This work was supported by the Knut and Alice Wallenberg Foundation, the Swedish Research Council and the Chalmers Area of Advance Nanoscience and Nanotechnology.

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The authors have nothing to disclose.

The optical trapping setup described in this protocol is built around a commercial inverted microscope and uses red laser light. However, the techniques outlined are versatile and can be used to construct circularly polarized optical tweezers around most commercial or home-built microscopes, both upright and inverted, with only slight alterations. The trapping laser wavelength can be chosen within a wide visible-NIR spectrum, as long as the rest of the optical components and detectors are functional at this specific wavelength. Nevertheless, when choosing a laser wavelength, the size and spectral vicinity to resonances of the particles to be manipulated should be considered because this will affect the optical trapping forces and rotation performance2,5, the magnitude of photothermal effects1, and the trapping stability26. We have previously successfully worked with circularly polarized laser tweezers using laser wavelengths of 660, 785, 830, and 1064 nm.
One of the most important components of the optical trapping setup is the microscope objective. The objective in this protocol is a dry objective with NA = 0.95. The use of a dry objective is experimentally a simpler realization of the setup; however, it does lead to optical aberrations due to refraction in the sample cell interfaces. In the present case, the result is a slightly enlarged focus spot (~1.2 &#181;m) compared to the diffraction limit (~0.4 &#181;m), but this does not significantly change the general or rotary performance of the platform. In principal, a wide range of microscope objectives can be used, provided they have good transmission at the trapping wavelength, good polarization maintenance and long enough working distance to perform trapping through a microscope cover slip and layer of water. In case of 2D trapping, the NA can be relatively low, which makes the entire experiment simpler and provides cleaner circular polarization in the focus. However, higher laser powers might be required than in case of a high NA objective. In our experience, the best performance for trapping, rotation and dark-field spectroscopy is obtained with objectives with NA 0.7-0.95, but it is possible to use lower as well as higher NA objectives.
To obtain good photon correlation measurements of rotary motion, a fast single-pixel detector is needed. Choose a detector with a bandwidth at least two, preferably ten, times higher than the expected rotation frequency multiplied by the shape degeneracy factor and high sensitivity at the trapping wavelength used. Amplified Si photodetectors, single photon counting APDs, and PMTs have been used with success in different setups in our laboratories. Additional information, for example on trap stiffness, can be obtained by measuring and analyzing particle translational displacement using well-established techniques such as power spectral analysis5. A number of previous publications describe different variants of this technique38,39. DF spectroscopy can be performed using a wide range free-space or fiber coupled spectrometers and the choice should be based on the spectral range and wavelength and temporal resolution needed for the planned study.
When performing a trapping experiment, additional particles may accidentally enter the trap. This can be detected by monitoring the rotation frequency, which will fluctuate strongly due to the disturbance. Visual inspection by DF microscopy can be used to verify the presence of an additional particle, in which case the stage can be moved to avoid further disturbance or the experiment needs to be restarted.
The system described above is a simple and efficient way to realize 2D confinement and rotation of metallic nanoparticles. However, for some applications, the extra degree of freedom for manipulation that comes with 3D trapping is important, and the current configuration is therefore a limitation. However, 3D confinement and rotation might be achievable by utilizing counter propagating laser tweezers or more exotic trapping configurations.
Although the particle and system parameters discussed here can be optimized to reduce photothermal heating to below ~15 K4, the temperature increase associated with plasmonic excitation of metal nanoparticles can be problematic in certain applications. A possible route towards further heat reduction is to use high-index dielectric nanoparticles instead of plasmonic particles. Such particles support strong Mie-type scattering resonances but at the same time exhibit low intrinsic absorption coefficients. We have recently been able to manufacture colloidal resonant Si nanoparticles that might prove useful in this respect40,41.

