T, Genoud, C, Engel, A, Kefalov, VJ, and Palczewski, K. 2013. Autosomal recessive retinitis pigmentosa E150K opsin mice exhibit photoreceptor disorganization. J. Clin. Invest. 123:121‐137.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library