Accurate morphological description of filamentous structures requires the ultimate resolution available from light microscopy today with structures often of the size that is near or at the theoretical limit of resolution of the best objective lens. The best possible imaging is needed to capture the finest of structures of interest. These are fundamental reasons why the analysis of neuron and filament morphology is so challenging. Purely manual or purely automated strategies are therefore likely to fail. Imaris FilamentTracer uses strategies that involve an optimal combination of automated analysis and operator decision. The choice of the best method is an essential element in optimizing efficiency. FilamentTracer allows the choice between four complementary tracing strategies ranging from manual to automatic. FilamentTracer utilizes a creation wizard to easily guide the user through the creations steps.

Equally essential to optimizing efficiency is the ability to overlay the segmented structure (tracing) and the 3D original data in real time. FilamentTracer is fully integrated with Imaris, enabling users to leverage its impressive range of 3D and 4D visualization capabilities as well as its image editing and processing functions. With FilamentTracer there is no need to switch between programs, windows, or views, costing the user valuable time. Instead FilamentTracer allows the creation, visualization, editing, and analysis all in one window. By having an interactive overlay of the original image and the resulting tracing the user can easily assess the validity of the tracing and make any necessary changes in 3D space.

FilamentTracer has been specifically designed to deal with the complex problem of spine analysis over time. This is a challenging process that to date has been a time-consuming task that often did not produce a reliable 4D analysis of spines’ fate. There is no need to manually analyze spines anymore: With just a few clicks of a button, FilamentTracer allows 4D tracking and classification of detected structures such as spines and dendrites. Researchers can examine statistics that change over time for both dendrites and spines including information on counts, branching, size, shape, and intensity.   Researchers can being to explore spine evolution, e.g. during brain development, memory formation, stroke and other vital life processes.
 
Together with ImarisXT, analysis can be taken a step further and spines can be classified by diameter, size, shape, or any other parameter that is calculated by FilamentTracer. The ImarisXT module allows custom configuration of any user defined classification rules. The ideal combination for FilamentTracer is Imaris, Imaris MeasurementPro (measurement of volume and intensity changes over time and manual object segmentation), ImarisTrack (for tracing the temporal changes in spines and dendrites) and ImarisXT (automated spine classification).

The FilamentTracer functionality requires Imaris.

(1) Detection Methods

FilamentTracer offers the largest choice of tracing methods on the market to ensure the fast and accurate tracing of filament like structures in 2D and 3D microscopy images. The creation wizard helps the user choose the optimal parameters for any detection method. Detection and segmentation methods can be freely combined on any image.

• Fully Automatic – FilamentTracer offers the choice between two fully automatic detection methods; “AutoPath” (creates trees without loops) or automatic detection based on intensity thresholds (may contain loops). Just put simple parameters into the creation wizard and FilamentTracer completes the tracing.

• Semi Automatic – FilamentTracer allows semi automatic tracing (using “AutoPath”) by manually selecting starting and end points. Click on a starting location in 3D space or choose a segment or group of segments as the starting point. FilamentTracer performs a calculation. Then you can simply click on end points and FilamentTracer instantly provides a preview tracing along the best path back to the starting point as you move the mouse in the image. Clicking the end point sets the tracing.

• Advanced Manual – FilamentTracer’s advanced manual tracing mode allows you to directly trace onto a 3D volume image with automatic z depth placement (“AutoDepth”) and achieve a fast, accurate and reproducible manual tracing result. This enables you to quickly trace in 3D space.

• Manual – FilamentTracer provides a tool to fully trace structures manually when focusing on specific details in complex structures. Tracing can be completed on individual planes.

• Region of interest processing (ROI) – FilamentTracer allows you to process single or multiple regions of interest to target specific areas of the image or to test tracing parameters which then can be used on automatically on the entire image.

• Iterative Processing – FilamentTracer allows the user to trace a small section of the dataset automatically or manually and then the user can “build” upon the existing data in an iterative fashion using the automatic methods. This provides excellent functionality for data sets that are extremely large or where there is no clear starting point. 

• Rebuild – Choose between keeping and deleting existing data when re-entering the automatic creation process of the FilamentTracer Creation Wizard. This allows you to keep the structures you have traced manually while adjusting the automatic parameters that were originally set.

