AutoTrack
by
Doug Root
INTRODUCTION
	AutoTrack is a set of macros for Image version 1.55 that assists the determination of distances traveled by actin filaments during in vitro  motility assays. (It may be used to measure the velocity any image that leaves a track of uniform width, e.g. sperm.) The "Track" macro sums binary images of filaments into a track, and the "TrackLength" macro measures the contour length of the track to determine the distance the actin filament traveled. Average velocities (speed) are determined by dividing the measured distance by the elapsed time. This method works best, if noise is removed from the images by frame averaging and erosion of the binary images. The determination of accurate filament velocities requires human interaction, but tracks can be easily edited with standard Macintosh paint  tools. The virtues of this method are that the algorithm is fast, the software is free, and the equipment for using this software is relatively inexpensive compared to commercial tracking systems.

HINTS AND EXPLANATIONS
	Realtime frameaveraging and background subtraction are not required but may speed up the analysis. A VideoSpigot framegrabber (SuperMac) is sufficient for most applications. I recommend grabbing 320x240 frames at a fixed near maximum frame rate (10-15 frames per second with a VideoSpigotNubus card) using ScreenPlay (Supplied with VideoSpigot). Choose Save As and Options to save the movie as compression=None and 256 Grays. Then import the movie into NIH Image using the Average Quicktime Movie macro in AutoTrack. I suggest averaging about 8 frames to remove noise. The acquisition frequency determines the frame rate and the number of frames that will be used for the analysis; for instance, a 160 frame 16 second long movie imported with an acquisition frequency of 4 will give a 40 frame 16 second long stack in NIH Image. The final frame rate must be high enough so that individual filament positions overlap each other. However, if the frame rate is too high then processing time may be increased unnecessarily. I find that about 20-50 total frames are optimal for analyzing. Analysis can be continued as described in the basic procedure above.
	If the initial image quality is not high, then the most time consuming step can be editing the tracks (step 7 of the basic procedure). Track editing requires understanding of the processes of AutoTrack. The tracks should ideally be smooth, have uniform short width, come from filaments moving continuously during the sequence, and not overlap with other tracks or itself. Any tracks not fitting these criteria must be deleted or edited.
	A track that overlaps itself (figure on left) can be edited for use by opening enclosed areas (figure on right).


	Tracks that overlap can be edited by separating them (but you must erase the initial filament length if it is long).


	NIH Image's animation funtions are useful to determine if filaments move uniformly throughout the sequence. It is possible keep track by hand of how much time individual filaments moved during the sequence being analyzed. Then filaments that did not move during the entire sequence can still be quantified. A minimum of 20 frames is commonly the standard for velocity determinations. 

GRABBING A QUICKTIME MOVIE WITH VIDEOSPIGOT
1. Refer to manual of quicktime framegrabber card for more details.
2. Open the application "ScreenPlay."
3. Change preferences to grab at a specific framerate (about 12 fps for 320x240 up to 29 fps for 160x120 quicktime movie sizes.
4. Drag bottom right hand corner of viewing window to resize it for the desired capture size (maximum is 320x240).
5. When the vcr plays the sequence of interest, click the circular button to record, and the square button to stop.
6. Choose "Save" from the file menu to save the movie.
7. Then choose "Save As" from the file menu and set the compression to "None" and "256 Grays" and resave the movie (with a different name). This quicktime movie can now be imported into NIH Image.


QUICKTIME BASIC PROCEDURE
1. Open NIH Image (versions 1.54 or higher) with at least 4 megabytes (more is always better!) of free memory (change memory allocation if necessary).
2. Close all open windows.
3. Choose "Load Macros" from the Special menu and load "AutoTrack-Quicktime".
4. Choose "Average quicktime movie" from the Special menu and import a quicktime movie that was saved with compression=None, 256 Grays format.
5. The following sequence of commands from the special menu gives the best image quality: InvertStack (for fluorescent images), SubtrackBackground, EnhanceContrastStack, ThresholdStack (you must determine the proper threshold value by using the "Threshold" command from the options menu), and ErodeStack (repeat this command as needed to remove noise and smooth filament contours).
6. Choose "Track" from the Special menu.
7. Edit tracks using paint tools.
8. Choose "TrackLength" from the Special menu ("FilamentLength" does the same as "TrackLength" except that it does not subtract out the length of the first frame filament and returns a distance measurement only) and set the minimum particle size high enough to exclude any noise from being measured.
9. Save the results. Speed is the contour length of the tracks divided by time in units per second.

QUICKTIME AUTOMATED PROCEDURE
1. A Quicktime Movie (compression=None, 256 Grays) that has been captured at high frame rates can be automatically frame averaged and tracked by using first  "AutoTrack Quicktime," editing the tracks, and then using "TrackLength Quicktime."
2. PLEASE NOTE THAT AUTOTRACK MAY NEED ADJUSTMENTS FOR INDIVIDUAL IMAGING SETUPS. Pay particular attention to thresholding values, gain and brightness on framegrabber, scale calibration, and image inversion functions. Also, the macros assume that you are using NTSC (30 frames per second) standard video rate; if you use PAL/SECAM or slow or fast play modes of the VCR, the macros will need modification. The speed calculations are also based on a 21 frame stack, so to use larger stacks, adjustments in the macros will have to be made accordingly.

