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.

1. Installation of 980 nm Wavelength Single Optical Trap
<ol>
	<li>Optical trapping at 980 nm wavelength is often optimal for biophysics experiments and inexpensive laser diodes are readily available with power output as high as 300 mW. It is preferable for a diode laser to be pigtailed with polarization-preserving single mode fiber with a known mode field diameter. The fiber needs to be sufficiently long to act as a mode filter and is typically terminated with either a FC/PC or FC/APC connector. Of these, FC/APC is preferable to minimize back reflection of light and potential feedback instabilities.</li>
	<li>Secure the 980 nm laser diode in a mount which allows for power and temperature control. It is best to fix the mount to an optical table directly to maximize passive heat sinking and thereby minimize the danger of diode failure due to a malfunction of temperature controller.</li>
	<li>Mount the PC/APC fiber connector to the beam collimating optics. It is critical to assure that the collimated beam have minimal divergence so adjustable fiber ports are most useful. Make sure that the chosen fiber port matches the mode field diameter of the diode pigtail fiber. If the beam is to be rastered using acousto-optic deflectors (AOD) or electro-optic deflectors (EOD) then the collimated laser beam waist must also be slightly less than the size of the deflector aperture.</li>
	<li>Secure the collimating adapter to the optical table at sufficient distance from the microscope to allow for beam routing, expansion, and placement of other desired components. Adjust the fiber port to ensure consistent beam waist over distances comparable to overall beam path to the microscope.</li>
	<li>Install mirrors indicated in Figure 1. Remove the objective from the microscope and use the mirrors to route the beam through the aperture in the objective mounting stage. If preferred, placement of the dichroic mirrors DM1 and DM3 can be omitted until later. DM2 and DM3 are both shortpass and transmit visible light while reflecting near IR and above.</li>
	<li>It is helpful to temporarily mount a red laser pointer in place of the objective, aligned on the optical axis of the microscope. A custom mechanical adapter is necessary to assure centration of the laser pointer. Visible beam from the laser pointer can then be routed back to the center of the aperture of the fiber port and can then be used to install the lenses (see below).</li>
	<li>Install the 980 nm beam expander (L8 and L9) at a proper distance from the fiber port to allow future insertion of steering components (AOD or EOD) as necessary 1. The expanded beam must slightly overfill the back focal aperture of the objective. (Here, lenses with focal lengths of 125 mm and 60 mm are in a Keplerian arrangement to approximately double the beam waist). Use visible laser pointer beam (see section 1.6) to ensure proper lens placement and rough alignment.</li>
	<li>Install the 980 nm steering lenses (L2 and L3) in a telescope arrangement as indicated (here both have 60 mm focal length) 1. L3 is mounted in a plane conjugate to the back-focal plane of the objective. Mount L3 on a precision XYZ positioning stage to allow for beam steering. It is helpful for the XYZ stage to have digital indicators for its micrometers, allowing for repeatable positioning and repositioning of the trap. The 0.5" range of travel is usually sufficient, however longer travel for L3 positioning along the optical axis may be helpful. Use visible laser pointer beam (see section 1.6) to ensure proper lens placement and rough alignment.</li>
</ol>
2. Installation of Laser Detector
<ol>
	<li>Install the dichroic mirror DM3 above the condenser as shown in Figure 1. A custom mount is typically required. Secure the quad photo diode (QPD) or a position sensitive detector (PSD) 8 to the side of the condenser assembly and ensure that the 980 nm laser beam reflected by DM3 is hitting it roughly on center. When using QPD, ensure that it is mounted on a small XY stage to allow for precise centering of the sensor on the laser beam.</li>
	<li>Install L1 (typically a 30 mm lens) between DM3 and the sensor. Position L1 so as to focus the beam to a single spot on the sensor.</li>
	<li>Install the notch filter just before L1 to block the 1,064 nm beam as well as any stray visible light reflections from the microscope illuminator and ambient lighting.</li>
</ol>
3. Installation of 1,064 nm Wavelength Holographic Trap
<ol>
	<li>The holographic part of the setup is built around a commercially available hardware/software package The holographic mirrors used in this package are rated to a maximum incident power of 5 or 10 W/cm2. Single mode TEM00 beams in this power range can be easily sourced from a DPSS laser at 1,064 nm wavelength.</li>