The methods presented in this article replicates those used in our previous work1 to study nanoscale photothermal effects influencing light-driven gold nanorod rotary motors. Variants of the experimental platform has been used in several related publications2,3,4,5,6,7,8,9.
Optical tweezers are widely used for controlling position, force and linear momentum transfer at small length scales in physics, biology, and engineering10,11,12,13,14. Angular momentum carried by circularly polarized light can be employed for additional motion control because it continuously transfer torque to trapped objects15. By combining optical linear and angular momentum transfer, it is then possible to construct non-invasive rotary nanomotors with potential for diverse applications, such as drug delivery into single cells16,17, nanoscale surgery18, and active nanofluidics19, amongst others.
By using metallic nanoparticles as the subject of light driven manipulation, one can exploit the advantages of localized surface plasmon resonances (LSPR&#39;s), which provide large optical cross sections, high sensitivity to environmental changes, and large field enhancements20,21,22,23. This has led to a wealth of studies at the boundary between plasmonics and optical manipulation8,24,25,26,27. The strong light-matter interaction provided by LSPR has enabled us to design a platform where circularly polarized laser tweezers are capable of driving gold nanorods to spin at record rotation frequencies in water2. By tracking the Brownian motion of a rotating nanorod, detailed information about its environment and temperature can be obtained3,5. Simultaneous spectroscopic analysis provides an additional independent information channel for analysing local temperature and the morphological stability of the rotating nanorod1. A range of systems and configurations have been used for studying and applying rotary motion in optical tweezers, generating important insights within the field15,28,29,30,31,32. However, most of these studies have dealt with objects several micrometers in diameter while a single nanorod gives access to the nanometer size regime. Furthermore, when gold nanorods are used as the rotary nanomotor, torque is efficiently transferred mainly via scattering2,33. This decreases the risk of overheating the trapped particle3,34,35.
In the following method, we outline the steps required to build a system capable of efficient optical trapping and rotation of metal nanoparticles. The gold nanorods considered in these studies have high scattering cross sections, and the radiation pressure turns out to be stronger than the counteracting gradient force in the propagation direction. To still confine the particles in 3D, we utilize the force balance between Coulomb repulsion from a glass surface and the laser scattering force in the propagation direction. This 2D-trapping configuration greatly expands the range of trappable particles, as compared to standard 3D optical tweezers, and it can be easily combined with dark-field optical imaging and spectroscopy.
A trapped and rotating metal nanoparticle interacts with its environment, and detailed information about this interaction is contained in its motion and spectral properties. After describing how to construct the circularly polarized optical tweezers, we therefore also outline how to integrate instrumentation for probing rotational dynamics and for measuring Rayleigh scattering spectra in the experimental setup. The result is a versatile platform for studies of nanoscale rotation phenomena in physics, chemistry, and biology.
This protocol assumes that the researcher has access to suitable colloidal metal nanoparticles, preferably single crystalline gold nanorods. Gold nanorods can be purchased from specialized companies or synthesized in house using wet-chemistry methods. The nanorods used in our experiments were made by the seed-mediated growth method described in Ye et al. 201336. It is advantageous if the morphology and optical properties of the nanoparticles are well characterized, for example using scanning electron microscopy (SEM) and optical extinction measurements. Figure 1 displays data recorded from such measurements for representative nanorod types1.
An outline of the protocol is as follows: In the first section, we describe the construction of the optical tweezers based on circular polarization. In the second section, we describe how to extract information from the nanomotor by recording its rotational dynamics and scattering properties. The rotation frequency and the rotational Brownian motion of the trapped particle is measured using photon correlation spectroscopy by projecting backscattered laser light filtered through a linear polarizer on a fast single-pixel detector3. By fitting the data to a theoretical autocorrelation function, both the rotation frequency and the decay time of the rotational Brownian diffusion can be extracted2,3. The optical properties of the trapped and rotating nanoparticle are measured using dark field spectroscopy, which provides complementary information on the particle and its environment. In the third section, we describe the experimental procedure for the trapping and rotation of gold nanorods.
The protocol described up to this point is a straightforward path to a functioning circularly polarized optical tweezers system for nanoparticle rotation. However, sometimes issues arise that demand additional attention. In the fourth section, we outline a few of the common problems that we have encountered and how to address them. These include issues related to nanoparticle optical properties leading to poor trap stability (4.1), low rotation frequencies due to suboptimal circular polarization caused by beamsplitter birefringence (4.2), sticking of nanoparticles at the glass surface due to insufficient Coulomb repulsion (4.3), and deviation from the characteristic autocorrelation signal (4.4).

