Optical Trapping in Plant Cells - Plant Cell Morphogenesis: Methods and Protocols - page 254

3.4 Preparation
of Slides

Only use coverslips with a thickness of 1.0 (equivalent to 0.13-
0.16 mm), prepare slides with plant material just before trapping,
and mount the sample as close and fl at to the coverslip as possible,
since the strength of the tweezers drops rapidly with distance from
the coverslip.
We use custom-made slides covered with gas-permeable Biofoil
([ 6 ]; for Biofoil ordering details, see the root hair growth chapter
in this volume) and use VALAP (1:1:1 vaseline:lanolin:paraffi n) to
prevent slides from drying out by sticking a glass Pasteur pipette
approximately 1 cm into solidifi ed VALAP at room temperature.
When the tip of the Pasteur pipette containing the VALAP is briefl y
heated in a fl ame, it melts. By tracing the outline of the coverslip
with the tip of the pipette with the molten VALAP, the slide can
be sealed.
We noticed that not only transparent refractive bodies but also
light-absorbing structures such as chloroplasts or colored lipid
droplets can be trapped. Trapping of these structures leads to rapid
local heating due to light absorption. In cells that show signs of
vitality loss or degradation, for example, by increased contrast and
cytoplasmic clumping, it is more diffi cult or not possible at all to
trap or move structures.
Interactions of organelles with the actin cytoskeleton, such as
cytoplasmic streaming, produce forces in the same range as the
optical tweezers and can interfere with trapping in some cell
types. These interactions can be inhibited by actin depolymeriza-
tion when it does not interfere with the research questions ( see ,
e.g., ref. [ 4 ]).
Objects that are irregularly shaped are diffi cult to trap because
they are often repulsed from the focal point of the trapping laser.
This occurs, for example, with polystyrene beads that have been
stored under non-sterile conditions and have acquired an irregular
coating of bacteria. Elongate micron-sized objects can be trapped
but tilt with their long axis in the z -axis of the focal point of the
optical tweezers laser. Such reorientations in the trap focus should
be considered in experiments.
Since the amount of trapping force correlates with the differ-
ence in refractive index of the trapped structure and the surround-
ing medium, the visibility of such a structure when using Nomarski
optics often is an indication whether an object can be trapped or
not, although we have successfully trapped objects that could not
be detected at all using Nomarski imaging. An excessively high
intensity of the trapping laser can cause accumulation of multiple
refractile bodies in the focal point of the trap. In plant cells this is
manifested by an accumulation of organelles around the position
of the trap. To avoid this, the minimal power that is required for
trapping the desired organelle should be determined.