The system described herein employs a traditional optical trap as well as an independent holographic optical trapping line, capable of creating and manipulating multiple traps. This allows for the creation of complex geometric arrangements of refractive particles while also permitting simultaneous high-speed, high-resolution measurements of the activity of biological enzymes.
High resolution microscope systems with optical traps allow for precise manipulation of various refractive objects, such as dielectric beads 1 or cellular organelles 2,3, as well as for high spatial and temporal resolution readout of their position relative to the center of the trap. The system described herein has one such “traditional” trap operating at 980 nm. It additionally provides a second optical trapping system that uses a commercially available holographic package to simultaneously create and manipulate complex trapping patterns in the field of view of the microscope 4,5 at a wavelength of 1,064 nm. The combination of the two systems allows for the manipulation of multiple refractive objects at the same time while simultaneously conducting high speed and high resolution measurements of motion and force production at nanometer and piconewton scale.
Optical trapping is one of the key techniques in biophysics 6. A crucial advancement in optical trapping has been the development of holographic traps which allow for the creation of three-dimensional trapping patterns rather than conventional point traps 7. Such holographic traps possess the advantage of versatility in positioning of refractive objects. However conventional traps can be easily aligned to be more symmetric than commercially available holographic kits. They also allow for fast precise tracking of the trapped objects. Here we describe a system (Figure 1) which combines the two trapping approaches in one instrument and allows the user to exploit the benefits of both as appropriate.
The general considerations of constructing optical traps (based on single or multiple laser beams) are discussed in detail elsewhere 8-10. Here, we outline the considerations specific to our setup and provide detail of our alignment procedure. For instance, systems with two optical trapping beams have been described before (e.g. ref. 11), typically using one laser beam for trapping a refractive object and using the other (intentionally low power beam) for decoupled readout of the position of the trapped object. Here however, both laser beams need to be high powered (300 mW or higher) because both are to be used for trapping. For measurements of biological systems, the lasers used for trapping should optimally fall within a specific NIR window of wavelength to minimize light induced protein degradation 1. Here we have chosen to use 980 nm diode and 1,064 nm DPSS lasers because of their low cost, high availability and ease of operation.
We have also chosen to use a spatial light modulator (SLM) to create and manipulate multiple traps simultaneously in real time 4,5. These devices are commercially available however their integration into a complete setup presents unique challenges. Here we describe a practical approach which addresses these potential difficulties and provides a highly versatile instrument. We provide an explicit example for the specific setup described which can be used as a guide for modified designs.
1. Installation of 980 nm Wavelength Single Optical Trap
2. Installation of Laser Detector
3. Installation of 1,064 nm Wavelength Holographic Trap
4. System Installation and Alignment Notes
The assembled setup allows the operator to trap multiple refractive objects in real time and position them in all three dimensions within the field of view. We illustrate the holographic capabilities of the instrument by trapping 11 microspheres (Figure 2). The trap confining each object is manually re-positioned upon trapping so that the final arrangement depicts the logo of the University of Utah where this experiment was performed. A combined function of holographic and conventional trap is shown in Figure 3. The conventional trap moves central bead progressively faster (trap speeds of 1.3, 10 and 82 μm/sec are shown), while holographically defined traps remain stationary. At the highest speed, the entire motion of the bead occurs during the recording of one frame of video and thus appears as extreme motion blur. It is possible to move the conventional trap fast enough that beads are forced from the trapping potential by hydrodynamic drag (not shown).
Note that the assembly of complex shapes utilizing multiple microspheres may lead to a case where the number of microspheres in the field of view is insufficient for full assembly (as is evident in Figure 2). In such cases, the operator needs to physically move the field of view relative to the sample (i.e. reposition the sample stage in the microscope) to expose additional microspheres while retaining the objects already trapped.
Figure 1. Schematic of the high resolution microscope system with two trapping beams. Components labeled L1-L9 are basic lenses. Components labeled DM1-DM3 are dichroic mirrors. Lenses L2 and L3 are used for steering. Lenses L4 and L5 act as a beam reducer and spacer. Lenses L6/L7 and L8/L9 are beam expander pairs for their respective laser beams. Unlabeled components depicted as solid black rectangles are basic mirrors. Components labeled MC and MO are the microscope condenser and object, respectively. Other components are a quad photo diode (QPD), notch filter (NF), Peltier temperature controller stage (PTC), hot filter (HF), spatial light modulator (SLM), acousto-optic deflector (AOD), shutters (S1 and S2), half-wave plate (HWP) and polarizing beam splitter (PBS).
Figure 2. A spatial arrangement representing a University of Utah logo is made using 11 operator defined and controlled holographic traps. The objects trapped are refractive beads (see Table of Materials for more details) suspended in de-ionized water. Red and green circles show trap positions. Frames (a)-(f) represent successive stages in logo construction.
Figure 3. Two rows of traps are made using 6 operator defined and controlled holographic traps. An additional conventional trap is defined between the two rows and its position is adjusted at various speeds as indicated. The bead is moved to a maximal spatial displacement of 4.1 μm and then back to the original location. A video of bead motion is recorded at 47 fps. As trap repositioning speed is increased, progressively larger motion blur is observed in the video. The objects trapped are refractive beads (see Table of Materials for more details) suspended in de-ionized water. Frame timings are shown in red. Trap repositioning speed is shown for each row. Green scale bars correspond to 5 μm in each direction.
