Neuron Reconstruction and
Analysis Workshop
Julie Korich, Ph.D.
Susan Tappan, Ph.D.
mbfbioscience.com
Workshop Outline
• Neurolucida manual neuronal reconstructions
• Tools for automatic neuronal reconstructions
• AutoNeuron, AutoSpine and AutoSynapse modules
• Imaging considerations
• Morphometric analysis in Neurolucida Explorer
• 3D Visualization of neuron reconstructions
• Preview of Neurolucida 360
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• Reconstruction of neuronal structures
• Quantify neuronal outgrowth in response to
growth factors, drugs, etc.
• Calculate spine and synaptic densities
• Quantification of anatomical regions and
cells
• Calculate volume of infarct or tumor
• Map stem cell migration in the spinal cord
• Identification of neuronal networks and
connectivity within an anatomical region
Introduction to Neurolucida
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Spectrum
Cholera Toxin
Transgenic
Transfection
Injection/Fill
Golgi
Specificity
Neurolucida
Explorer
Blue Brain
NeuroMorpho
Whole Brain
Biolucida
NEURON
.asc .dat .xml .obj
ANALYZING
Neurolucida
AutoNeuron
AutoSpine
AutoSynapse
TRACING &
RECONSTRUCTING
Images
Image stacks
Virtual slides
2D/3D
IMAGING
confocal
two-photon
EM
brightfield
LABELING
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Manual Neuron Reconstruction: • Directly on the scope
• From images and image stacks
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Motorized stage
focus encoder, and stage
controller
High
resolution
digital camera
Computer with
MicroBrightField software
and video capture card
Microscope
with high
quality optics
Reconstructing Neurons Directly
from Slides
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Reconstruct Neurons Directly from
Slides (cont.)
• The full extent of the
dendrites and axons
usually extend across
multiple fields-of-view
150 serial sections
• A motorized stage
moves the specimen
when tracing outside
the field-of-view
• The x,y,z information
is stored to create a
3D reconstruction
Courtesy of Dr. Rosa Cossart
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Reconstructing from Images
• Load 2D images, 3D image
stacks or montages into NL
for 2D or 3D reconstruction
• Trace through the entire stack
or montage while focusing
through the stack
• Stacks can be acquired on a
MBF system, on a confocal,
or a 2-photon scope
Image courtesy of MBF Bioscience
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Image Montage Module
• A number of overlapping image stacks were acquired that
need to be aligned
• Image Montage Module will automatically align confocal stacks
in XYZ
Image Stacks Courtesy of Dr. Rosa Cossart
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Image Montage Module
Image Stacks Courtesy of Dr. Rosa Cossart
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Adding Spines and Varicosities
• Marked while tracing
or once the dendrite is
reconstructed
• Use the spine toolbar
to add spines
• Use the marker tool
bar to add varicosities
or other features
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Reconstructing Anatomical
Regions and Neurons
• Trace contours across serial sections to reconstruct an
anatomical region of interest, lesions, etc.
• Map neuronal projections and cells
• From live video or images collected throughout the ROI
http://www.mbfbioscience.com/brain-mapping/cytoarchitectonics
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Changing Tracing Colors
• Change the display of neurons, marker, and contours
• Prior to Tracing:
• Options>Display Preferences> Neuron, Marker, or Contour
tab
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Editing
• While tracing, hit CTRL Z to delete the last point placed
• After tracing, use the editing tool to:
• Modify fibers:
• Delete trees (fibers)
• Modify thickness along the tree
• Add branch points
• Modify colors
• Correct z errors
• Modify contours and markers
• Delete
• Modify thickness
• Resize
• Modify colors
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Automatic Reconstruction: AutoNeuron
AutoSpine
AutoSynapse
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AutoNeuron module
• Automatic reconstruction of neuronal processes and cell somas in
2D and 3D
• Uses fully automatic or interactive modes
• Recommend high magnification images with a small Z step
(around 0.5µm)
20mm
http://www.mbfbioscience.com/image-gallery
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AutoNeuron Advanced Options
• Step 5 of the AutoNeuron workflow
• Seed detection:
• Adjust sampling density to ensure uniform sampling
and seed coverage
• Tracing:
• AN sets the most optimal tracing settings based on
the type of image: low magnification confocal, high
magnification confocal and brightfield
• Branch connections:
• Ignore traces shorter than user defined amount
• Adjust tolerance to gaps in staining
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DWORK
Images courtesy of Andrew Dwork Images courtesy of Dr. Andrew Dwork
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AutoSpine Module: Spine
