This presentation is from a workshop about neuron reconstruction. It reviews the process of neuron tracing directly from tissue specimens at the microscopy to study neuron morphology.

1. Neuron Reconstruction and
Analysis Workshop
Jose Maldonado, Ph.D.
Head of Operations, Latin America &
Africa

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Workshop Outline
• Neurolucida manual neuronal reconstructions
• Imaging considerations
• Setting up a microscope for neuron tracing
• Tracing a neuron using a digital camera
• Morphometric analysis in Neurolucida Explorer
• 3D Visualization of neuron reconstructions
• Preview of Neurolucida 360

3. Introduction to Neurolucida
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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

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Historical Perspective
1963 – first computer microscope developed
Glaser and Van de Loos - used analog
computer and oscilloscopes
(note the slide rule)
1971
1986 – commercial implementation

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Historical Perspective
This figure represents one of the first
neuron reconstructions (circa 1964)
Pyramidal cell in rat cortex
The first neuron reconstructions were
performed to obtain 3D quantification
information. It was seen as “cute but
unimportant.” Note simple vectors

6. What is neuronal tracing?
Computer assisted neuron tracing
 The user traces by placing points along a neuron and
this can be done in both 2D and 3D
 Trace cell body, neurites and place spines
 Editing function allows user to erase or add branch points, entire trees
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and/or points
 While tracing, you can set neurite diameter
 Assign trees as axons or dendrites
 3D tracing
 The user traces while focusing through the Z axis
 Also trace neuronal projections through multiple sections
 Saving data
 Live tracing – the user can save both the image with the tracing or save
separately
 Tracing itself is saved as .dat or .asc file

7. Which type of microscopy do I use for
my neuron tracing study?
How much resolution do you need to resolve the
data your wish to quantify?
 High magnification lens for neuronal reconstruction and low
magnification for anatomical reconstruction.
 How small a focal plane do you need to resolve two
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structures on the same cell?
 Focal plane reduction by NA and removal of out of focus light:
 air condenser (0.9 NA) : 1 μm resolution
 oil condenser (1.4 NA): 0.5 μm resolution
 Must be able to visualize the neuron or region of interest in three
dimensions.

8. ANALYZING
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Spectrum
Golgi
Transgenic
Transfection
Injection/Fill
Cholera Toxin
Specificity
 Neurolucida
Explorer
 Blue Brain
 NeuroMorpho
 Whole Brain
 Biolucida
 NEURON
.asc .dat .xml .obj
TRACING &
RECONSTRUCTING
Neurolucida
AutoNeuron
AutoSpine
AutoSynapse
IMAGING
brightfield
confocal
two-photon
EM
Images
Image stacks
Virtual slides
2D/3D
LABELING

9. “I have decided to use bright field
microscopy for neuron reconstructions.”
Benefits of manual neuron
reconstruction:
• Low cost of microscopy hardware
• Can be used to generate high resolution 3D
models for quantifying neuronal cell
morphology.
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• Easy to learn.

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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

11. Tracing Neurons in 3D

12. Changing Tracing Colors
– Change the display of neurons, marker, and contours
– Prior to Tracing:
• Options>Display Preferences> Neuron, Marker, or Contour tab

13. Reconstructing neurons
larger than a single field-of-
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view
Here a motorized stage is used to move the specimen
when the area of interest is large
Note the circular
cursor is used to
measure the process
diameter
The x,y,z points of
the tracing are
stored to create the
reconstruction

14. Axial Resolution Matters
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Image captured by MBF

15. Importance of the Objective Lens
To achieve a thin depth of field
 High numerical aperture oil
objective lenses
 Koehler illumination (for
brightfield)
 Confocal (for fluorescence)
High resolution and a thin depth of
field aid in the ability to discriminate
between objects on top of each
other.
Objective Approx. Depth of Field
40 x (NA 0.65) 1.84 m
40 x (NA 0.95) 0.98 m
60 x (NA 1.0) 0.68 m
100 x (NA 1.4) 0.58 m
Image courtesy of Chandra Avinash, http://photography.learnhub.com/lesson/page/41-understanding-depth-of-field

