This work was supported by EPSRC Career Acceleration grant EP/J002402/1.

CAUTION: Please consult all relevant material safety data sheets before use. Hydrogen peroxide used in this protocol is harmful, and the evolution of oxygen gas when exposed to platinum poses an explosion risk. Use all appropriate safety controls during this protocol including engineering controls while handling peroxide solutions (fume hood) and personal protective equipment (safety glasses, gloves and lab coat).
1. Making Catalytic Janus Particles
<ol>
	<li>Prepare glass slide substrate
	<ol>
		<li>Clean an unused standard microscope slide using sequential washing for a few minutes each in deionized (DI) water, glass decontamination solution and DI water. Then wash with a 70:30 v:v mixture of ethanol:DI water, and finally blow dry in a clean air/nitrogen stream.</li>
		<li>Examine the glass slide under a microscope to verify the surface is clean and free from evidence of particulate contamination. Repeat step 1.1.1 if required.</li>
	</ol>
	</li>
	<li>Prepare colloidal dispersion for deposition
	<ol>
		<li>Pipette 10 &#181;l of aqueous stock fluorescent colloid solution (10% wt.) into 990 &#181;l of ethanol. Adjust volumes as required depending on stock solution concentration used to arrive at approximately 0.1% wt colloidal suspension.</li>
		<li>Vortex mix for 10 sec.</li>
	</ol>
	</li>
	<li>Spin coat colloidal dispersion onto glass slide substrate
	<ol>
		<li>Equip a spin coater with the clean glass slide. Fill a pipette tip with 100 &#181;l of the diluted colloidal solution prepared above. Program spin coater to execute a 30 sec, 2,000 rpm cycle. Start spin coater, and when up to speed continuously pipette the prepared solution onto the center of the spinning glass slide.</li>
		<li>Remove glass slide from the spin coater, return to the optical microscope and verify that an even dispersion of mainly non-touching separate colloids covers the central region of the glass slide.</li>
	</ol>
	</li>
	<li>Vacuum evaporate platinum metal onto colloid decorated glass slide
	<ol>
		<li>Insert the colloid coated glass slide into a metal evaporator. Ensure the colloid decorated side is facing the evaporation source. Install a platinum metal evaporation source and deposit 15 nm of platinum metal. 
		Note: Following metal deposition, samples should be stored under an inert atmosphere.</li>
	</ol>
	</li>
</ol>
2. &#34;Swimming&#34; Janus Particles
<ol>
	<li>Re-suspend Janus Colloids into a peroxide containing solution
	<ol>
		<li>Cut a 1 x 1 cm square of lens tissue, and dampen the end with 10 &#181;l of DI water. Hold with tweezers, and gently rub along the surface of the platinum coated colloid decorated glass slide (this step physically removes colloids from the substrate).</li>
		<li>Insert the lens tissue into 1.5 ml of DI water and manually shake vigorously for 30 sec in a sealed tube. Remove lens tissue.</li>
		<li>Pipette 1 ml of the colloid containing solution into a new container, filled with 1 ml of 30% w/v H2O2 stock solution. Gently mix the solutions, and then place in an ultrasonic bath at room temperature for 5 min, followed by another 25 min unstirred incubation period. 
		CAUTION: This solution may evolve oxygen; do not seal.</li>
		<li>Dry 100 &#181;l of the remaining aqueous colloidal solution onto a scanning electron microscope (SEM) stub to enable SEM verification of the Janus colloid structure.14</li>
	</ol>
	</li>
	<li>Prepare analysis cuvette
	<ol>
		<li>Add an additional 1 ml of DI water to the incubated solution containing peroxide and platinum coated colloids to arrive at a suitable fuel strength (10%) to allow fast propulsion. 
		Note: The prior incubation stages were carried out in higher concentration fuel concentrations to clean the platinum catalyst surface.</li>
		<li>Fill a low volume rectangular quartz glass cuvette with the incubated solution. Loosely fit a push-in cap. 
		CAUTION: Explosion risk &#8212; do not use a screw cap.</li>
	</ol>
	</li>
</ol>
3. Microscopic Observation
<ol>
	<li>Locate particle of interest
	<ol>
		<li>Load cuvette into a fluorescent microscope equipped with a suitable objective (e.g., 20X) and excite fluorophore emission using a suitable combination of filters (excitation 450-490 nm, emission &#62;515 nm).</li>
		<li>Manually search for fluorescent colloids within the cuvette. 