<table><tbody><tr><td>Gold nanoparticles</td><td></td><td></td><td>Purchased or home-grown</td></tr><tr><td>Commersial inverted microscope</td><td>Nikon</td><td>Eclipse TI</td><td></td></tr><tr><td>Trapping laser</td><td>Cobolt</td><td>Flamenco 05-01&amp;nbsp;</td><td>660 nm</td></tr><tr><td>Objective</td><td>Nikon</td><td>CFI Plan Apo Lambda 40X</td><td></td></tr><tr><td>Laser safety googles</td><td>Thorlabs</td><td>LG4</td><td></td></tr><tr><td>Assorted optomechanical components for mounting optics.</td><td></td><td></td><td>A range of mounts, posts and components from any company</td></tr><tr><td>Lens 1 Keplarian telescope</td><td>Thorlabs</td><td>AC254-035-A-ML</td><td></td></tr><tr><td>Lens 2 Keplarian telescope</td><td>Thorlabs</td><td>LA1725-A-ML</td><td></td></tr><tr><td>Silver coated mirrors</td><td>Thorlabs</td><td>PF10-03-P01</td><td></td></tr><tr><td>Kinematic mirror mounts</td><td>Thorlabs</td><td>KM100</td><td></td></tr><tr><td>Translation stage</td><td>Thorlabs</td><td>PT1/M</td><td>Quantity: 2</td></tr><tr><td>50/50 R/T Beamsplitter</td><td>Chroma</td><td>21000</td><td></td></tr><tr><td>CMOS camera</td><td>Andor</td><td>Zyla 5.5</td><td></td></tr><tr><td>Quarter waveplate (QWP, &amp;lambda;/4)</td><td>Thorlabs</td><td>AQWP05M-600</td><td></td></tr><tr><td>Power meter</td><td>Thorlabs</td><td>PM100USB</td><td></td></tr><tr><td>Photodiode Power Sensors</td><td>Thorlabs</td><td>S121C</td><td></td></tr><tr><td>Linear polarizer</td><td>Thorlabs</td><td>LPVIS050</td><td>For laser polarization measurement</td></tr><tr><td>360&amp;deg; rotation mount</td><td>Thorlabs</td><td>RSP1/M</td><td></td></tr><tr><td>Half waveplate (HWP, &amp;lambda;/2)</td><td>Thorlabs</td><td>AHWP05M-600</td><td>Used if polarization is not sufficient with only QWP</td></tr><tr><td>Oil DF condenser</td><td>Nikon</td><td>C-DO Dark Field Condenser Oil&amp;nbsp;</td><td></td></tr><tr><td>30/70 R/T Beamsplitter</td><td>Chroma</td><td>21009</td><td></td></tr><tr><td>Fast Si detector</td><td>Thorlabs</td><td>PDA36A-EC</td><td></td></tr><tr><td>Data Acquisition Module</td><td>National Instruments</td><td>USB-6361</td><td></td></tr><tr><td>Fiber 400 &amp;micro;m core size</td><td>Thorlabs</td><td>M74L01</td><td></td></tr><tr><td>xy-translation mount</td><td>Thorlabs</td><td>LM1XY/M</td><td></td></tr><tr><td>Linear polarizer</td><td>Thorlabs</td><td>LPVIS050</td><td></td></tr><tr><td>Spectrometer</td><td>Princeton Instruments&amp;nbsp;</td><td>IsoPlane SCT320&amp;nbsp;</td><td></td></tr><tr><td>CCD camera for spectrometer</td><td>Princeton Instruments&amp;nbsp;</td><td>PyLoN&amp;nbsp;</td><td></td></tr><tr><td>Notch filter</td><td>Semrock</td><td>NF03-658E-25</td><td></td></tr><tr><td>Notch filter</td><td>Thorlabs</td><td>NF658-26</td><td></td></tr><tr><td>Ultrasonic cleaner bath</td><td>Branson</td><td>Branson 3510&amp;nbsp;</td><td></td></tr><tr><td>Microscope slide</td><td>Ted Pella</td><td>260202</td><td></td></tr><tr><td>No. 1.5 Coverslips</td><td>VWR</td><td>630-2873</td><td></td></tr><tr><td>Aceton</td><td></td><td></td><td></td></tr><tr><td>Isopropanol</td><td></td><td></td><td></td></tr><tr><td>Basic detergent</td><td>Hellma</td><td>Hellmanex III</td><td>Cleaning if particle sticking is an issue</td></tr><tr><td>Secure-Seal Spacer</td><td>Thermo Fisher</td><td>S24735</td><td>Spacer tape with hole, for making sample cell</td></tr><tr><td>Immersion Oil</td><td>Zeiss</td><td>444960-0000-000&amp;nbsp;</td><td></td></tr><tr><td>PS beads</td><td>Microparticles GmbH</td><td>PS-R-5.0</td><td></td></tr><tr><td>Spectrophotometer</td><td>Agilent</td><td>Cary 5000 UV-Vis-NIR</td><td></td></tr><tr><td>SEM</td><td>Zeiss</td><td>Ultra 55 FEG SEM</td><td></td></tr><tr><td>Tweezers</td><td></td><td></td><td>Any brand</td></tr></tbody></table>

