Chapter 22 Optical Trapping in Plant Cells Tijs Ketelaar, Norbert de Ruijter, and Stefan Niehren Abstract Optical tweezers allow noninvasive manipulation of subcellular compartments to study their physical interactions and attachments. By measuring (delay of) displacements, (semi-)quantitative force measurements within a living cell can be performed. In this chapter, we provide practical tips for setting up such experiments paying special attention to the technical considerations for integrating optical tweezers into a confocal microscope. Next, we describe some working protocols to trap intracellular structures in plant cells. Key words Optical tweezers, Optical trap, Confocal microscope, Noninvasive manipulation, Plant cell, Cytoskeleton, Endomembrane system
1
Introduction Optical tweezers use gradient light forces for attracting (sub-) micrometer-sized particles in a highly focused laser beam. Differences in refractive indices cause a total force pointing towards the center of the diffraction-limited spot. If the diffraction index of the particle is higher than the index of the surrounding medium, the force is attractive and creates an optical trap. Generally, infrared laser radiation is used for optical trapping since the absorbance of light with wavelengths in the infrared range is extremely low in most biological materials. For more information about optical trapping, see ref. [1]. Optical trapping is often used in in vitro experiments in which conditions can be controlled. Nevertheless, optical trapping of subcellular compartments with a high refractive index is also possible in intact, live cells. In plant cells, these types of experiments have greatly improved our understanding of physical aspects of intracellular organization [2, 3]. In in vitro studies, optical trapping is often combined with wide-field fluorescence microscopy to obtain positional information of fluorescently tagged molecules or beads. Plant cells are often large, embedded in tissues, and cell walls or other cell constituents can give autofluorescence. Thus, imaging of fluorescence
Viktor Žárský and Fatima Cvrcˇková (eds.), Plant Cell Morphogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 1080, DOI 10.1007/978-1-62703-643-6_22, © Springer Science+Business Media New York 2014
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in plant cells highly benefits 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 fluorescently 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 manipulation 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 confocal microscopy. Even so, this type of experimentation opens fantastic 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 specifications 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 follow 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 fill the back focal plane of a 100×/N.A. 1.45 α-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 connected. 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 fluorescent 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 reflected into the optical path for confocal imaging. This is achieved by placing a beam splitter in the Axiovert 200 M reflector module that combines maximal transmittance for excitation and emission at
Optical Trapping in Plant Cells
TD
261
BF
CCD
100x QPD2 HBO QPD1
IR
Ar diode Ar He/Ne
Scan module (PMTs)
He/Ne
Fig. 1 Schematic overview of the described confocal microscope (Zeiss LSM510 META) with integrated optical tweezers (MMI CellManipulator). The grey structure 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-field halogen (BF) and wide-field fluorescence (HBO) illumination sources. CCD, charge-coupled device camera for collection of wide-field 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 reflection for optical trapping at 1,064 nm (positioned at the back port). The hardware is operated by two separate computers, one running the Zeiss LSM510 operating software and the other computer 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 field 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 additional 13× magnification at the left-side port to allow accurate bright-field (DIC) position detection for force calibration.
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Tijs Ketelaar et al.
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 trapping at identical z-planes. Afterwards, the x,y position of the trapping 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-field 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 sufficient 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 continue to adjust the x and y positions using the CellTools software by first repositioning the trapping laser to the center position of the field 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/calibration (see Note 4).
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 reflection on glass surfaces such as the coverslip. To position the trapping laser, we collect a bright-field or a wide-field fluorescence 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 positional 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 α-Plan Fluar and a Zeiss 63×/N.A. 1.4 PlanApochromat objective. At lower magnifications or numerical
3.2
Optical Trapping in Plant Cells
263
Fig. 2 Moving a trapped, unidentified organelle into the vacuolar lumen by relocating the position of the optical tweezers causes the formation of a cytoplasmic strand that acquires ER. The arrows indicate the position of the tweezers in the Nomarski images, and the arrowhead points to the ER that has appeared in the tweezersformed strand after 10 s. The first row of images shows the confocal fluorescence images, the second row the Nomarski images, and the third row a merge of the upper two rows in which the fluorescence image is displayed in green. Bar: 10 μm
apertures, we were not able to trap structures within plant cells. During the trapping experiments, the IR-laser power was varied between 25 and 100 %, which corresponds to a laser intensity in the sample that lies in the 25–130 mW range. Several considerations for sample preparation are given below. As an example of a typical imaging sequence, we have included three images taken from a time series in which an unidentified organelle is trapped in a Tobacco BY-2 cell expressing the live cell ER marker HDELGFP. When the organelle is moved into the vacuolar lumen, a tweezers-formed cytoplasmic strand is produced that is surrounded by a tonoplast membrane. This particular experiment shows that an ER strand rapidly appears in the tweezers-formed strand (Fig. 2).
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Tijs Ketelaar et al.