	<li>Mount the 1,064 nm laser on an elevated platform to roughly match the height of the beam path for the 980 line (see section 1).</li>
	<li>If not directly controllable, laser power can be manually adjusted by installing a half wave plate (HWP) and a polarizer (PBS) right after laser output aperture. It is helpful to mount the polarizer in a rotary stage to be able to match holographic mirror requirement for beam polarization.</li>
	<li>Install the 1,064 nm beam expander (L6 and L7). The laser beam waist must be expanded to match the diagonal size of the holographic mirror. For large expansion ratios (above 10X) it may be a concern to keep the size of the expander small. Thus it may be desirable to use lenses with unusually small focal length (here: 16 mm and 175 mm).</li>
	<li>Install the other mirrors as indicated to direct the 1,064 nm beam through the objective.
	<ol>
		<li>Secure DM1 dichroic (45&deg; angle of incidence) in a kinematic mount and place the assembly in the 980 nm beam path so that it allows for undiminished transmission of that beam.</li>
		<li>Activate laser pointer light. The DM1 mirror should reflect sufficient amount of visible light to properly position the spatial light modulator (SLM) in the path of this beam. The SLM also needs to be angled so that the incoming and outgoing laser beams are as close as possible to normal incidence. However the angle of incidence must be sufficiently large to ensure that the laser beam is not clipped by lens mounts and other optical components. A 5&deg; angle should be easily achievable and is sufficiently small. Finally the distance from DM1 to the SLM must be accurately measured so that the insertion of lenses L4 and L5 (see 3.6 below) can conjugate the SLM mirror plane and the back-focal plane of the objective.</li>
		<li>Install a mirror to direct the light from 1,064 nm beam expander to the SLM. Ensure that the laser pointer light hits the beam expander aperture on center.</li>
	</ol>
	</li>
	<li>Install lenses L4 and L5 (here: lenses with 125 mm and 200 mm respectively). This telescope pair conjugates the SLM mirror plane to the back focal plane of the objective and also reduces the beam waist to only slightly overfill the back aperture of the objective. We chose lenses with long focal lengths to space the SLM away from DM1. This not only clears room for the second laser line but also tends to make alignment easier.</li>
	<li>Remove the laser pointer. Leave the mounting adapter to serve as coarse alignment aperture.</li>
</ol>
4. System Installation and Alignment Notes
<ol>
	<li>Lens L3 and SLM must be positioned so as to be optically conjugated to the back focal plane of the objective. The common focal point of L4 and L5 is optically conjugated to the sample plane if the optical trapping beams are injected into the infinity space of the microscope.</li>
	<li>sing IR card viewer align the 980 nm beam to go along the center axis of the aperture in the laser pointer adapter.</li>
	<li>Use IR card to ensure that the 1,064 nm beam hits the same spot as the 980 nm beam on DM1, L2, and L3 and that the 1,064 nm beam goes along the center axis of the aperture in the laser pointer adapter.</li>
	<li>Replace the laser pointer mounting adapter with an objective. High numerical aperture oil or water objective is typical.</li>
	<li>Align the 980 nm trap as described in 9 by "walking" the laser beam until a radially symmetric interference pattern is seen on the camera.</li>
	<li>With the holographic mirror off (i.e. acting as a passive mirror) use SLM and DM1 to "walk" the undiffracted 1,064 nm beam to align the 1,064 nm trap.</li>
	<li>The SLM produces a significant undiffracted beam which results in a strong unmovable laser trap in the field of view. This is useful for alignment but may be undesirable for experiments. To block this trap one can insert a small opaque object in the path of undiffracted light at the location conjugate to the sample plane (e.g. the common focal point of L4 and L5). The size of this central spot blocker needs to be somewhat larger than the diameter of the Airy disk for the focused light (a blocker with 100-300 &mu;m diameter for the system described).</li>
	<li>djust 1,064 nm beam polarization using the polarizer to match SLM orientation. Rotate the half-wave plate to set the output power of the beam as desired.</li>
	<li>If desired, insert AOD or EOD beam steering elements into the 980 nm laser line. Ensure proper conjugation of these elements to the back-focal plane of the objective and re-align the trap. It is helpful to mount the steering elements on a goniometric stage.</li>
</ol>

