Construction of a High Resolution Microscope with Conventional and Holographic Optical Trapping Capabilities
April 22nd, 2013
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.
In this procedure, a conventional and holographic optical trapping line are assembled and aligned To accomplish this first, measure out the locations for optical components, then mount the components securely in their proper locations. Next, align the optics to allow trapping. The final step is to block the TED beam from the holographic mirror.
Ultimately, the finished system allows simultaneous holographic and ordinary trapping. The main advantage of this technique over existing methods like ordinary trapping alone, is that it allows simultaneous positioning of multiple refractive objects in three dimensions and fast object manipulation measurements. Generally, individuals new to this method will struggle because design, layout and alignment can be challenging.
The starting point of this video is an optical bench with the microscope in place to create an optical trap at 980 nanometers. Start with one of the readily available laser diodes of that wavelength. Use a pig tilt laser with a polarization, preserving single mode fiber of known mode field diameter.
The fiber should be long enough to act as a mode filter and should be terminated with a fiber channel connector with either an FCPC or an F-C-A-P-C connector. The single mode optical fiber should be handled with care twists should be avoided. Secure the laser diode and mount that allows for power and temperature control.
The mount should be directly affixed to the optical bench to maximize passive heat sinking. Next, mount the fiber connector to the beam collating optics. Ensure that the chosen fiber port matches the mode field diameter of the diode.
Pigtail fiber attach the collating adapter to the optical table at a sufficient distance from the microscope to allow for beam routing expansion and placement of other components. Rod colors can be used to fix the lens spacing for robust repeatable positioning. Adjust the fiber port to ensure consistent beam waste over distances comparable to the overall beam path to the microscope.
There are five mirrors in the path of the DDE laser beam. Starting from the laser dde, there are two mirrors to direct its output into the microscope housing. The beam also has to pass through a dichroic mirror, which allows undiminished transmission of the laser diode beam at 45 degrees to the angle of incidents.
Finally, two additional short pass diic mirrors that transmit visible light, but reflect near infrared and above. Route the beam into and out of the microscope. Infinity space one is within the microscope housing to adjust the mirrors, remove the microscope objective.
Use the mirrors and an infrared viewing card to route the beam through the aperture in the objective mounting stage. If the beam is off center, adjust the mirrors to fix the alignment so that the beam is on center. When mounting lenses, a cage system can result in a rigid assembly.
Calipers can be used to pre-measure distances between lenses along cage rods prior to installation. Temporarily mount a red laser pointer with a custom adapter in place of the objective so that its beam is along the optical axis of the microscope. Use the visible laser pointer beam to place and roughly align the 980 nanometer beam expander.
This setup uses lenses with focal length of 60 millimeters and 125 millimeters in a keary arrangement to approximately double the beam waist. Next, install the 980 nanometer steering lenses using the pointer beam as a guide here. Each lens has a 60 millimeter focal length mount, the first lens in the beam path on a precision X, Y, Z stage with at least half an inch range of travel to allow for beam steering.
The second lens can now be positioned to form a one-to-one keary arrangement with the first lens. This lens must be positioned so as to be optically conjugated to the back focal plane of the objective. To prepare the holographic trap mount the 1064 nanometer laser on an elevated platform at roughly the height of the 980 nanometer beam.
Place a half wave plate and polarizer right after the laser output. To allow manual adjustment of laser power. Install the beam expander for this laser so the beam waste is expanded to match the diagonal size of the holographic mirror.
If constrained by space, use lenses with small focal lengths here, 16 millimeters and 175 millimeters. Now use the laser pointer beam to position the spatial light modulator or SLM, which is part of the commercial holographic package used. In this experiment, the spatial light modulator must be positioned so as to be optically conjugated to the back focal plane of the objective.
Place it so that the laser pointer beam has the minimal possible angle of incidents, but so that its reflection safely clears all the hardware on the optical table. Place another mirror in front of the 1064 nanometer laser beam expander to direct its light to the spatial light modulator. Ensure that the laser pointer light hits the center of the beam expander aperture.