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.

The rotation and rotational Brownian motion of a gold nanorod that is properly trapped in the circularly polarized laser tweezers can be probed by recording light scattering intensity fluctuations (Figure 3a) using a single-pixel detector. An autocorrelation spectrum of this signal contains an oscillatory component, similar to the one shown in Figure 3b. which can be fit to a theoretical autocorrelation function. The fitting allows extraction of the rotation frequency and the autocorrelation decay time, which is related to the rotational Brownian fluctuations, of the nanorod.
As mentioned in the protocol (instruction 4.4.2), it is essential to use a sufficiently thick fiber core to collect the backscattered laser light for photon correlation spectroscopy. If this is not the case, an additional decay term related to particle translation in and out of the probe volume will be present in the correlation function, see Figure 4. Through careful analysis, this could provide more information about the system; however, it complicates the analysis of the rotational Brownian dynamics contained in the data.
To obtain correct DF scattering spectra from trapped nanoparticles, as described in section 3.5, the raw spectral data needs to be calibrated. This is done by recording the illumination lamp spectrum as well as a background spectrum (Figure 5a). When focusing intense laser light at a glass surface, such as the substrate against which the nanorods are trapped, some fluorescence might be generated (see the red spectral contribution in the background spectrum of Figure 5a). This fluorescence contamination can be reduced by using fused silica substrates. However, it is anyway highly recommended to record a background spectrum with empty optical tweezers at the correct laser power. When a scattering spectrum is recorded and all spectral components not related to the actual nanoparticle scattering have been compensated for, the spectrum can be fitted in energy scale with a bi-Lorentzian fitting function to extract information related to the LSPR peak positions (Figure 5b).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57947/57947fig1.jpg" /> 
Figure 1: SEM images and ensemble extinction spectra for two representative nanoparticle batches. a) Scale bar is 200 nm. b) The blue/red bordered SEM images in a) correspond to the red/blue spectrum, respectively. The spectral peaks related to the transverse and longitudinal LSPRs are clearly distinguishable. <a href="https://www.jove.com/files/ftp_upload/57947/57947fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57947/57947fig2.jpg" /> 
Figure 2: Schematic illustration of optical tweezers setup for nanoparticle rotation measurements. Laser light is collimated and expanded through a Keplerian telescope, and subsequently guided to the objective through the use of two movable mirrors (M1, M2) and a beamsplitter (BS). Two waveplates in the laser path optimize the circular polarization of the optical tweezers (&#955;/2, &#955;/4). Backscattered laser light can be collected after a linear polarizer for photon correlation spectroscopy and rotational dynamics measurements. After removing the laser light, scattered white light is guided to a spectrometer or a camera. <a href="https://www.jove.com/files/ftp_upload/57947/57947fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57947/57947fig3.jpg" /> 
Figure 3: Representative intensity and autocorrelation data with curve fit for a trapped and rotating nanorod. a) Intensity fluctuations recorded by the single pixel detector after a linear polarizer for 1s, and a zoomed in plot of the fluctuations. b) Autocorrelated data of intensity fluctuation for a rotating gold nanorod (blue points), collected from backscattered laser light. The data shows an oscillation that decays after a few periods. The oscillation is related to the rotation frequency of the nanorod, whereas the decay is due to rotational Brownian motion. A fit to the theoretical autocorrelation function is performed (red line) to extract a rotation frequency of f = 24285 &#177; 45 Hz and a correlation decay time of &#964;0 = 40.9 &#177; 1.06 &#181;s. The f and &#964;0 uncertainties represent 95% confidence intervals of the fit, which has a coefficient of determination (R2) of 0.9877. <a href="https://www.jove.com/files/ftp_upload/57947/57947fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57947/57947fig4.jpg" /> 
Figure 4: Issue with a too small probe volume in photon correlation spectroscopy measurements. a) Autocorrelation data for a rotating gold nanorod, collected using a thick (400 &#181;m, blue data) and a thin (62.5 &#181;m, red data) fiber. Collection using a thick fiber ensures that the nanorod is always confined within the probe volume and that the autocorrelation function measures rotational dynamics only. An additional decay term due to translational Brownian motion is present when the probe volume is insufficient. In b) and c), schematic illustrations of the effect and images of the back-illuminated collection region are shown. Scale bars are 2 &#181;m. <a href="https://www.jove.com/files/ftp_upload/57947/57947fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57947/57947fig5.jpg" /> 
Figure 5: Exemplary dark field scattering spectra recorded for a gold nanorod optically trapped by 660 nm laser light. The spectral region 630-670 nm (1.85-1.97 eV) is distorted due to notch filters needed to block the trapping laser light. a) Raw scattering spectra (dark blue) displaying features that are not inherent to the scattering of the particle and should be calibrated for. These include the background spectrum (red), which contains autofluorescence excited by the highly focused laser light, and the white light excitation spectrum (orange, recorded without notch filter). After calibration, the corrected scattering spectrum (light blue) shows two distinct LSPR peaks as expected. The arrows indicate the scale for each spectrum. b) Scattering spectrum for a trapped nanorod (blue points) together with a fit to the bi-Lorentzian model function (red) with its components (light blue and orange). The distorted spectral region is disregarded in the fitting of the data and the fit has an R2 of 0.9975. <a href="https://www.jove.com/files/ftp_upload/57947/57947fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

