Neuron Reconstruction and

Analysis Workshop

Julie Korich, Ph.D.

Susan Tappan, Ph.D.

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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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