Chiyoung Kim

4/27/15

Extavour Lab Meeting

Agabros: The Multi-Egg Holder

One of the biggest drawbacks to live-imaging of biological specimens is that most current methods allow for only one specimen at a time,

making the acquisition of large data sets unfeasible. The agarose mold solves this problem by allowing multiple embryos to be imaged at once, leading to

more timelapse data per trial. With the data gleaned through use of this mold, I hope to get a better insight into the inner workings of the embryo and quantify cell flows during cell migration.

Goals: Create an agarose mold must be user friendly, adaptable, reliable Analyze data created as a result direction of velocities, % of cells in positive x direction

Methods: Modeling egg troughs

Creating egg molds using trough design

Analysis in MATLAB Circular sector

Positive x

Egg Troughs, Creation of Egg Molds

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Fig. 1: 1: Seth's prototype of the egg troughs

2: Subsequent creation of egg troughs of different depths with different acrylic shapes 3: Final product for version 1 trough length = 2.93 mm, trough depth = .57 mm

Fig. 2: From left to right: The first version of the egg mold. Left shows bottom of the mold with small handle and right shows top with agar pour hole

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Fig.3: 1: The bottom of the agar mold V2. The mold hole is very deep in this version.

2: The top of the agar mold V2. Note the deep circular buffer well.

3: The accessory parts of V2 from left to right: Buffer well cap, plug, mold cap 4: The fully assembled V2, with plug removed.

Addressed problems: The mold did not always stay on, and alignment ended up being very inaccurate. Additionally, the old design wasted agar because it depended

on overflow of agar to fill up the cavity. Because of this, I switched to

a version where you pour from the top so as to reduce agar waste.

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Fig.4: 1: The bottom of the agar mold V2.2. The mold hole has been made shallower. 2: The top of the agar mold V2.2. The buffer well has been made shallower.

3: The accessory parts of V2.2 from left to right: Buffer well cap, mold cap. Note the mold cap's coordinate system. 4: The fully assembled V2.2

Addressed problems: The previous version used too much agar and material in general. This version attempted to

cut down on agar and acrylic usage as much as possible.

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Fig.5: 1. The agar mold V2.2 Mini with the mold cap. Note that the insert's agar hole is not sufficiently large enough.

2. The assembled agar mold V2.2 Mini with mold cap

Addressed Problems: Made the mold insert part much smaller so as to minimize on acrylic usage and fit the profile of the microscope.

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Fig.6: 1. The new V2.3 inserts. Note the wider agar hole.

2. The assembled V2.3 inserts using the V2.2 mold caps.

Addressed Problems: Made the agar hole bigger because the previous trough mold did not fit into the holes of the previous versions.

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Fig.7: 1. The new body insert. Note the

rounded corners of the mold. 2, 4: The new mold cap. Note the

slot on the bottom for alignment.

2 = Top view, 4 = Bottom view 3, 5: The new mold insert. Note the

rectangular tab on the bottom

for alignment. 3 = Top view, 5 = Bottom view

Addressed Problems: Corners of the resulting

agar block interfered

with usage of the mold.

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Fig.8: 1. The fully assembled mold. The mold insert goes into the mold cap. The body insert goes into this combination.

2. The mold insert and mold cap combination. This is equivalent to the mold cap of the previous versions.

Lacking are photos of the newest generation which has a thinner mold cap and body insert. This allows the body insert to completely fit inside the petri

dish and allows a cap to be placed over the entire combination.

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Fig.9: 1. The failed vertical mold. The tight spacing of the comb caused the agar to stick to the mold and stay there forever.

2. The petri dish used for imaging the embryos.

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Fig.10: 1. Setting the agar.

2. The mold in action. Note the embryos in a neat 2x4 arrangement. 3. Timelapse image of embryo in the mold. The lower cluster of cells was analyzed in the following MATLAB graphs.

Fig.11: Top: A graph of the ratios of cells moving in the positive x direction.

Bottom: A graph of the proportions of different directions the cells velocities took.

Things to work on:

Try different PIV settings Fix problems with ATLAB currently getting unexpected results that seem to be wrong Possibly a problem in the PIV method try using bigger timesteps Masking of the black non-embryo region