		Note: Adjusting colloid density while maintaining the peroxide concentration may be required. For example, dilution is recommended if the colloid density is high, and numerous oxygen bubbles producing flows are present. A colloid volume concentration of about 0.003% is a recommended starting point.</li>
		<li>Optimize optical settings for 3D tracking: under suitable illumination conditions, in focus colloids will appear as sharp circular objects. However, as propulsion moves the colloids in and out of the focal plane a distinctive size changing bright ring centered around the sphere will be observed, this is used to determine the z coordinate to enable 3D tracking.</li>
	</ol>
	</li>
	<li>Record video
	<ol>
		<li>Before starting video capture, focus the microscope so that the particle of interest produces a concentric ring, with the particle &#34;under&#34; the focus position. Do not move the focus plane during video capture.</li>
		<li>Record videos of particles of interest. Use 30 sec video durations with frame rates in excess of 30 Hz to allow detailed trajectory reconstruction.</li>
	</ol>
	</li>
	<li>3D trajectory reconstruction
	<ol>
		<li>Calibrate z-axis
		<ol>
			<li>Make up a 2% wt. gellan gum solution in water containing a suspension of fluorescent Janus particles at 60 &#176;C, place in an equivalent cuvette to that used above, and allow to set to form a stiff transparent gelled sample containing fixed static colloids.</li>
			<li>Focus on a single fixed colloid using the same illumination conditions selected above, now record a series of still images as the z-focus is raised by known displacements relative to this plane.</li>
			<li>Determine the radius of the ring at each known focus position11. 
			Note: This is most efficiently performed by using an image analysis algorithm that can be applied as a batch process to all the calibration still frames, and videos. A typical approach involves smoothing the image, thresholding to identify an approximate location of the objects&#39; center, and then locating the actual x and y coordinates of the ring center by measuring the distance between intensity peaks either side of the ring. The mean radial distance to the intensity peaks from the ring center can then be found.11 This allows both the radius of the bright ring and the x-y coordinate to be determined with sub-pixel accuracy. By locating the x, y, z position of a Janus sphere fixed in gellan gum 30 &#181;m from the focal plane, in a time sequence of images, particles can be located with an error of &#177; 25 nm along each axis. The error can be attributed to noise in the images. The signal to noise ratio and therefore, the accuracy of the location algorithms is dependent upon the intensity of the detected fluorescence light. When a Janus sphere is far from the focal plane its intensity becomes too weak to accurately track it, e.g., for a 4.8 &#181;m diameter colloid a z-range of around 200 &#181;m is possible. An alternative non-algorithmic method is to use simple manual measurement of x,y center and radius, however this will reduce accuracy.</li>
			<li>Plot a calibration curve to relate radius to z-position, and fit to an appropriate function (e.g., cubic equation) to allow interpolation.11</li>
		</ol>
		</li>
		<li>Calibrate x,y axis
		<ol>
			<li>Record a still frame optical microscope image of a spatial calibration graticule using the same microscope conditions chosen in 3.2.</li>
			<li>Measure the &#34;in pixels&#34; dimension of an object with known real world size from the image of the spatial calibration graticule and use this to establish a pixels to micron conversion factor for the x,y image plane.</li>
		</ol>
		</li>
		<li>Reconstruct trajectory
		<ol>
			<li>Determine the x and y coordinates and radius for each frame of the video sequence as described in 3.3.1.3, use the function found in 3.3.1.4 to convert radius into z, and the calibration factor found in 3.3.2.2 to convert x and y pixel coordinates into microns. This procedure will result in an accurate x, y, z coordinate for the propulsive particles location as a function of time.11 This procedure can be implemented using an algorithm, or manually.</li>
			<li>Determine the derived properties such as average velocity to quantify the extent of observed catalytic swimming.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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E., Sen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+power+:+Autonomous+nano-+and+micromotors+propelled+by+self-generated+gradients.">Small power : Autonomous nano- and micromotors propelled by self-generated gradients.</a> Nano Today. 8 (5), 531-554 (2013).</li><li>Gao, W., Dong, 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=Artificial+Micromotors+in+the+Mouse's+Stomach+:+A+Step+toward+in+Vivo+Use+of+Synthetic+Motors.">Artificial Micromotors in the Mouse's Stomach : A Step toward in Vivo Use of Synthetic Motors.</a> ACS nano. 9 (1), 117-123 (2015).</li><li>Restrepo-P&#233;rez, L., Soler, L., Mart&#236;nez-Cisneros, C., S&#225;nchez, S., Schmidt, O. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofunctionalized+self-propelled+micromotors+as+an+alternative+on-chip+concentrating+system.">Biofunctionalized self-propelled micromotors as an alternative on-chip concentrating system.</a> Lab Chip. 14 (16), 2914-2917 (2014).</li><li>Soler, L., S&#225;nchez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+nanomotors+for+environmental+monitoring+and+water+remediation.">Catalytic nanomotors for environmental monitoring and water remediation.</a> Nanoscale. 6 (13), 7175-7182 (2014).</li><li>Howse, J., Jones, R., Ryan, A., Gough, T., Vafabakhsh, R., Golestanian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Motile+Colloidal+Particles:+From+Directed+Propulsion+to+Random+Walk.">Self-Motile Colloidal Particles: From Directed Propulsion to Random Walk.</a> Phys. Rev. Letts. 99 (4), 8-11 (2007).