We have constructed an instrument which combines two optical traps of different types (Figure 1) to provide separate trapping facilities for object manipulation and measurement. The “conventional” optical trap is built around a 980 nm diode laser. This beam is expanded, steered and then injected into our inverted microscope (“light red” beam in Figure 1). The holographic optical trap is built around a 1,064 nm DPSS laser. The beam is expanded to fit the size of the spatial light modulator (SLM), reflected off the SLM at low angle of incidence, reduced to slightly overfill the back focal aperture of the objective, combined with the “conventional” trapping line using a dichroic mirror, and finally injected into the our microscope (“dark red” beam in Figure 1). Note that the SLM must be placed in a plane which is optically conjugate to the back focal plane of the objective.
In the protocol section, we describe the design and alignment considerations which allow us to minimize spatial footprint of the setup and still enable relatively easy construction. We also describe the blocking of the undiffracted component produced by the SLM, which may be necessary for a commercial package like the system used here but is somewhat challenging and to date poorly documented.
The design described here is highly customizable. We have included brief mentions of several popular high level customizations for optical traps and how one would integrate those into our design. For instance, a single trap can be steered in multiple ways, including acousto-optic deflectors (AOD), electro-optic deflectors (EOD)12, movable or deformable reflectors or simply rastering the steering lens (L3 in our setup) 1. Similarly, the position of a trapped object can be determined using many schemes and sensors. In such cases, the typical placement and alignment of relevant components is briefly described. We expect that this work may provide a template for more complex designs in the future.
Several practical considerations and usage limitations are of note. First, the optical traps should not be positioned too close to each other so as not to interfere with their attractive potentials near trap center. If close positioning of two traps is needed, then it is possible to define a line trap connecting the two points so that the attractive potential of the trap extends along the entire line. Another practical issue is that the trapped objects cannot be moved so fast that they experience excessive hydrodynamic drag (the exact threshold depends on trap strength) otherwise the drag can push the objects out of the trap.
The authors have nothing to disclose.
Funding was provided by the University of Utah. We would like to thank Dr. J. Xu (UC Merced) and Dr. B.J.N Reddy (UC Irvine) for useful discussions.
Equipment | Company | Catalog Number | コメント |
Optical table | Newport corporation | ST-UT2-56-8 | Irvine, CA |
Microscope, Inverted, Eclipse Ti | Nikon USA | MEA53220 | Melville, NY |
Plan apo 100X oil objective (1.4 NA) | Nikon USA | MRD01901 | Melville, NY |
Oil condenser Lens 1.4 NA | Nikon USA | MEL41410 | Melville, NY |
EMCCD camera | Andor technology USA | Ixon DU897 | South Windsor, CT |
1/3″ CCD IEEE1394 camera | NET USA Inc | Foculus FO124SC | Highland IN |
Laser, TEM00, SLM, 1,064 nm wavelength | Klastech Laser Technologies | Senza-1064-1000 | Dortmund; Germany |
laser diode, TEM00, SLM, 980 nm | Axcel Photonics | BF-979-0300-P5A | Marlborough, MA |
laser diode mount | ILX Lightwave | LDX-3545, LDT-5525, and LDM-4984 | Bozeman, MT |
adjustable fiber ports | Thorlabs | PAF-X-11-B | Newton, NJ |
holographic system | Arryx | HOTKIT-ADV-1064 | Chicago, IL |
holographic mirror | Boulder Non-linear Systems | this is a part of HOTKIT-ADV-1064 | Lafayette, CO |
Calcite polarizer | Thorlabs | GL10-B | Newton, NJ |
half-wave plate | Thorlabs | WPH05M-1064 | Newton, NJ |
Polarizer rotation mount | Thorlabs | PRM1 | Newton, NJ |
half-wave plate rotation mount | Thorlabs | RSP1 | Newton, NJ |
Shutter | Thorlabs | SH05 | Newton, NJ |
dichroic mirrors (DM2 & DM3); 45° AOI | Chroma Technology | t750spxrxt | Bellows Falls, VT |
dichroic mirror (DM1); 45° AOI | Thorlabs | DMSP1000R | Newton, NJ |
custom mechanical adapter | Thorlabs | SM1A11 and AD12F with enlarged inner bore | Newton, NJ |
notch filter | Semrock | FF01-850/310-25 | Rochester, NY |
Acousto-Optic deflector (2-axis) | intraAction | DTD-584CA28 | Bellwood, IL |
goniometric stage | New Focus | 9081 | Santa Clara, CA |
60 mm steering lenses | Thorlabs | LA1134-B | Newton, NJ |
16 mm aspherical expander lens | Thorlabs | AC080-016-C | Newton, NJ |
175 mm expander lens | Thorlabs | LA1229-C | Newton, NJ |
Spot blocker (cabron-steel sphere) | Bal-Tec | 0.0100″ diameter | Los Angeles, CA |
Microspheres (Carboxyl-polystyrene) | Spherotech | CP-45-10 | Lake Forest, IL |