Biology Reference
In-Depth Information
7.1
DESIGN OF THE PHOTOMASK
The micropatterns which define different cell sizes and shapes are typically drawn
using software such as L-Edit (
www.tannereda.com
)
. The files containing the micro-
patterns are then sent to a company such as Toppan (
www.photomask.com
)
which
laser-etches the micropatterns onto a synthetic quartz plate, transparent to deep UV
light (wavelength below 200 nm). This is the “photomask” which will be used sub-
sequently in lab to produce the micropatterned substrate to adhere cells. Photomasks
vary in size and composition. We find that a typical 12.5 cm
12.5 cm photomask is
useful and can fit many different micropatterns.
The size of individual micropatterns is an important parameter, for example, if the
pattern is too big, the cells will not be able to spread completely, and if it is too small,
the cells will not spread enough for visualizing the mitochondria properly. Examina-
tion of cells spread on a regular coverslip can give reasonable estimates of cell size
prior to designing the micropatterns. It is important to keep in mind that cells respond
to the size of the micropatterns. Really small or really big patterns change the tension
of the underlying actin cytoskeleton, which has been shown to change, for example,
centrosome positioning (
Rodr´guez-Fraticelli, Auzan, Alonso, Bornens, & Mart´n-
Belmonte, 2012
). A too small size might even induce apoptosis (
Chen, Mrksich,
Huang, Whitesides, & Ingber, 1997
). It is a good idea to design several sizes to opti-
mize cell spreading for specific studies. Well-spread cells will help visualize the indi-
vidual mitochondrion (
Chevrollier et al., 2012
), whereas a more compact spreading
may be good for making mitochondria density maps (
Schauer et al., 2010
).
Another parameter to consider is the shape of the micropatterns, as the adhesion
of cell to its substrate profoundly influences cytoskeletal organization, cell polarity,
and cell shape (
Th´ry et al., 2006
). For example, a round micropattern leads to round
cells which have no polarity compared to a crossbow-shaped micropattern, which
produces highly polarized cells. In this context, one can design micropatterns
where the whole area of the shape will be filled with adhesive substrate, or only
the outline of the shape will be adhesive, or any combination of adhesive and non-
adhesive regions, etc. The distance separating individual micropatterns should be big
enough to prevent a cell from spreading over two patterns or to contact another cell
on an adjacent pattern. This distance is dependent on cell size and how mobile the
cells are, and therefore on cell types, but 50-100
m is a good reference.
Finally, the transfer of the micropatterns from the photomask onto individual glass
coverslip can be optimized. A typical photomask has dimension 12.5 cm
m
12.5 cm,
and a typical coverslip has dimension 2 cm
2 cm. Therefore,
the photomask
can be sectored into 5
5 squared regions, fitting 25 different coverslips simulta-
neously. Each region can have about 1000 identical micropatterns or 1000 different
individual micropatterns, or any other combinations. It is useful to create 1-mm wide
borders and to introduce numbering/lettering visible to the eye to distinguish the
different regions. Inside each region, further markings may help differentiate different
areas of the region which may help subsequent navigation through the coverslip
under the microscope, if the patterns are stained with a fluorescent protein.
