Whole Mouse Brain Image Reconstruction from Serial Coronal Sections Using FIJI (ImageJ)

Ronald Paletzki1, Charles R. Gerfen1

1 Laboratory of Systems Neuroscience, NIMH, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.25
DOI:  10.1002/0471142301.ns0125s73
Online Posting Date:  October, 2015
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Abstract

Whole‐brain reconstruction of the mouse enables comprehensive analysis of the distribution of neurochemical markers, the distribution of anterogradely labeled axonal projections or retrogradely labeled neurons projecting to a specific brain site, or the distribution of neurons displaying activity‐related markers in behavioral paradigms. This unit describes a method to produce whole‐brain reconstruction image sets from coronal brain sections with up to four fluorescent markers using the freely available image‐processing program FIJI (ImageJ). © 2015 by John Wiley & Sons, Inc.

Keywords: neuroanatomy; imaging; brain reconstruction

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Histologic Preparation of Brain Sections
  • Basic Protocol 2: Imaging Brain Sections
  • Basic Protocol 3: Organizing Coronal Sections in Serial Order
  • Basic Protocol 4: Application of FIJI Macros to Produce Whole‐Brain Reconstructions (WBR)
  • Support Protocol 1: Optional FIJI Macros
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Histologic Preparation of Brain Sections

  Materials
  • Mouse brains (see unit 1.1; Gerfen, )
  • Phosphate‐buffered saline (PBS; KD Medical; cat. no. RGF‐3210)
  • Primary antibodies directed against brain proteins or axonal tracers
  • Secondary antibodies to visualize primary antibodies with distinct fluorescent fluorophores (e.g., Alexa 488; 555; 647, Cy3, Cy5)
  • Fluorescent Nissl stain: (Neurotrace 435/455; Life Technologies)
  • Fluorescent mounting medium
  • 24‐well cell culture plate (Corning Costar, cat. no. 3524)
  • 10‐well plate with mesh bottom
  • Paint brush for transferring sections
  • Gelatin‐coated 1‐ × 3–in. microscope slides (prepared as in unit 1.1; Gerfen, )
  • Glass coverslips
  • Additional reagents and equipment for microtome sectioning (unit 1.1; Gerfen, ) and immunohistochemical localization of proteins (unit 1.2; Volpicelli‐Daley and Levey, )

Basic Protocol 2: Imaging Brain Sections

  Materials
  • Slides with brain sections ( protocol 1)
  • Microscope equipped with:
    • Fluorescent illumination
    • Computer controlled motorized stage
    • z‐axis control
    • Camera
  • Software to run microscope imaging system with capacity to:
    • Capture image tiles across microscope slide and “stitch” them together
    • Capture multiple fluorescent channels for each imaged tile
    • Capture multiple focal planes through brain section for each imaged tile

Basic Protocol 3: Organizing Coronal Sections in Serial Order

  Materials
  • Software to manage image files (optional):
    • BrainMaker ( MBF Bioscience)
    • Adobe Photoshop Bridge

Basic Protocol 4: Application of FIJI Macros to Produce Whole‐Brain Reconstructions (WBR)

  Materials
  • The following FIJI macros are used for whole‐brain reconstruction:
    • WBR_Macro_01_TIFF_format (converts the images into TIFF format)
    • WBR_Macro_02_determine_autothresholding_method (this macro is optional and is used to determine the autothresholding methods to be used in Macro_03)
    • WBR_Macro_03_align_sections (image alignment)
    • WBR_Macro_04_adjust_alignment (manual adjustment of alignment as needed)
    • WBR_Macro_05_flatten_cell_stain_image (flattens Nissl or other cell stains across the brain section)
  • The following optional image‐processing FIJI macros are used to optimize labeled structures:
    • WBR_Macro_06_normalize_background (normalizes uneven background produced by immunohistochemical staining)
    • WBR_Macro_07_remove_nonspecific_cell_labeling (removes “bleed through” of some Nissl stains)
    • WBR_Macro_08_fiber_enhancement (increases contrast of labeled fibers and processes)
    • WBR_Macro_09_extract_individual_channels (splits TIFF files into individual files for each channel to allow image processing in Photoshop or other image‐processing software)
  • These FIJI macros are used to complete image‐processing steps to produce a whole‐brain reconstruction image series:
    • WBR_Macro_01_TIFF_format (combine channels into a single image file)
    • WBR_Macro_10_display_stack (allows for display and analysis of whole‐brain reconstruction files)
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Figures

Videos

Literature Cited

Literature Cited
  Gerfen, C.R. 2003. Basic neuroanatomical methods. Curr. Protoc. Neurosci. 23:1.1.1‐1.1.11.
  Gerfen, C.R., Paletzki, R., and Heintz, N. 2013. GENSAT BAC Cre‐recombinase driver lines to study the functional organization of cerebral cortical and basal ganglia circuits. Neuron 80:1368‐1383. doi: 10.1016/j.neuron.2013.10.016.
  O'Connor, N., Tappan, S., and Glaser, J. 2014. How to prepare neuroanatomical image data. Curr. Protoc. Neurosci. 69:1.21.1‐1.21.14.
  Oh, S.W., Harris, J.A., Ng, L., Winslow, B., Cain, N., Mihalas, S., Wang, Q., Lau, C., Kuan, L., Henry, A.M., Mortrud, M.T., Ouellette, B., Nguyen, T.N., Sorensen, S.A., Slaughterbeck, C.R., Wakeman, W., Li, Y., Feng, D., Ho, A., Nicholas, E., Hirokawa, K.E., Bohn, P., Joines, K.M., Peng, H., Hawrylycz, M.J., Phillips, J.W., Hohmann, J.G., Wohnoutka, P., Gerfen, C.R., Koch, C., Bernard, A., Dang, C., Jones, A.R., and Zeng, H. 2014. A mesoscale connectome of the mouse brain. Nature 508:207‐214. doi: 10.1038/nature13186.
  Schindelin, J., Arganda‐Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.‐Y., White, D.J., Hartenstein, V., Eliceiri, K., Tomancak, P., and Cardona, A. 2012. Fiji: An open‐source platform for biological‐image analysis. Nat. Methods 9:676‐682. doi: 10.1038/nmeth.2019.
  Volpicelli‐Daley, L.A. and Levey, A. 2004. Immunohistochemical localization of proteins in the nervous system. Curr. Protoc. Neurosci. 25:1.2.1‐1.2.17.
Key References
  Burger, W. and Burge, M.J. 2010. Digital Image Processing: an Algorithmic Introduction Using Java. Springer, New York.
  General references related to the use of FIJI and JAVA for image processing.
  Mateos‐Pérez, J.M. and Pascau, J. 2013. Image Processing with ImageJ. Packt Publishing, Birmingham, U.K.
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Supplementary Material