<ol>
	<li>Svoboda, K., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+applications+of+optical+forces.">Biological applications of optical forces.</a> Annu. Rev. Biophys. Biomol. Struct. 23, 247-285 (1994).</li><li>Ashkin, A., Schutze, K., Dziedzic, J. M., Euteneuer, U., Schliwa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+generation+of+organelle+transport+measured+in+vivo+by+an+infrared+laser+trap.">Force generation of organelle transport measured in vivo by an infrared laser trap.</a> Nature. 348, 346-348 (1990).</li><li>Shubeita, G. T., Tran, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+motor+copy+number+on+the+intracellular+transport+of+kinesin-1-driven+lipid+droplets.">Consequences of motor copy number on the intracellular transport of kinesin-1-driven lipid droplets.</a> Cell. 135, 1098-1107 (2008).</li><li>Polin, M., Ladavac, K., Lee, S. H., Roichman, Y., Grier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+holographic+optical+traps.">Optimized holographic optical traps.</a> Opt Express. 13, 5831-5845 (2005).</li><li>Sun, B., Roichman, Y., Grier, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+holographic+optical+trapping.">Theory of holographic optical trapping.</a> Opt. Express. 16, 15765-15776 (2008).</li><li>Moffitt, J. R., Chemla, Y. R., Smith, S. B., Bustamante, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+optical+tweezers.">Recent advances in optical tweezers.</a> Annu. Rev. Biochem. 77, 205-228 (2008).</li><li>Grier, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revolution+in+optical+manipulation.">A revolution in optical manipulation.</a> Nature. 424, 810-816 (2003).</li><li>Neuman, K. C., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping.">Optical trapping.</a> Rev. Sci. Instrum. 75, 2787-2809 (2004).</li><li>Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+tweezers+in+cell+biology.">Laser tweezers in cell biology.</a> Introduction. Methods Cell Biol. 55, xi-xii (1998).</li><li>Spudich, J. A., Rice, S. E., Rock, R. S., Purcell, T. J., Warrick, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+traps+to+study+properties+of+molecular+motors.">Optical traps to study properties of molecular motors.</a> Cold Spring Harb. Protoc. 2011, 1305-1318 (2011).</li><li>Visscher, K., Gross, S. P., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+multiple-beam+optical+traps+with+nanometer-resolution+position+sensing.+Selected+Topics+in+Quantum+Electronics.">Construction of multiple-beam optical traps with nanometer-resolution position sensing. Selected Topics in Quantum Electronics.</a> IEEE Journal of. 2, 1066-1076 (1996).</li><li>Valentine, M. T., Guydosh, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+steering+of+an+optical+trap+by+electro-optic+deflection.">Precision steering of an optical trap by electro-optic deflection.</a> Opt Lett. 33, 599-601 (2008).</li></ol>

The authors have nothing to disclose.

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 modulato...

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.