16. Manual tracing live vs manual
tracing from image stacks?
How does manual tracing from image
stacks work?
• Acquire image data in 3D
• Manually trace from image stacks using
keyboard and mouse.
• Image data resolution limits analytical
resolution!
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17. Summary
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18. Hands on Demonstration
• Lets use the microscope to learn
how to set up Kohler Illumination
• How to create 3D Virtual Tissue
using serial section manager
• Loading files and tracing from a
Virtual Image
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19. How To: Setting up Serial Section
Manager
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 Enter new section into serial
section manager
 To trace contours:
Enter information about cut
thickness of your tissue
To reconstruct neuronal
projections:
Enter information about the
thickness of tissue post processing
Need to apply shrinkage correction
factor to account for tissue
shrinkage

20. Tracing in Serial Sections
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•
Trace contour and neuron in first section defined in serial section manager
Switch to a second section
 Match contour and tracing from 1st section to 2nd section
 Focus at the top of the second section (-50m in schematic)
 Focusing down through tissue - Z is moving in the negative direction
 Draw the contour in the second section
 Continue tracing the neuronal processes from the 1st section into the 2nd
section Tissue
Section 1 Section 2
Top of
section 1 = 0m
Bottom of
section 1 = -50m
Top of
section 2 = -50m
Bottom of
section 2 = -100m
0m -50m -100m

21. Editing and cleaning up
reconstructions
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22. Editing Neuronal Tracing
Without adjustment With adjustment
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• Fix branch node errors
• Eliminate erroneous node
• Splice segments
• Insert node
• Splice from node to segment
• Remove spurious branch
• Delete branch
• Detach branch from tree
• Splice segments
• May require changing ending types
• Z value adjustment
x
y z z z
Commonly
used when
tracing
between
sections

23. Axial resolution impacts reconstruction
granularity
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Reconstruction courtesy of Bob Jacobs

24. Adding Spines and Varicosities
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• 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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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

26. Tips for better reconstructions
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Brightfield:
• Select:
• Coverglass (#1.5)
• Mounting medium
• Objective
• Immersion medium
• Koehler Illumination
• Fully open condenser
If mapping live:
• Place points often
as you focus
Image courtesy of Dan Peruzzi
If imaging:
• Use small step sizes (0.5
μm or less)
• Create a virtual tissue

27. Morphometric Analysis in
Neurolucida Explorer
MORPHOM3D VISUALIZATION
AND
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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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Neurolucida Explorer
 Analyses for
hundreds of
parameters
Branch analysis
Sholl analysis
Fan-in analysis
Vertex analysis
Dendritic spine
distribution
Generate this
information for:
2D and 3D neuron
tracing
Serial reconstruction

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Spine Analysis

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Synapse Analysis

32. Reconstructing Serial Sections and
Neuronal Projections
 3D visualization and reconstruction neuronal projections
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over multiple serial sections
 Also trace contours within serial sections for anatomical
reconstruction of your region of interest
 Depth of separation between samples can range from
fractions of microns to hundreds of microns
Neurolucida includes tools for section rotation, alignment
and comprehensive morphometric analysis

33. Reconstructing Anatomical
Regions and Neurons
• Trace contours across serial sections to reconstruct an
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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

34. Marker and Regional Analysis
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• Calculate marker number
within entire region and
per section
• Nearest neighbor
analysis
• Determine cellular
distribution
• Marker distance to
contour

35. Reconstructing Serial Sections
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 Each anatomical
region within the
brain is traced using a
different contour
labeled for that region
 Analyze individual
contours as well as
entire reconstruction
 Also could have
traced individual
neurons in this
reconstruction