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 flat 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:paraffin) to prevent slides from drying out by sticking a glass Pasteur pipette approximately 1 cm into solidified VALAP at room temperature. When the tip of the Pasteur pipette containing the VALAP is briefly heated in a flame, 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 difficult 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 depolymerization when it does not interfere with the research questions (see, e.g., ref. [4]). Objects that are irregularly shaped are difficult 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 difference in refractive index of the trapped structure and the surrounding 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.
Optical Trapping in Plant Cells
4
265
Notes 1. It is important that the coverslip surface is flat. This can be achieved by fixing the coverslip with super glue or vacuum grease on a metal frame. 2. Safety considerations: a 3,000 mW infrared laser beam is both invisible and very dangerous; especially exposure to the eyes should be avoided at all times. Although most optical trapping systems require the use of infrared-blocking safety goggles, we have equipped our system with safety detectors that automatically switch off the infrared laser when the appropriate filters are not in place, when the trapping laser is not properly connected to the back port, or when the arm of the microscope is tilted backwards. 3. If desired, the output intensity of the trapping laser can be measured by placing a photon flux sensor, adjusted to sensitivity at 1,064 nm, in a flat position in the focal plane of an objective lens. The output intensity should be linear in a large output range of the laser (25–100 % output). 4. If the trap does not appear to trap anything in plant samples, a slide with fluorescent beads can be prepared to further test the tweezers. Freshly prepared fluorescent, polystyrene beads of 0.5–5 μm should be easy to trap. Freshly prepare a dilution of 1 drop polystyrene beads (Polysciences, carboxylate fluorescent microspheres) in 5 ml water. Prepare a slide with diluted beads. A fraction of the beads will attach to the charged glass surface and cannot be trapped.
References 1. Neumann KC, Block SM (2004) Optical trapping. Rev Sci Instrum 75:2787–2810 2. Grabski S, Xie XG, Holland JF et al (1994) Lipids trigger changes in the elasticity of the cytoskeleton in plant cells: a cell optical displacement assay for live cell measurements. J Cell Biol 126:713–726 3. Grabski S, Arnoys E, Busch B et al (1998) Regulation of actin tension in plant cells by kinases and phosphatases. Plant Physiol 116:279–290 4. Sparkes IA, Ketelaar T, de Ruijter NC et al (2010) Grab a Golgi: laser trapping of Golgi
bodies reveals in vivo interactions with the endoplasmic reticulum. Traffic 10:567–571 5. van der Honing HS, de Ruijter NC, Emons AM et al (2010) Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers. New Phytol 185:90–102 6. Vos JW, Dogterom M, Emons AMC (2004) Microtubules become more dynamic but not shorter during preprophase band formation: a possible ‘search-and-capture’ mechanism for microtubule translocation. Cell Motil Cytoskeleton 57:246–258
1
Introduction Optical tweezers use gradient light forces for attracting (sub-) micrometer-sized particles in a highly focused laser beam. Differences in refractive indices cause a total force pointing towards the center of the diffraction-limited spot. If the diffraction index of the particle is higher than the index of the surrounding medium, the force is attractive and creates an optical trap. Generally, infrared laser radiation is used for optical trapping since the absorbance of light with wavelengths in the infrared range is extremely low in most biological materials. For more information about optical trapping, see ref. [1]. Optical trapping is often used in in vitro experiments in which conditions can be controlled. Nevertheless, optical trapping of subcellular compartments with a high refractive index is also possible in intact, live cells. In plant cells, these types of experiments have greatly improved our understanding of physical aspects of intracellular organization [2, 3]. In in vitro studies, optical trapping is often combined with wide-field fluorescence microscopy to obtain positional information of fluorescently tagged molecules or beads. Plant cells are often large, embedded in tissues, and cell walls or other cell constituents can give autofluorescence. Thus, imaging of fluorescence
Viktor Žárský and Fatima Cvrcˇková (eds.), Plant Cell Morphogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 1080, DOI 10.1007/978-1-62703-643-6_22, © Springer Science+Business Media New York 2014
259
260
Tijs Ketelaar et al.
in plant cells highly benefits 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 fluorescently 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 manipulation 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 confocal microscopy. Even so, this type of experimentation opens fantastic 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 specifications 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 follow 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 fill the back focal plane of a 100×/N.A. 1.45 α-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 connected. 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 fluorescent 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 reflected into the optical path for confocal imaging. This is achieved by placing a beam splitter in the Axiovert 200 M reflector module that combines maximal transmittance for excitation and emission at
Optical Trapping in Plant Cells
TD
261
BF
CCD
100x QPD2 HBO QPD1
IR
Ar diode Ar He/Ne
Scan module (PMTs)
He/Ne
Fig. 1 Schematic overview of the described confocal microscope (Zeiss LSM510 META) with integrated optical tweezers (MMI CellManipulator). The grey structure 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-field halogen (BF) and wide-field fluorescence (HBO) illumination sources. CCD, charge-coupled device camera for collection of wide-field 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 reflection for optical trapping at 1,064 nm (positioned at the back port). The hardware is operated by two separate computers, one running the Zeiss LSM510 operating software and the other computer 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 field 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 additional 13× magnification at the left-side port to allow accurate bright-field (DIC) position detection for force calibration.