<table><tbody><tr><td>Optical table</td><td>Newport corporation</td><td>ST-UT2-56-8</td><td>Irvine, CA</td></tr><tr><td>Microscope, Inverted, Eclipse Ti</td><td>Nikon USA</td><td>MEA53220</td><td>Melville, NY</td></tr><tr><td>Plan apo 100X oil objective (1.4 NA)</td><td>Nikon USA</td><td>MRD01901</td><td>Melville, NY</td></tr><tr><td>Oil condenser Lens 1.4 NA</td><td>Nikon USA</td><td>MEL41410</td><td>Melville, NY</td></tr><tr><td>EMCCD camera</td><td>Andor technology USA</td><td>Ixon DU897</td><td>South Windsor, CT</td></tr><tr><td>1/3&quot; CCD IEEE1394 camera</td><td>NET USA Inc</td><td>Foculus FO124SC</td><td>Highland IN</td></tr><tr><td>Laser, TEM00, SLM, 1,064 nm wavelength</td><td>Klastech Laser Technologies</td><td>Senza-1064-1000</td><td>Dortmund; Germany</td></tr><tr><td>laser diode, TEM00, SLM, 980 nm</td><td>Axcel Photonics</td><td>BF-979-0300-P5A</td><td>Marlborough, MA</td></tr><tr><td>laser diode mount</td><td>ILX Lightwave</td><td>LDX-3545, LDT-5525, and LDM-4984</td><td>Bozeman, MT</td></tr><tr><td>adjustable fiber ports</td><td>Thorlabs</td><td>PAF-X-11-B</td><td>Newton, NJ</td></tr><tr><td>holographic system</td><td>Arryx</td><td>HOTKIT-ADV-1064</td><td>Chicago, IL</td></tr><tr><td>holographic mirror</td><td>Boulder Non-linear Systems</td><td>this is a part of HOTKIT-ADV-1064</td><td>Lafayette, CO</td></tr><tr><td>Calcite polarizer</td><td>Thorlabs</td><td>GL10-B</td><td>Newton, NJ</td></tr><tr><td>half-wave plate</td><td>Thorlabs</td><td>WPH05M-1064</td><td>Newton, NJ</td></tr><tr><td>Polarizer rotation mount</td><td>Thorlabs</td><td>PRM1</td><td>Newton, NJ</td></tr><tr><td>half-wave plate rotation mount</td><td>Thorlabs</td><td>RSP1</td><td>Newton, NJ</td></tr><tr><td>Shutter</td><td>Thorlabs</td><td>SH05</td><td>Newton, NJ</td></tr><tr><td>dichroic mirrors (DM2 &amp;amp; DM3); 45 &amp;deg; AOI</td><td>Chroma Technology</td><td>t750spxrxt</td><td>Bellows Falls, VT</td></tr><tr><td>dichroic mirror (DM1); 45 &amp;deg; AOI</td><td>Thorlabs</td><td>DMSP1000R</td><td>Newton, NJ</td></tr><tr><td>custom mechanical adapter</td><td>Thorlabs</td><td>SM1A11 and AD12F with enlarged inner bore</td><td>Newton, NJ</td></tr><tr><td>notch filter</td><td>Semrock</td><td>FF01-850/310-25</td><td>Rochester, NY</td></tr><tr><td>Acousto-Optic deflector (2-axis)</td><td>intraAction</td><td>DTD-584CA28</td><td>Bellwood, IL</td></tr><tr><td>goniometric stage</td><td>New Focus</td><td>9081</td><td>Santa Clara, CA</td></tr><tr><td>60 mm steering lenses</td><td>Thorlabs</td><td>LA1134-B</td><td>Newton, NJ</td></tr><tr><td>16 mm aspherical expander lens</td><td>Thorlabs</td><td>AC080-016-C</td><td>Newton, NJ</td></tr><tr><td>175 mm expander lens</td><td>Thorlabs</td><td>LA1229-C</td><td>Newton, NJ</td></tr><tr><td>Spot blocker (cabron-steel sphere)</td><td>Bal-Tec</td><td>0.0100&quot; diameter</td><td>Los Angeles, CA</td></tr><tr><td>Microspheres (Carboxyl-polystyrene)</td><td>Spherotech</td><td>CP-45-10</td><td>Lake Forest, IL</td></tr></tbody></table>

construction of a high resolution microscope with conventional and holographic optical trapping capabilities

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.

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 <...

Watch this Scientific Journal Video about Construction of a High Resolution Microscope with Conventional and Holographic Optical Trapping Capabilities at JoVE.com

Construction of a High Resolution Microscope with Conventional and Holographic Optical Trapping Capabilities

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 ...

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Research

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Engineering

12.1K Views.  University of Utah. 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.

