Three‐Dimensional Reconstruction of Tissues

Francisco Cruz1, Richard T. Lee1, Hayden Huang1

1 Brigham and Women's Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.16
DOI:  10.1002/0471142727.mb1416s74
Online Posting Date:  May, 2006
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Abstract

Cellular morphology is inherently three‐dimensional. However, most histological techniques for tissue analysis focus on extracting information from two‐dimensional slices of fixed samples or dissociated cells. These techniques result in a significant loss of the three‐dimensional information of the tissue, including true cell volume, orientation, and whole cell shape. This unit discusses various options for three‐dimensional imaging, provides a protocol for performing post‐processing reconstruction based on serial slicing, and discusses the current advantages and limitations of the three‐dimensional approach to quantitative tissue analysis. The focus of this protocol is on cardiac tissue, but the techniques can be applied to any solid tissue.

Keywords: fluorescence microscopy; image registration; histology; cell morphology

     
 
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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Labeled tissue, or animal to be labeled and appropriate anesthesia
  • Labeling compounds (e.g., Invitrogen): 10 mg/ml Hoechst stain; 5 mg/136 µl Texas red maleimide in DMEM; 500 µg/ml Alexa Fluor 488 in PBS with Ca2+ and Mg2+; or other label of interest
  • Fixative (e.g., 4% paraformaldehyde)
  • 70% ethanol
  • Paraffin
  • 1‐ml or smaller syringes
  • Small‐gauge needle
  • Microtome
  • Glass slides and coverslips
  • Fluorescence microscope with appropriate objectives, filters, cameras, and acquisition software
  • Computer (minimum requirements are Pentium 4 with 1 GHz or faster processor, 512 MB or higher RAM, and hard drive of 100‐GB capacity or greater, depending on size and number of image files, running MacOS X or Windows 2000)
  • Mathematical processing package (e.g., MatLab, Mathworks)
  • Additional reagents and equipment for fixation, embedding and sectioning of tissues (e.g., unit 14.1)
NOTE: Using other programming languages, such as Visual C, requires having the appropriate compiler and knowledge of the programming syntax, which is not provided here. For the code presented in this protocol (available for download at http://vascular.bwh.harvard.edu/cpmb3d/cpmb3d.html), Matlab 7.0 or higher with Image Processing and Curve Fitting toolboxes is required.
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Figures

Videos

Literature Cited

Literature Cited
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