Finally, install a telescope pair of lenses between the spatial light modulator and the dichroic mirror. These lenses have focal lengths of 125 millimeters and 200 millimeters. Remove the laser pointer, leave the custom adapter mount to serve as a course alignment aperture.
Start checking alignment using only the 980 nanometer beam. Use an infrared card viewer to align the 980 nanometer beam to go along the center axis of the aperture in the custom adapter. Then use the infrared card to ensure the 1064 nanometer beam hits the same spots as the 980 nanometer beam along their shared beam path.
Once this is done, replace the custom laser pointer mounting adapter with a high numerical aperture oil or water objective. Next, align the 980 nanometer trap by walking the laser beam until a radially symmetric interference pattern is seen on the camera with the holographic mirror off. Align the 1064 nanometer trap by using the spatial light modulator and the first dichroic mirror to walk the UN DEFRACTED 1064 nanometer beam.
In this example, the aligned image for both traps indicates good alignment for efficient trapping, but can be further refined. There are also minor aberrations associated with the round 1064 beam, hitting a square SLM mirror, which cannot be easily eliminated by refining lens alignment. In order to block the trap produced by the underacted beam from the spatial light modulator, insert a small opaque object in the path of the underacted light at a location conjugate to the sample plane for this system.
An opaque microsphere of 100 to 300 microns diameter, which is metal, is glued to an infrared transparent round glass cover slip or window. The block is placed at the common focal point of the lenses, which is optically conjugated to the sample plane. If the optical trapping beams are injected into the infinity space of the microscope, the beam blocker can be positioned to block unraced light or can be moved to allow all light to pass.
The assembled setup allows the operator to trap multiple refractive objects in real time and position them in all three dimensions in the field of view. Here, the holographic capabilities of the instrument are shown by trapping microspheres, suspended in deionized water, red and green circles show trap positions with green indicating a trap selected for repositioning in this clip, playback is sped up after an object is trapped. Its trap is manually repositioned.
The final arrangement has 11 beads depicting the logo of the University of Utah where this experiment was performed. The system also allows the use of both the holographic and conventional traps together. Here two rows of holographic traps are defined and controlled by the operator.
An additional conventional trap is defined between the two rows. Microsphere suspended in deionized water are bound by the traps. The center bead is moved to a maximum spatial displacement of 4.1 micrometers and then backed to its original location under the same circumstances.
But with the center bead moving at 82 micrometers per second, this sequence of frames is produced. Note the motion blur in the second and third images. After watching this video, you should have a good understanding of how to lay out and align a dual optical trap system with independent holographic and normal trap in the same setup.
Don't forget that working with high power lasers can be extremely hazardous and take precautions such as wearing proper protective eyewear.
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Overview
This article describes a system that integrates conventional and holographic optical trapping techniques, enabling the manipulation of multiple traps. This innovative approach facilitates the creation of complex geometric arrangements of refractive particles while allowing for high-speed, high-resolution measurements of biological enzyme activity.
Key Study Components
Area of Science
- Optical trapping
- Biophysics
- Enzyme activity measurement
Background
- Traditional optical trapping methods are limited in their capability to manipulate multiple objects simultaneously.
- Holographic optical trapping offers enhanced flexibility in positioning and manipulating particles.
- Combining these techniques can improve measurement accuracy and speed.
- New users may face challenges in design and alignment of optical components.
Purpose of Study
- To develop a system that allows for simultaneous conventional and holographic optical trapping.
- To enable the manipulation of multiple refractive objects in three dimensions.
- To facilitate fast measurements of biological enzyme activity.
Methods Used
- Assembly and alignment of optical trapping components.
- Measurement of locations for optical components.
- Secure mounting of components in designated positions.
- Alignment of optics to enable effective trapping.
Main Results
- The system successfully integrates conventional and holographic trapping techniques.
- It allows for the simultaneous positioning of multiple refractive objects.
- High-speed measurements of enzyme activity were achieved.
- Users reported challenges in initial setup but recognized the system's advantages.
Conclusions
- The developed system enhances the capabilities of optical trapping techniques.
- It provides significant advantages for researchers studying biological processes.
- Further training may be necessary for new users to master the setup.
