Building a tracker: our current version

We recently have built several new video tracking setups for microfluidic behavior experiments. By using inexpensive optics, LED backlighting, and machine vision cameras, we have reduced costs to under $1000 each.  Here we use off-the-shelf support materials, although some custom machining may be required to attach the camera.

Our current setup:

Parts:

Vendor	Item Description	Catalog #	Qty	Price ea	Total	Notes
Edmund Optics	Ring Mount with 0.75" Inner Diameter (ID)	Stock No. #54-264	2	16.50	33.00
Edmund Optics	Small Platform Base	Stock No. #53-009	1		219.00
Edmund Optics	3/4" Dia Stainless Posts, Vertical Post 24"	Stock No. #52-100	1		60.00
Edmund Optics	3/4" Dia Stainless Posts, Horizontal Arm 18"	Stock No. #39-354	1		55.00
Edmund Optics	Steel Post, 4" Length, 1/4-20 Stud	Stock No. #59-000	4	9.70	38.80
Edmund Optics	90 Degree Angle Mount	Stock No. #39-355	1		49.00
McMaster Carr	Two Conductor Power Cord NEMA 1-15 Plug, SPT-1-Flat, 18/2 AWG, 6' Length	7248K21	1		2.29
McMaster Carr	Heat-resistant Borosilicate Glass Square, 1/4" Thick, 10" X 10"	8476K225	1		16.82
McMaster Carr	Push-in Flexible Rubber Grommet Ms 35489-91, An 931-c3-5, 3/16" Id, 7/16" Od	9307K37	1 pk		4.84
McMaster Carr	18-8 Ss Button Head Socket Cap Screw 8-32 Thread, 5/8" Length	92949A196	1 pk		5.87
McMaster Carr	Standard Rubber Edge Trim 1/4" Opening, 19/32" Inside Depth	8507K32	10 ft		2.49
McMaster Carr	Plastic-Head Thumb Screw Black Rosette Head, 1/4"-20 Thread, 5/8" Length, packs of 10	91185A805	1 pk		11.01
Automate Scientific	Syringe Bracket (holds 8x 35ml and 60ml syringes)	sybrk8	1		48.00
Ebay	Pixelink PL-A741 (or similar)		1		150.00	range 150-300
Ebay	Zoom lens 18-108/2.5 No. 4520988 (or similar)		1		15.00	range 15-75
Ebay	12W Panel SMD LED Ceiling Down Light Bulb Lamp square		1		22.99	range 23-28
Amazon	"3M Gold Privacy Filter-3M GPF19.0W Widescreen Monitor Privacy Screen"		1		74.24	enough for three
Amazon	Dremel 7134 diamond bit, or 3/16" diamond drill bit		1			to drill glass
					808.35	TOTAL

Building the tracker:

1. Assemble 24" vertical post, 18" post, and four 4" steel posts onto small platform base.
2. Drill four 3/16" holes in the 10x10" glass plate and insert grommets.
3. Attach glass plate to steel posts and add optional rubber edge trim.
4. Solder power cord to LED backlight.  Cut privacy filter to fit backlight, 2 pieces at 90 degrees rotation.
5. Screw syringe bracket to ring mount and add thumb screw.
6. Attach zoom lens to camera and mount to 90 degree angle mount.

Microfluidic tubing connections

There are many ways to interface from the macroscale (tubing, reservoirs, etc) to a microfluidic device.  Commonly, this involves flexible tubing and rigid hollow pins that insert into the PDMS device.

We typically use the following materials:

• Pins: New England Small Tube, P/N: NE-1027-12, 0.042OD x 0.027ID x 0.500long
• Tubing: Tygon S-54-HL 0.020" ID, 0.020" wall, Fisher cat# 1417015B
• Punch: Acuderm 1mm dermal punch

Note that the ID of the tubing is less than that of the pin, so I heat the tubing up (brief sweep above a flame until soft), open with fine forceps, and wiggle in the pin. As it cools it shrinks and grabs tightly on the pin. Best is to insert ~1/3 of the pin.

Video formats

Tracking code currently works with videos that are in .AVI format and compressed with Indeo Video 5 (or uncompressed).  Videos should be 24-bit color depth.

If your video is in another format, many free video converters are available online.  One that I use regularly is VideoMach.  Note that in this program, the video format (or wrapper), in this case .AVI, is chosen when selecting a filename for the converted video.  The compression codec (Indeo Video 5, or IV50) is selected separately under the video tab.

Getting started with the Arena Worm Tracker

For getting started with the software package, I've added a sample raw video file and the results of tracking on the ArenaWormTracker site.  The video shows wild-type (N2) C. elegans navigating a microfluidic arena and responding to two stripes of isoamyl alcohol over 15 mins.

Get the sample video and analysis here. Warning: it's a big file (>70Mb, .zip).

Once the tracker suite is installed, begin analysis by running TrackerScript.

A frame of the video should appear.  The following user inputs are required, according to the prompts shown in the figure:

1. Click a region outside the arena to record background light intensity for each frame.
2. Drag a box around the arena to indicate its position.  Choose the left/right edges to exclude at least one whole circular post, to exclude behaviors when the animal touches the upstream/downstream barriers.  Choose upper/lower boundaries just outside of the border.  The selected region turns red.  Click once inside the red box to confirm; otherwise, click outside and drag a new box.
3. Get scaling.  The sample video shows a 22 mm vertical device.  Pick two points on the upper and lower boundaries of the arena where they are horizontal (for example, upper-left and lower-left corners).

Analysis should begin and the figure should update every 60 frames, showing the current frame and tracked animals (blue + indicates centroid; red line is prior path).

At the end of analysis, a single multipage PDF should appear in the same folder as the raw video.

Version 1.0 scripts were tested on Win XP and Windows 7 running MATLAB v7.0.1.