(2) Dendrite and Spine Tracking

With its novel Spine and Dendrite tracking in 3D over time, FilamentTracer offers you a powerful tool with which you can analyze images of neurons undergoing continual changes. Utilizing FilamentTracer's intuitive wizard-driven detection algorithms, the user can easily define which spines and dendrites are going to be segmented, then which terminal, branch or attachment points will be tracked and finally visualized in 4D.

• Automatic Tracking - After segmentation with one of the automatic methods described above, choose to track dendrites and / or spines within the creation wizard.
  - Dendrites may be tracked based on the position of dendrite branch points or dendrite terminal points.
  - Spines may be tracked based on the position of spine attachment points, terminal points, or branch points.
  

• Autoregressive Motion Model - For the most efficient tracking of directional objects like Spines and Dendrites the autoregressive motion model is automatically used. The user simply has to input the maximum distance they believe a spine and / or dendrite attachment point will move between  two adjacent time points.  If due to imaging problems a spine or dendrite  attachment point is not detected at a particular time point, the user has the option to allow a specified number of time frame to pass where the attachment point is missing and still complete the tracking properly.  In this mode,  FilamentTracer can automatically deal with changes in acceleration / deceleration of growth or shrinkage of spines and dendrites and improves tracking accuracy for those objects.

• Manual Track Editing – Once segmentation is complete and the image is automatically tracked, the user can use the track editor to manually correct any errors that may have occurred during automatic tracking.  The track editor for filaments performs the same way that is does for spot(s), surface(s), and cell(s) objects making learning the software easier.

(3) Best-In-Class Visualization

A rich selection of interactive 3D display modes is important for the creation, editing, and presentation of tracings. This allows multiple viewing combinations in which the tracing can be displayed individually as an overlay on the original data or together with other cellular components that may have been imaged in other channels

• Display – FilamentTracer allows the display of traced segments as lines, constant-width cylinders or real-diameter cylinders (showing the actual diameter measured from the structure). Each part of a filament tracing can be displayed with any color or transparency.

• Surpass View - Combine Filament tracings with any Imaris “Surpass Object” to show surrounding cellular or tissue components and superimpose filaments over the original volume rendering to verify your segmentation.

(4) Graph-Selection / Editing / Classification

FilamentTracer has a versatile range of methods to allow you to select parts of the tracing for editing and classification following the creation process.

• Selection – FilamentTracer provides for selection of individual segments, entire branches, or single points as well as for the selection of loops or segments based on absolute or relative size. Multiple selections, inverse selections, and clearing selections can also be completed.

• Selection Processing – Once selected, traced components can be deleted or joined. More importantly any segment (such as manually or semi-manually drawn segments) can be properly centered to the original data and can also be “grown” so the tracing has the accurate diameter and shape when compared to the original data.

• Classifications – Any selected segment can be classified as a dendrite or spine. This is important for proper analysis of neuroscience images. When combined with Bitplane’s ImarisXT spine classifier, spines can be classified into different groups. Both spines and dendrites can also be classified into beginning points, branches, branching points and terminal points.

• Filament Processing – Choose between manual and automatic merging of Filaments, split filaments into unconnected sub-filaments and manually connect broken or unconnected segments.

(5) Measurements

FilamentTracer provides interactive measurements and data export. This feature requires Imaris MeasurementPro.

• Statistical Values – FilamentTracer provides a huge selection of Neuroscience specific statistical values: Dendrite Volume, Dendrite Length, Number of Dendrite Segments, Number of Dendrite Branches, Dendrite Area, Spine Volume, Spine Length, Number of Spines, Spine Density, Branch Level, Scholl Analysis, and many more. Now in addition to these parameters the users can select specific values related to the 3D tracking over time of Dendrite and Spine's edges, branch and attachment points This tracking data allows researchers to examine changes over time in any statistical parameter calculated by FilamentTracer.  These statistics can also be used on non-neuroscience related filament like structures.

• Data Export – Easy export of data to selected modeling programs (Neuron) and to spreadsheet programs (Excel, CSV format).

• Filtering – Utilize the identical filtering methods with FilamentTracer as described in Imaris MeasurementPro.

Automatic Dendrite and Spine Detection

 This video tutorial provides an overview of automatic dendrite and spine detection.

Movie: Automatic Dendrite and Spine Detection Tutorial movie (8:21)
Download the Movie

Semi Automatic and Manual Filament Tracing Methods

This video tutorial provides an overview of AutoPath, AutoDepth and Manual filament tracing methods. 

Movie: Semi Automatic and Manual Filament Tracing Methods Tutorial movie (7:26)
Download the Movie