Lab-on-a-Chip for Studying Growing Pollen Tubes - Plant Cell Morphogenesis: Methods and Protocols - page 228

developed an experimental platform based on microfl uidics and
microelectromechanical systems (MEMS) technology, the TipChip.
The TipChip is a lab-on-a-chip device with planar geometry
that allows for high-resolution optical microscopy and fl uorescence
imaging. It consists of a microfl uidic network with limited thick-
ness in order to restrain any interactions between two cells or cell
and microstructure to a two-dimensional space, to avoid the accu-
mulation of pollen grains into stacks, and to ensure that all growth
activity occurs in one focal plane. The design meets several criteria:
(1) several cells can be treated and observed simultaneously; (2)
positioning of pollen grains occurs through defi ned fl uid fl ow; (3)
the experimental environment is enclosed from all sides, thus pre-
venting evaporation of the growth medium, while allowing con-
tinuous fl ow of medium to supply fresh nutrients and oxygen to
pollen tubes; and (4) optical compatibility must allow monitoring
of pollen tube growth in bright-fi eld and fl uorescence mode.
The fabrication of the design starts with the planning of its
layout to ensure the proper, fl uid-fl ow-mediated positioning of the
pollen grains at the entrance of the microchannels and the incorpo-
ration of the experimental tests within the microchannel. The
design pattern is drawn in a CAD software, reproduced on a pho-
tomask, and transferred to a silicon/SU-8 mold through photoli-
thography. Next, the microfl uidic network is fabricated from
polydimethylsiloxane (PDMS) by creating replicas using the sili-
con/SU-8 mold [ 8 , 9 ]. The choice of PDMS as material is moti-
vated by its biocompatibility (nontoxicity), optical transparency,
relative low cost, and ease of use. Conventional planar microfabri-
cation techniques and soft lithography make redesign loops
straightforward since fabrication is systematically performed.
Furthermore, the fabrication procedure can be modifi ed easily to
include more sophisticated structures, layers, or features.
Using the TipChip in various implementations [ 10 , 11 ], we
obtained successful pollen germination and properly elongating
tubes displaying growth morphology and behavior that are indistin-
guishable from conventional in vitro setups. Pollen tubes grow along
the microchannels in the direction enforced by their shape attaining
total lengths over 1 mm. Pollen germination and growth rate within
the device are consistent with those observed under conventional in
vitro conditions confi rming that the spatial confi nement and associ-
ated limitation of the volume of the surrounding growth medium
does not interfere negatively with cellular behavior. The interaction
of pollen tubes with the microchannel features can elucidate many
aspects of pollen tube behavior as demonstrated here through a sim-
ple mechanical obstacle test. More importantly, the presented micro-
device allows for the design and easy integration of different kinds of
microsensors within the microfl uidic network to measure various
biological parameters at the level of a single pollen tube. This opens
multiple new avenues for experimental assays that have not been
possible to conduct in conventional bulk experiments.