What is neuron tracing and why is it so important?
Neuron tracing is the process of creating digital reconstructions of the entire (or sometimes only specific portions) of neurons. Neuron tracing includes delineating and reconstructing the axon, dendrites soma, and other sub-cellular components of a neuron, thereby creating a digital, geometric model of the neuron. Researchers use neuron tracing techniques for reconstruction and morphological analysis of neurons in tissue sections, tissue slabs, intact brains, and cell culture. Neuron tracing is typically performed using light microscopy imaging, sometimes it is performed using electron microscopy imaging.
The resulting 3D reconstructions are used to visualize and analyze not only overall neuronal process length, but also other morphological quantities such as branching frequency, segment length, branch angle, spine or receptor density, and segment diameter. These digital reconstructions can be morphometrically analyzed to understand more about intricate neuronal structures. The reconstructions can also be used with electrotonic modeling software to model the electrical properties of neurons. The morphological structure and connectivity of somas, dendrites, axons, spines, varicosities and synapses provide extremely valuable information on the workings of neurons throughout the body, ranging from brain circuits to the peripheral nervous system.
Santiago Ramón y Cajal, was the first to perform neuron tracing. He did so with a pencil and paper, creating graphic representations of neurons he observed. Edmund Glaser, MBF Bioscience co-founder, and Hendrick van der Loos performed the first digital neuron tracing in the early 1960’s. Today, neuron tracing in performed in 1000’s of laboratories around the world using techniques that range from computer assisted, to fully-automatic.
Why do researchers trace neurons?
An accurate digital reconstruction of neuron morphology is critical for the most accurate quantitative analyses. Neuron tracing is an important facet of researching in learning, memory, and behavior. It is also used in research into all major neurodevelopmental, neuropsychiatric, neurodegenerative and neurological disorders, including: intellectual disability, traumatic brain injury, neuroAIDS, ischemia, mild cognitive impairment, vascular dementia, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, epilepsy, schizophrenia, autism spectrum disorder, attention deficit hyperactivity syndrome, depression, drug addiction, neuroinflammation, multiple sclerosis, spinal cord injury and neuropathic pain.
How do you trace neurons?
There are two general methods for tracing neurons. One popular method is to obtain images (2D or 3D) from a microscope and then use software that can import the image data and provide you with a set of tracing and segmentation tools. Some software may also have some built-in analyses tools.
Three important factors with this method are:
1. Image data quality: The XY and Z resolution are very important as well as high contrast and low noise in the images
2. Neuron tracing tools: These tools can vary substantially from very basic drawing tools that come in free software to highly specialized tools that are designed specifically to reconstruct as accurate and detailed digital model of the neuron as possible
3. Analyses tools: These tools can also very substantially from very basic measurement tools to highly sophisticated tools designed specifically to provide quantitative morphometric data that are relevant to neuroscience researchers
A second popular method for tracing neurons does not require any previously acquired image data at all. This method requires a computer-controlled microscope-based system with software that provides neuron tracing tools to trace from a live camera image. This method is very popular for tracing Golgi stained neurons or other brightfield staining methods as there is no concern for tissue bleaching.
Three important factors with this method are:
1. The software used must be capable of fully-controlling all motorized functions of the microscope to create one fully integrated system and the motorized stage must be off a high enough resolution in order to record accurate XYZ position data
2. The software must include neuron tracing tools that allow the user to trace over a live camera image and across multiple fields of view and even multiple tissue sections
3. The software must provide similar tools for image analysis that would be used in the first method.
What makes a good, accurate reconstruction?
- Accuracy of digitizing small discrete positions in all 3 axes, with proper calibration
- Accurate thickness traced and determine for all axon and dendritic processes
- Accurate soma reconstruction
- All branching structures are traced and connected properly and nodes are identified
- Branching structures can be segmented and color coded
- Reconstruction matches the image data when viewed in 3D
- Spines are identified and classified
- Varicosities and boutons are identified
- Neuron in serial sections are joined into a comprehensive digital reconstruction
What makes some neuron tracing software better than others?