36. Volume Analysis Area Analysis
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Regional Analysis
Name Qty of Contours Enclosed Volume(μm³) Surface Area(μm²)
Left Hemisphere 37 5.98831E+14 46149100000
Right Hemisphere 37 5.45442E+14 73043300000
Optic L 18 5.33316E+11 843032000
Optic R 18 4.89997E+11 807050000
Lateral Ventricle L 19 6.42725E+12 4157860000
Lateral Ventricle R 19 6.31731E+12 4057540000
Cingulum 17 1.06517E+12 1241400000
Corpus/callosum 17 1.87231E+13 7571140000
Caudate L 5 3.81237E+12 1474400000
Caudate R 5 4.25086E+12 1505010000
Ant Horn of Lat Vent 14 2.14729E+12 1513890000
Caudate 12 1.30445E+12 1114080000
Surface 4 6.21773E+12 2379950000
Basal Ganglia L 12 8.66572E+12 2676360000
Basal Ganglia R 13 9.37228E+12 2716710000
Cerebellum 13 1.36236E+14 19343200000
Thalamus L 7 4.63984E+12 1847880000
Thalamus R 7 4.91661E+12 1937630000
Optic 2 83874300000 139533000
Lat Vent R 6 5.99702E+11 801104000
Lat Vent L 6 5.47828E+11 836201000
Fimbria L 5 3.19795E+11 695797000
Fimbria R 6 3.34206E+11 653764000
IV Ventricle 5 8.67936E+11 510980000
Cerebellum Left 3 7.56976E+12 3384100000
Cerebellum Right 3 7.77047E+12 3432440000
Name Open Closed Tot Len(μm) Mean Len(μm) Tot Area(μm²) Mean Area(μm²)
Left Hemisphere 0 37 10213400 276037 1.33503E+11 3608200000
Right Hemisphere 0 37 16170100 437030 1.21895E+11 3294450000
Optic L 0 18 197898 10994.3 128544000 7141320
Optic R 0 18 186320 10351.1 115048000 6391580
Lateral Ventricle L 0 19 812508 42763.6 1266810000 66674000
Lateral Ventricle R 0 19 788147 41481.4 1259750000 66302800
Cingulum 0 17 294877 17345.7 265050000 15591200
Corpus/callosum 0 17 1684500 99088 4808460000 282850000
Caudate L 0 5 222165 44433.1 639143000 127829000
Caudate R 0 5 224576 44915.2 682183000 136437000
Ant Horn of Lat Vent 0 14 356964 25497.4 534354000 38168200
Caudate 0 12 243511 20292.6 309872000 25822600
Surface 0 4 489453 122363 2028470000 507116000
Basal Ganglia L 0 12 643612 53634.3 2302020000 191835000
Basal Ganglia R 0 13 677609 52123.7 2413530000 185656000
Cerebellum 0 13 3346800 257446 26589700000 2045360000
Thalamus L 0 7 367633 52519 1290630000 184375000
Thalamus R 0 7 381211 54458.7 1371270000 195896000
Optic 0 2 48798 24399 41937200 20968600
Lat Vent R 0 6 187512 31252.1 161570000 26928400
Lat Vent L 0 6 196044 32674.1 145628000 24271300
Fimbria L 0 5 167501 33500.2 76795800 15359200
Fimbria R 0 6 187566 31261 105056000 17509400
IV Ventricle 0 5 112344 22468.9 200446000 40089200
Cerebellum Left 0 3 431137 143712 2527880000 842628000
Cerebellum Right 0 3 451159 150386 2566910000 855638000

37. 3D Visualization
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38. 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
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animated movie for display (.avi)

39. Future Directions in Neuron
Tracing
Neurolucida 360
MORPHOM3D VISUALIZATION
AND
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40. Future Directions – Neurolucida
360
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• 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

41. Thanks!
NIMH grants MH076188, MH085337, MH93011
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National Institutes of Health
MBF Programmers, Staff, and Staff Scientists
All of you for attending our workshop
Current MBF Customers who provided the image data

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