Detection
• Automated reconstruction of
dendritic spines
• Dendritic branch can be traced
manually or automatically
• Dendritic spines modeled as a
3D mesh using defined
parameters
• Recommend high magnification
image stack with small Z step
(under 0.5µm)
Image courtesy of Dr. Jacob Jedynak
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AutoSynapse Module: Putative
Synapse Detection
• Putative synapes automatically
detected along a traced branch
& modeled as a 3D mesh using
defined parameters
• User determines detection
distance from dendrite
• Recommend high magnification
image stack with small Z step
(under 0.5µm)
• Future versions will support co-
localization
Images courtesy of Dr. Francisco Alvarez & Travis Rotterman
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The Spine/Synapse Detector is a
Toroid
Inside radius
Outside radius
Image courtesy of Dr. Jacob Jedynak
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Editing
• After tracing, use the editing
tool as you would for manual
traces
• AutoNeuron:
• The splice tool is most often
used
• AutoSpine:
• Delete and classify spines
• AutoSynapse
• Delete synapses
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Orthogonal View for Editing
• Displays
portion of the
image and
tracing in Z
• Make editing
complex
neurons
easier
Image courtesy of Dr. Jacob Jedynak
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Imaging for Reconstruction
• Reconstruction goals
• What to choose:
• At the scope or from images?
• Time vs Effort
• Imaging modality • Brightfield
• Fluorescence
– CFM or MPFM
Axial resolution
Depth of field
Step size
Lateral resolution
Objective choice
Improve image analysis with correct
capture and post-processing techniques.
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Axial Resolution Matters
Image captured by MBF
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Axial resolution impacts reconstruction
granularity
Reconstruction courtesy of Bob Jacobs
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Tips for better reconstructions
Brightfield:
• Select:
• Coverglass (#1.5)
• Mounting medium
• Objective
• Immersion medium
• Koehler Illumination
• Fully open condenser
Image courtesy of Dan Peruzzi
If mapping live:
• Place points often
as you focus
If imaging:
• Use small step sizes (0.5
µm or less)
• Create a virtual tissue
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Tips for better reconstructions
Fluorescent: • Select:
• Coverglass (#1.5)
• mounting medium
• Objective
• Immersion medium
• Small step sizes (0.5 µm or less)
• Create a virtual tissue for seamless fields of view
• Maximize Dynamic Range
• After acquisition, deconvolve if necessary
Image from Randy Bruno. Figure from Dumitru, Rodriguez and
Morrison Nat Protoc. 2011 August 25; 6(9): 1391–1411.
If using single or multiphoton microscope:
• Match the Pinhole Size for each fluorophore!
Adjust the dynamic
range.
Overexposure
exaggerates axial blur.
Image courtesy of Rosa Cosart
Image courtesy of Ryan Ash
Note circular profile.
Improve image analysis
with correct capture and
post-processing
techniques.
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MORPHOM3D VISUALIZATION
AND
Morphometric Analysis in
Neurolucida Explorer
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Data analysis
• Neuronal Analyses
• Spine Data
• Synapse Data
http://vadlo.com/cartoons.php?id=71
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Neuronal Analysis
Branching analysis
• Length per tree (dendrite/axon), per
neuron, and per branch order
Sholl Analysis
• Calculated per tree and branch
order
Layer Analysis
• Calculate length within cortical
layers
Branch Analysis
• Calculate branch angles and
numbers of branch points
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Spine Analysis
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Synapse Analysis
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3D Visualization
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3D Visualization Module
• Integrated within MBF software
• Display 3D rendering of objects built from
reconstructions
• Rotate and zoom
• Place a “skin” around wireframe and adjust opacity
• Display the tracing and image data simultaneously
• Save solids view as a TIFF or JPEG2000 or create an
animated movie for display (.avi)
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MORPHOM3D VISUALIZATION
AND
Neurolucida 360
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Future Directions – Neurolucida
360 & SpineStudio
• Partnership with
Dr. Patrick Hof
and original
developers of
Neuron Studio
• Full 3D interactive
tracing and
editing
• Open API for 3rd
party algorithm
plug-ins
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NIMH grants MH076188, MH085337, MH93011
National Institutes of Health
MBF Programmers, Staff, and Staff Scientists
Thanks!
All of you for attending our workshop
Current MBF Customers who provided the image data
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