262
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Tijs Ketelaar et al.
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 trapping at identical z-planes. Afterwards, the x,y position of the trapping 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-field 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 sufficient 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 continue to adjust the x and y positions using the CellTools software by first repositioning the trapping laser to the center position of the field 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/calibration (see Note 4).
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 reflection on glass surfaces such as the coverslip. To position the trapping laser, we collect a bright-field or a wide-field fluorescence 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 positional 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 α-Plan Fluar and a Zeiss 63×/N.A. 1.4 PlanApochromat objective. At lower magnifications or numerical
3.2
Optical Trapping in Plant Cells
263
Fig. 2 Moving a trapped, unidentified organelle into the vacuolar lumen by relocating the position of the optical tweezers causes the formation of a cytoplasmic strand that acquires ER. The arrows indicate the position of the tweezers in the Nomarski images, and the arrowhead points to the ER that has appeared in the tweezersformed strand after 10 s. The first row of images shows the confocal fluorescence images, the second row the Nomarski images, and the third row a merge of the upper two rows in which the fluorescence image is displayed in green. Bar: 10 μm
apertures, we were not able to trap structures within plant cells. During the trapping experiments, the IR-laser power was varied between 25 and 100 %, which corresponds to a laser intensity in the sample that lies in the 25–130 mW range. Several considerations for sample preparation are given below. As an example of a typical imaging sequence, we have included three images taken from a time series in which an unidentified organelle is trapped in a Tobacco BY-2 cell expressing the live cell ER marker HDELGFP. When the organelle is moved into the vacuolar lumen, a tweezers-formed cytoplasmic strand is produced that is surrounded by a tonoplast membrane. This particular experiment shows that an ER strand rapidly appears in the tweezers-formed strand (Fig. 2).
264
Tijs Ketelaar et al.
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 flat 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:paraffin) to prevent slides from drying out by sticking a glass Pasteur pipette approximately 1 cm into solidified VALAP at room temperature. When the tip of the Pasteur pipette containing the VALAP is briefly heated in a flame, 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 difficult 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 depolymerization when it does not interfere with the research questions (see, e.g., ref. [4]). Objects that are irregularly shaped are difficult 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 difference in refractive index of the trapped structure and the surrounding 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.
Optical Trapping in Plant Cells
4
265
Notes 1. It is important that the coverslip surface is flat. This can be achieved by fixing the coverslip with super glue or vacuum grease on a metal frame. 2. Safety considerations: a 3,000 mW infrared laser beam is both invisible and very dangerous; especially exposure to the eyes should be avoided at all times. Although most optical trapping systems require the use of infrared-blocking safety goggles, we have equipped our system with safety detectors that automatically switch off the infrared laser when the appropriate filters are not in place, when the trapping laser is not properly connected to the back port, or when the arm of the microscope is tilted backwards. 3. If desired, the output intensity of the trapping laser can be measured by placing a photon flux sensor, adjusted to sensitivity at 1,064 nm, in a flat position in the focal plane of an objective lens. The output intensity should be linear in a large output range of the laser (25–100 % output). 4. If the trap does not appear to trap anything in plant samples, a slide with fluorescent beads can be prepared to further test the tweezers. Freshly prepared fluorescent, polystyrene beads of 0.5–5 μm should be easy to trap. Freshly prepare a dilution of 1 drop polystyrene beads (Polysciences, carboxylate fluorescent microspheres) in 5 ml water. Prepare a slide with diluted beads. A fraction of the beads will attach to the charged glass surface and cannot be trapped.
References 1. Neumann KC, Block SM (2004) Optical trapping. Rev Sci Instrum 75:2787–2810 2. Grabski S, Xie XG, Holland JF et al (1994) Lipids trigger changes in the elasticity of the cytoskeleton in plant cells: a cell optical displacement assay for live cell measurements. J Cell Biol 126:713–726 3. Grabski S, Arnoys E, Busch B et al (1998) Regulation of actin tension in plant cells by kinases and phosphatases. Plant Physiol 116:279–290 4. Sparkes IA, Ketelaar T, de Ruijter NC et al (2010) Grab a Golgi: laser trapping of Golgi
bodies reveals in vivo interactions with the endoplasmic reticulum. Traffic 10:567–571 5. van der Honing HS, de Ruijter NC, Emons AM et al (2010) Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers. New Phytol 185:90–102 6. Vos JW, Dogterom M, Emons AMC (2004) Microtubules become more dynamic but not shorter during preprophase band formation: a possible ‘search-and-capture’ mechanism for microtubule translocation. Cell Motil Cytoskeleton 57:246–258