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Moment of Inertia

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8.8K Views.  Chalmers University of Technology. Enterprise gold nanostaafjes kan worden gevangen in vloeistoffen en gedraaid bij kHz frequenties met behulp van cirkel-gepolariseerde optisch pincet. Invoering van instrumenten voor Brownian dynamics analyse en lichte scatteringspectroscopy leidt tot een krachtig systeem voor onderzoek en toepassing op talrijke gebieden van de wetenschap.

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

Fabrication and Operation of a Nano-Optical Conveyor Belt

The technique of using focused laser beams to trap and exert forces on small particles has enabled many pivotal discoveries in the nanoscale biological and physical sciences over the past few decades. The progress made in this field invites further study of even smaller systems and at a larger scale, with tools that could be distributed more easily and made more widely available. Unfortunately, the fundamental laws of diffraction limit the minimum size of the focal spot of a laser beam, which makes particles smaller than a half-wavelength in diameter hard to trap and generally prevents an operator from discriminating between particles which are closer together than one half-wavelength. This precludes the optical manipulation of many closely-spaced nanoparticles and&#160;limits the&#160;resolution of optical-mechanical systems. Furthermore, manipulation using focused beams requires beam-forming or steering optics, which can be very bulky and expensive. To address these limitations in the system scalability of conventional optical trapping our lab has devised an alternative technique which utilizes near-field optics to move particles across a chip. Instead of focusing laser beams in the far-field, the optical near field of plasmonic resonators produces the necessary local optical intensity enhancement to overcome the restrictions of diffraction and manipulate particles at higher resolution. Closely-spaced resonators produce strong optical traps which can be addressed to mediate the hand-off of particles from one to the next in a conveyor-belt-like fashion. Here, we describe how to design and produce a conveyor belt using a gold surface patterned with plasmonic C-shaped resonators and how to operate it with polarized laser light to achieve super-resolution nanoparticle manipulation and transport. The nano-optical conveyor belt chip can be produced using lithography techniques and easily packaged and distributed.

The scalability and resolution of conventional optical manipulation techniques are limited by diffraction. We circumvent the diffraction limit and describe a method of optically transporting nanoparticles across a chip using a gold surface patterned with a path of closely spaced C-shaped plasmonic resonators.

The technique of using focused laser beams to trap and exert forces on small particles has enabled many pivotal discoveries in the nanoscale biological ...

