Optical Trapping in Plant Cells - Plant Cell Morphogenesis: Methods and Protocols - page 251

TD

BF

CCD

100x

QPD2

HBO

QPD1 IR

Ar diode

Ar

Scan module
(PMTs)

He/Ne

He/Ne

Fig. 1 Schematic overview of the described confocal microscope (Zeiss LSM510
META) with integrated optical tweezers (MMI CellManipulator). The grey struc-
ture represents a Zeiss Axiovert 200 M inverted microscope. The colored boxes
represent lasers, as described in the text, and the boxes marked with BF and
HBO represent, respectively, the bright-fi eld halogen (BF) and wide-fi eld fl uores-
cence (HBO) illumination sources. CCD, charge-coupled device camera for col-
lection of wide-fi eld images during tweezers experiments; TD, transillumination
detector of the LSM510 system; QPD1 and QPD2, quadrant photo detectors;
PMTs, photomultiplier tubes. The dichroic mirror that integrates confocal with
trapping lasers (see description system) is positioned at the HBO/IR optical path

400-800 nm from/to the LSM510 scan module (positioned at the
base port) with maximal refl ection for optical trapping at 1,064 nm
(positioned at the back port).
The hardware is operated by two separate computers, one run-
ning the Zeiss LSM510 operating software and the other com-
puter running the CellTools (MMI) software that controls the
optical tweezers. A switch box links both systems to a Märzhauzer
XY SCAN IM 120-100 stage (Märzhauzer stage, Wetzlar,
Germany) to control x,y positioning with step sizes down to 75 nm.
After calibration ( see Subheading 3 ) the CellTools software can
position up to ten quasi-simultaneous, timeshared traps to any
location in the fi eld of view taking advantage of the ultrafast galvos
that position the laser.
The system is equipped with two quadrant photo detectors
(QPDs; Spectral Applied Research, Ontario, Canada) and an addi-
tional 13× magnifi cation at the left-side port to allow accurate
bright-fi eld (DIC) position detection for force calibration.