Optical Trapping in Plant Cells - Plant Cell Morphogenesis: Methods and Protocols - page 252

3 Methods

3.1 Calibration of the
Optical Tweezers

Prior to trapping experiments, the focal point of the trapping laser
is adjusted in the z direction to the visual focus of the microscope
using the CellTools software (MMI), to allow imaging and trap-
ping at identical z -planes. Afterwards, the x,y position of the trap-
ping laser is calibrated such that the position on the screen and the
real position correspond. We use the following approach:
1. Paint one face of a large (50 × 24 mm or similar) coverslip with
a black marker and mount it dry on a slide with the painted
face towards the slide and focus with bright-fi eld optics on the
ink using the objective that requires calibration ( see Note 1 ).
The optical tweezers laser is switched on and the focus and
intensity of the laser are adjusted using the CellTools software
until the ink absorbs suffi cient energy to locally decompose
the ink ( see Note2 ).
2. Once the laser position has been detected, iterate adjustment
of the laser power and z position at different x,y positions by
moving the stage, until the laser produces a small and focused
point using minimal laser power. Keep this z position and con-
tinue to adjust the x and y positions using the CellTools soft-
ware by fi rst repositioning the trapping laser to the center
position of the fi eld of view and subsequently calibrating the
x,y direction and amplitude ( see Note 3 ).
3. When the trapping laser has been focused and its position has
been calibrated for the objective lenses that will be used, switch
to your biological sample or perform additional testing/cali-
bration ( see Note 4 ).

3.2 Laser Alignment

It is possible to align a low-intensity laser beam in the visible light
range with the trapping laser to detect its position while imaging.
After successfully trapping a structure, the visible laser can be
switched off during confocal imaging. Our system is not equipped
with such a laser. However, a CCD camera can be used to detect the
infrared trapping laser that gives a green refl ection on glass surfaces
such as the coverslip. To position the trapping laser, we collect a
bright-fi eld or a wide-fi eld fl uorescence image using the CellTools
software prior to a trapping experiment. By displaying this image on
the monitor of the computer that runs CellTools, we use it as a posi-
tional reference for confocal imaging. Then we switch to confocal
mode (imaging settings should be adjusted before the experiment)
and acquire confocal time series during a trapping experiment.

3.3 Trapping in Plant
Samples

High-numerical-aperture objectives maximize the focusing of the
trapping laser. We have obtained good results with a Zeiss
100×/N.A. 1.45 -Plan Fluar and a Zeiss 63×/N.A. 1.4 Plan-

Apochromat objective. At lower magnifi cations or numerical

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