Video: Construction of a High Resolution Microscope with Conventional and Holographic Optical Trapping Capabilities

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Immunology and Infection

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

Bioengineering

Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and automotive. However, effective inspection and characterization metrology systems are needed to ensure the functional reliability of MEMS. This study presents a system based on digital holography as a tool for MEMS metrology. Digital holography has gained increasing attention in the past 20 years. With the fast development and decreasing cost of sensor arrays, resolution of such systems has increased broadening potential applications. Thus, it has attracted attention from both research and industry sides as a potential reliable tool for industrial metrology. Indeed, by recording the interference pattern between an object beam (which contains sample height information) and a reference beam on a CCD camera, one can retrieve the quantitative phase information of an object. However, most of digital holographic systems are bulky and thus not easy to implement on industry production lines. The novelty of the system presented is that it is lens-less and thus very compact. In this study, it is shown that the Compact Digital Holographic Microscope (CDHM) can be used to evaluate several characteristics typically consider as criteria in MEMS inspections. The surface profiles of MEMS in both static and dynamic conditions are presented. Comparison with AFM is investigated to validate the accuracy of the CDHM.

We present a compact reflection digital holographic system (CDHM) for inspection and characterization of MEMS devices. A lens-less design using a diverging input wave providing natural geometrical magnification is demonstrated. Both static and dynamic studies are presented.

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and ...

Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and nanotechnology. Although near-field scanning microscopy and superlenses have improved the resolution in the near-field region, far-field imaging in real-time remains a significant challenge. Recently, the hyperlens, which magnifies and converts evanescent waves into propagating waves, has emerged as a novel approach to far-field imaging. Here, we report the fabrication of a spherical hyperlens composed of alternating silver (Ag) and titanium oxide (TiO2) thin layers. Unlike a conventional cylindrical hyperlens, the spherical hyperlens allows for two-dimensional magnification. Thus, incorporation into conventional microscopy is straightforward. A new optical system integrated with the hyperlens is proposed, allowing for a sub-wavelength image to be obtained in the far-field region in real time. In this study, the fabrication and imaging setup methods are explained in detail. This work also describes the accessibility and possibility of the hyperlens, as well as practical applications of real-time imaging in living cells, which can lead to a revolution in biology and nanotechnology.

The use of a hyperlens has been regarded as a novel super-resolution imaging technique due to its advantages in real-time imaging and its simple implementation with conventional optics. Here, we present a protocol describing the fabrication and imaging applications of a spherical hyperlens.

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and ...

Construction and Operation of a Light-driven Gold Nanorod Rotary Motor System

The possibility to generate and measure rotation and torque at the nanoscale is of fundamental interest to the study and application of biological and artificial nanomotors and may provide new routes towards single cell analysis, studies of non-equilibrium thermodynamics, and mechanical actuation of nanoscale systems. A facile way to drive rotation is to use focused circularly polarized laser light in optical tweezers. Using this approach, metallic nanoparticles can be operated as highly efficient scattering-driven rotary motors spinning at unprecedented rotation frequencies in water.
In this protocol, we outline the construction and operation of circularly-polarized optical tweezers for nanoparticle rotation and describe the instrumentation needed for recording the Brownian dynamics and Rayleigh scattering of the trapped particle. The rotational motion and the scattering spectra provides independent information on the properties of the nanoparticle and its immediate environment. The experimental platform has proven useful as a nanoscopic gauge of viscosity and local temperature, for tracking morphological changes of nanorods and molecular coatings, and as a transducer and probe of photothermal and thermodynamic processes.

Plasmonic gold nanorods can be trapped in liquids and rotated at kHz frequencies using circularly-polarized optical tweezers. Introducing tools for Brownian dynamics analysis and light scatteringspectroscopy leads to a powerful system for research and application in numerous fields of science.

The possibility to generate and measure rotation and torque at the nanoscale is of fundamental interest to the study and application of biological and ...

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

Developmental Biology

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

Biochemistry

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.

A detailed instruction is described on how to build a highly inclined swept tile (HIST) microscope and its usage for single-molecule imaging.

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain ...

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 &#181;m&#160;x 200 &#181;m&#160;x 200 &#181;m using LED illumination, to 5 mm&#160;x 5 mm&#160;x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...

Construction of a High Resolution Microscope with Conventional and Holographic Optical Trapping Capabilities

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Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization

Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging

Construction and Operation of a Light-driven Gold Nanorod Rotary Motor System

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects