Optical Trapping in Plant Cells - Plant Cell Morphogenesis: Methods and Protocols - page 250

in plant cells highly benefi ts from the reduced out-of-focus blur
obtained with confocal microscopy. Although the integration of
optical tweezers setup into a confocal microscope is nontrivial, it is
essential to perform simultaneous optical trapping experiments
and imaging of fl uorescently tagged structures in plant cells. In this
chapter, we describe our system, consisting of a Zeiss LSM510
META confocal microscope, mounted on a Zeiss Axiovert 200 M
inverted microscope stand with an optical tweezers setup
(Molecular Machines and Industries, Glattbrugg, Switzerland).
The combination of optical trapping and confocal imaging has
only become available in the last decade but has so far failed to
produce a large amount of published data of intracellular manipu-
lation of plant cells [ 4 , 5 ], mainly due to the limited availability of
suitable microscope systems and the challenges to prepare plant
samples that are suitable for combined optical trapping and confo-
cal microscopy. Even so, this type of experimentation opens fantas-
tic opportunities to gain insight in subcellular force generation. In
our work with the optical tweezers/confocal microscope system,
we have explored physical aspects of actin organization in Tobacco
Bright Yellow-2 (BY-2) cells [ 5 ], the connection between Golgi
bodies and the ER, and ER organization [ 4 ]. This chapter describes
the technical specifi cations of our integrated confocal microscope
and optical tweezers and provides hints to prepare plant tissues for
these experiments and a description of the subsequent steps we fol-
low during a typical experiment.

2 Materials

2.1 Description
of Our System

A Molecular Machines & Industries (MMI, Glattbrugg,
Switzerland) CellManipulator optical trap, consisting of an infrared
Nd-YAG solid-state laser (1,064 nm, 3,000 mW CW) and x-y galvo
scanner, is connected to the backport of an Axiovert 200 M inverted
microscope (Zeiss, Jena, Germany). At the same backport, a
Uniblitz shutter (Vincent Associates, Rochester, USA) is used to
control 100 W HBO illumination. An MMI expander is used to fi ll
the back focal plane of a 100×/N.A. 1.45

-Plan Fluar or a
63×/N.A. 1.4 Plan-Apochromat objective. At the Axiovert 200 M
base port a scan box of a Zeiss LSM510 META confocal is con-
nected. The confocal is equipped with a 25 mW 405 nM laser, a
30 mW Ar laser (458, 477, 488, 514 nm), a 1 mW green HeNe
laser (543 nm), and a 5 mM red HeNe laser (633 nm), which allows
simultaneous trapping and confocal imaging of various fl uorescent
probes. A schematic overview of our system is given in Fig. 1 .
During combined confocal imaging and optical trapping,
1,064 nm laser light from the optical tweezers is refl ected into the
optical path for confocal imaging. This is achieved by placing a
beam splitter in the Axiovert 200 M refl ector module that com-
bines maximal transmittance for excitation and emission at

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