- Accuracy and validity of the reconstruction produced
- Ease of use
- Speed of tracing and reconstruction
- Ability to detect, trace and reconstruct sub-cellular structures
- Automatic trading modes
- Fully Automatic
- User Guided
- Ability to handle big image data files
- Ability to handle multiple neurons
- Availability of technical support
- Built-in morphometric analyses tools
- Built-in tools for publishing data
What should you look for when purchasing a neuron tracing system?
If you are looking for a microscope-based system you should check whether it is able to control the newest models of microscopes, motorized stages and cameras as well as command internal microscope components like light sources, filter wheels and motorized optics to automatize imaging. When you look for an offline system it should be accurate and have its results validated. It should have a fast learning curve and yet be a powerful and flexible analysis tool with different algorithms and parameter configurations so you can trace a wide range of labels and image types, while having the ability to perform the specific analyses you need for your research. Additionally, it is essential to have support for many different microscope image formats so data can be quickly imported without the need of file format conversions that slow the process and often result in missing metadata.
An eight-channel fluorescence whole slide imaging and analysis system controlled by Neurolucida and built with a Zeiss AxioImager microscope with an ApoTome.
What is the best neuron tracing software?
Neurolucida and Neurolucida 360 are two different software applications that provide tools for 2D and 3D tracing dendrite, axons, spines, synapses, somas and varicosities. You can also use them to manually map the brain to give anatomical context to neuronal pathways, cells and synaptic distribution (for automatic brain nuclei mapping see NeuroInfo).
Neurolucida is primarily a manual computer-assisted tracing system that can be integrated directly with microscopes. In addition to reconstructing structures it can be used as your primary image acquisition software package so you can do brightfield and multichannel fluorescent stack imaging, multifocal tiling, image post-processing. Neurolucida is the most widely used neuron tracing system in the world due to its ability to accurately trace virtually any neuron in any specimen. It is the gold standard in neuron tracing.
Neurolucida 360 is designed for automatic 3D and 2D reconstruction from acquired images from brightfield, fluorescence, confocal, and light sheet microscopes. Using multiple algorithms and a 3D interactive tracing environment, it can easily detect and trace structures automatically. It is compatible with different image modalities (confocal, 2-photon, lightsheet) and many proprietary image formats (.czi, .lif, .oib, .nd2, etc.). Neurolucida 360 is the most advanced automatic neuron tracing software. It can be used on 2D and 3D images obtained from a wide variety of specimens. Its capabilities for tracing neurons and modeling dendritic spines are unparalleled.
Learn more about our neuron tracing software:
What kind of analysis I can obtain using neuron tracing?
All the data produced in Neurolucida and Neurolucida 360 is stored in a published (non-proprietary), vector format. It is extremely easy to edit on 3D or represent and scale graphics for your publications. The data can be analyzed with Neurolucida Explorer (included with Neurolucida and Neurolucida 360). This is an extremely powerful and comprehensive quantitative analysis package with more than one hundred types of morphometric analyses. They comprise multi-level morphometric analysis (length, diameters or volumes from population to segment level), topology and branch connectivity (degree, vertex analysis), spatial analysis (i.e., Sholl, convex hull), orientation and region-specific analysis (polar histogram, closed surface). If you don’t know the most suitable analysis for your experiment you can ask for help from our research support team which is composed of Ph.D. neuroscientists and experts in microscopy.
Contract Neuron Reconstruction Services
Our Neuron Reconstruction services gives your lab the ability to have industry-leading experts create a 3D digital reconstruction of individual or multiple neurons, for your research. MBF Labs will reconstruct your neurons giving you a breadth of quantitative information. The process is simple and practical. Provide us with slides or images, and MBF Labs experts do the rest.
When you call us you will speak with a person - not an automated system. Talk to us about your hardware, software, or experimental design questions. Our team includes Ph.D. neuroscientists and experts in microscopy, stereology, neuron tracing, and image processing; ready to help you over the phone or online.