SCION AG-5 BASIC PROCEDURE
1. Open Scion Image (versions 1.55 or higher) with at least 8 megabytes (more is always better!) of free memory (change memory allocation if necessary).
2. Close all open windows.
3. Choose "Load Macros" from the Special menu and load AutoTrack.
4. Choose "AG-5 Average Live," enter the nuber of frames to average (recommend about 8), then type "ð" and "." simultaneously to stop viewing the averaged image. Make a rectangular ROI selection. and select "Capture Averaged" to grab 21 averaged frames into a stack.
5. The following sequence of commands from the special menu gives the best image quality: SubtrackBackground, InvertStack (for fluorescent images), EnhanceContrastStack, ThresholdStack (you must determine the proper threshold value by using the "Threshold" command from the options menu), and ErodeStack (repeat this command as needed to remove noise and smooth filament contours).
6. Choose "Track" from the Special menu.
7. Edit tracks using paint tools.
8. Choose "TrackLength" from the Special menu ("FilamentLength" does the same as "TrackLength" except that it does not subtract out the length of the first frame filament and returns a distance measurement only) and set the minimum particle size high enough to exclude any noise from being measured.
9. Save the results. Speed is the contour length of the tracks divided by time in units per second.

SCION AG-5 AUTOMATED PROCEDURE
1. With an AG-5 framegrabber installed the processing time is substantially enhanced. Automated steps of the above procedure are illustrated by using first  "AG-5 Average Live," typing "ð" and "." simultaneously, making a rectangular ROI (region of interest) selection, using"AG-5 AutoTrack," editing the tracks, and then using "AG-5 TrackLength."
2. PLEASE NOTE THAT AUTOTRACK MAY NEED ADJUSTMENTS FOR INDIVIDUAL IMAGING SETUPS. Pay particular attention to thresholding values, gain and brightness on framegrabber, scale calibration, and image inversion functions. Also, the macros assume that you are using NTSC (30 frames per second) standard video rate; if you use PAL/SECAM or slow or fast play modes of the VCR, the macros will need modification. The speed calculations are also based on a 21 frame stack, so to use larger stacks, adjustments in the macros will have to be made accordingly.

LG3 BASIC PROCEDURE (Also works for AG-5)
1. Open NIH Image (versions 1.54 or higher) with at least 16 megabytes (more is always better!) of free memory (change memory allocation if necessary).
2. Close all open windows.
3. Choose "Load Macros" from the Special menu and load "AutoTrack-LG3/AG5".
4. Choose "Make Movie" from the Stacks menu. (Make certain that blind capture was selected in the Video Controls for optimum performance.) Capture a movie that has at least 21 * (Acquistion Frequency) + (the number of frames that you wish to average). For example, if you intend to average 8 frames and use an Acquisition frequency of 1 you must capture at least 29 frames in the movie.  A delay may be set to adjust the capture rate.  Save the movie as a TIFF Stack.
5. Choose "Average LG3-AG5 movie" from the Special menu and import a the saved TIFF Stack.
6. The following sequence of commands from the special menu gives the best image quality: InvertStack (for fluorescent images), SubtrackBackground, EnhanceContrastStack, ThresholdStack (you must determine the proper threshold value by using the "Threshold" command from the options menu), and ErodeStack (repeat this command as needed to remove noise and smooth filament contours).
7. Choose "Track" from the Special menu.
8. Edit tracks using paint tools.
9. Choose "TrackLength" from the Special menu ("FilamentLength" does the same as "TrackLength" except that it does not subtract out the length of the first frame filament and returns a distance measurement only) and set the minimum particle size high enough to exclude any noise from being measured.
10. Save the results. Speed is the contour length of the tracks divided by time in units per second.

QUICKTIME AUTOMATED PROCEDURE
1. Choose "Make Movie" from the Stacks menu. (Make certain that blind capture was selected in the Video Controls for optimum performance.) Capture a movie that has at least 21 * (Acquistion Frequency) + (the number of frames that you wish to average). For example, if you intend to average 8 frames and use an Acquisition frequency of 1 you must capture at least 29 frames in the movie.  A delay may be set to adjust the capture rate.  Save the movie as a TIFF Stack.
This movie that has been captured at high frame rates can be automatically frame averaged and tracked by using first  "AutoTrack LG3-AG5," editing the tracks, and then using "TrackLength LG3-AG5."
2. PLEASE NOTE THAT AUTOTRACK MAY NEED ADJUSTMENTS FOR INDIVIDUAL IMAGING SETUPS. Pay particular attention to thresholding values, gain and brightness on framegrabber, scale calibration, and image inversion functions. Also, the macros assume that you are using NTSC (30 frames per second) standard video rate; if you use PAL/SECAM or slow or fast play modes of the VCR, the macros will need modification. The speed calculations are also based on a 21 frame stack, so to use larger stacks, adjustments in the macros will have to be made accordingly.

	The FilamentLength macro can be used to determine the length of actin filaments in an image. 

Checkout: D. D. Root and K. Wang (1994) "Calmodulin-Sensitive Interactions of Human Nebulin Fragments with Actin and Myosin," Biochemistry 33(42):12581-12591, for an example of the use of AutoTrack (formerly Track1.47) in actin filament tracking.
	If there are any questions or bugs I may be contacted at ddr@emx.cc.utexas.edu. Have fun!
-Doug Root    University of Texas at Austin.