Preparation of ZnO Nanorod/Graphene/ZnO Nanorod Epitaxial Double Heterostructure for Piezoelectrical Nanogenerator by Using Preheating Hydrothermal

Well-aligned ZnO nanostructures have been intensively studied over the last decade for remarkable physical properties and enormous applications. Here, we describe a one-step fabrication technique to synthesis freestanding ZnO nanorod/graphene/ZnO nanorod double heterostructure. The preparation of the double heterostructure is performed by using thermal chemical vapor deposition (CVD) and preheating hydrothermal technique. In addition, the morphological properties were characterized by using the scanning electron microscopy (SEM). The utility of freestanding double heterostructure is demonstrated by fabricating the piezoelectric nanogenerator. The electrical output is improved up to 200% compared to that of a single heterostructure owing to the coupling effect of the piezoelectricity between the arrays of ZnO nanorods on the top and bottom of graphene. This unique double heterostructure have a tremendous potential for applications of electrical and optoelectrical devices where the high number density and specific surface area of nanorod are needed, such as pressure sensor, immuno-biosensor and dye-sensitized solar cells.

One-step fabrication method for obtaining freestanding epitaxial double heterostructure is presented. This approach could achieve ZnO coverage with a higher number density than that of the epitaxial single heterostructure, leading to a piezoelectric nanogenerator with an increased output electrical performance.

Well-aligned ZnO nanostructures have been intensively studied over the last decade for remarkable physical properties and enormous applications. Here, we ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Neuroscience

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Biochemistry

Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces are demonstrated. This work requires careful design, considerable synthetic effort, and proper analysis. The rotary motion of the molecular motor in solution is shown by 1H NMR and UV-vis absorption spectroscopy techniques. In addition, the method to graft the motor onto an amine-coated quartz is described. This method helps to gain more insight into molecular machines.

The manuscript describes how to synthesize and graft a molecular motor on surfaces for single molecular imaging.

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces ...

Chemistry

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Bioengineering

Construction of an Improved Multi-Tetrode Hyperdrive for Large-Scale Neural Recording in Behaving Rats

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems neuroscience. One key component of this technique consists of the precise placement of multiple electrodes into desired brain regions and the maintenance of their stability. Here, we describe a protocol for the construction of a 3D-printable hyperdrive, which includes eighteen independently adjustable tetrodes, and is specifically designed for in vivo extracellular neural recording in freely behaving rats. The tetrodes attached to the microdrives can either be individually advanced into multiple brain regions along the track, or can be used to place an array of electrodes into a smaller area. The multiple tetrodes allow for simultaneous examination of action potentials from dozens of individual neurons, as well as local field potentials from populations of neurons in the brain during active behavior. In addition, the design provides for simpler 3D drafting software that can easily be modified for differing experimental needs.

We present the construction of a 3D-printable hyperdrive with eighteen independently adjustable tetrodes. The hyperdrive is designed to record brain activity in freely behaving rats over a period of several weeks.

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems ...

Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, conventional microdrive designs often only allow use of one type of electrode to record from single or multiple regions, limiting the yield of single-unit or depth profile recordings. It also often limits the ability to combine electrode recordings with optogenetic tools to target pathway and/or cell type specific activity. Presented here is a hybrid microdrive array for freely moving mice to optimize yield and a description of its fabrication and reuse of the microdrive array. The current design employs nine tetrodes and one opto-silicon probe implanted in two different brain areas simultaneously in freely moving mice. The tetrodes and the opto-silicon probe are independently adjustable along the dorsoventral axis in the brain to maximize the yield of unit and oscillatory activities. This microdrive array also incorporates a set-up for light, mediating optogenetic manipulation to investigate the regional- or cell type-specific responses and functions of long-range neural circuits. In addition, the opto-silicon probe can be safely recovered and reused after each experiment. Because the microdrive array consists of 3D-printed parts, the design of microdrives can be easily modified to accommodate various settings. First described is the design of the microdrive array and how to attach the optical fiber to a silicon probe for optogenetics experiments, followed by fabrication of the tetrode bundle and implantation of the array into a mouse brain. The recording of local field potentials and unit spiking combined with optogenetic stimulation also demonstrate feasibility of the microdrive array system in freely moving mice.

This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes.

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, ...

Biophysical Characterization of Flagellar Motor Functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...

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

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