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

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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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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

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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 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

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High magnification lens for neuronal reconstruction and low

magnification for anatomical reconstruction.

How small a focal plane do you need to resolve two

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.

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?

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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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Benefits of manual neuron

reconstruction:• Low cost of microscopy hardware

• Can be used to generate high resolution 3D

models for quantifying neuronal cell

morphology.

• Easy to learn.

“I have decided to use bright field

microscopy for neuron reconstructions.”

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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

Tracing Neurons in 3D

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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Reconstructing neurons

larger than a single field-

of-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

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Axial Resolution Matters

Image captured by MBF

Importance of the Objective Lens

High numerical aperture oil

objective lenses

Koehler illumination (for

brightfield)

Confocal (for fluorescence)

To achieve a thin depth of field

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

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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Summary

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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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How To: Setting up Serial Section

Manager

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

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Tracing in Serial Sections

•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 (-50m 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 = 0m

Bottom of

section 1 = -50m

Top of

section 2 = -50m

Bottom of

section 2 = -100m

0m -50m -100m

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Editing and cleaning up

reconstructions

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Editing Neuronal Tracing

• 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 zzz

Without adjustment With adjustment

Commonly

used when

tracing

between

sections

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Axial resolution impacts reconstruction

granularity

Reconstruction courtesy of Bob Jacobs

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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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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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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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MORPHOM3D VISUALIZATION

AND

Morphometric Analysis in

Neurolucida Explorer

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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

parametersBranch 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

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Reconstructing Serial Sections and

Neuronal Projections

3D visualization and reconstruction neuronal projections

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

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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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Marker and Regional Analysis

• Calculate marker number

within entire region and

per section

• Nearest neighbor

analysis

• Determine cellular

distribution

• Marker distance to

contour

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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

Reconstructing Serial Sections

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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

Volume Analysis Area 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

Future Directions in Neuron

Tracing

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Future Directions – Neurolucida

360

• 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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