</li><li>Ebbens, S., Gregory, D. A., 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=Electrokinetic+effects+in+catalytic+platinum-insulator+Janus+swimmers.">Electrokinetic effects in catalytic platinum-insulator Janus swimmers.</a> EPL. 106 (5), 58003 (2014).</li><li>Orozco, J., Jurado-S&#225;nchez, B., 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=Bubble-propelled+micromotors+for+enhanced+transport+of+passive+tracers.">Bubble-propelled micromotors for enhanced transport of passive tracers.</a> Langmuir. 30 (18), 5082-5087 (2014).</li><li>Peterson, S. D., Chuang, H. -. S., Wereley, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gravitaxis+in+spherical+janus+swimming+devices.">Gravitaxis in spherical janus swimming devices.</a> Langmuir. 29 (46), 14066-14073 (2013).</li><li>Ebbens, S., Tu, M. -. H., Howse, J. R., Golestanian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+dependence+of+the+propulsion+velocity+for+catalytic+Janus-sphere+swimmers.">Size dependence of the propulsion velocity for catalytic Janus-sphere swimmers.</a> Physical Review. E Stat. Nonlin. Soft Matter Phys. 85, 020401 (2012).</li><li>Ebbens, S. J., Howse, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+the+direction+of+motion+for+spherical+catalytic+swimmers.">Direct observation of the direction of motion for spherical catalytic swimmers.</a> Langmuir. 27 (20), 12293-12296 (2011).</li><li>Archer, R. J., Campbell, a. I., Ebbens, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glancing+angle+metal+evaporation+synthesis+of+catalytic+swimming+Janus+colloids+with+well+defined+angular+velocity.">Glancing angle metal evaporation synthesis of catalytic swimming Janus colloids with well defined angular velocity.</a> Soft Matter. 11, 6872-6880 (2015).</li><li>Zhao, G., Sanchez, S., Schmidt, O. G., Pumera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poisoning+of+bubble+propelled+catalytic+micromotors:+the+chemical+environment+matters.">Poisoning of bubble propelled catalytic micromotors: the chemical environment matters.</a> Nanoscale. 5 (7), 2909-2914 (2013).</li><li>Wang, Y., Hernandez, R. M., 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=Bipolar+electrochemical+mechanism+for+the+propulsion+of+catalytic+nanomotors+in+hydrogen+peroxide+solutions.">Bipolar electrochemical mechanism for the propulsion of catalytic nanomotors in hydrogen peroxide solutions.</a> Langmuir. 22 (25), 10451-10456 (2006).</li><li>Dunderdale, G., Ebbens, S., Fairclough, P., Howse, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+particle+tracking+and+calculating+the+mean-squared+displacement+in+distinguishing+nanopropulsion+from+other+processes.">Importance of particle tracking and calculating the mean-squared displacement in distinguishing nanopropulsion from other processes.</a> Langmuir. 28 (30), 10997-11006 (2012).</li></ol>

The authors have nothing to disclose.

Many variables in the preparation protocol for platinum Janus particles will affect the observed trajectories. The parameters as described using 2 &#181;m diameter particles will give propulsion velocities in the order of 10 &#181;m per second. If smaller particles are used, velocities will increase, while increasing particle size will decrease propulsion velocity.12 The details of the evaporation protocol will also alter the trajectories observed. In this current protocol, a sparse distribution of colloids is recommended, together with metal evaporation normal to the slide orientation. These conditions result in symmetrical Janus structures as shown in Figure 2, which lead to linear trajectories within the limits of Brownian rotational diffusion.13 Conversely, if tight packed colloids are subject to glancing angle deposition, then the symmetry of the Janus cap can be broken, to induce spinning behavior.14 The particles produced here display relatively isotropic motion in all three dimensions; however if thicker platinum coatings, or larger particles are used, an upwards bias or gravitaxis can be imparted.11 Details of the storage of the Janus colloids after manufacture may also effect the swimming speeds observed. The high surface energy clean platinum surface emerging from the evaporation stage is susceptible to surface contamination for example from hydrocarbons, and in particular thiols.15
In addition, the solution properties in which the Janus colloids are re-suspended are critical for observing propulsion. Low peroxide concentrations will result in slower velocities, as the rate of the decomposition reaction producing motion reduces.6 In addition, low concentrations of salts will result in a dramatic reduction in propulsion velocity.7
A key feature of the colloids produced here is their neutral buoyancy, which makes them suitable for 3D tracking. In general the field of swimming devices has paid little attention to 3D effects, partly due to some prominent examples being made from dense metals, causing them to rapidly sediment,16 but also due to the difficulties and expense associated with making the required measurements. Clear drawbacks for some established 3D tracking methods exist for these rapidly moving colloids, for example, confocal scanning laser microscopy can lack the temporal resolution to record sufficient numbers of images to resolve trajectories. In this context, the method we present here has the significant advantage of only requiring a single frame to allow estimation of z-coordinate, which consequently allows high frame rates. Also, as z-coordinate reconstruction only relies on the relative contrast of the out-of-focus colloid in single frames, rather than the absolute fluorescence intensity, it is resilient to quenching and blinking effects in the fluorophore. These advantages are possible at the expense of a reduced depth of field over which 3D trajectory reconstruction is possible, and the requirement for well separated non-overlapping colloids. We hope that describing the protocol will allow other research groups with an interest in 3D behavior for their swimming devices to access this information straightforwardly and with a high degree of precision. It is clear that expanding the understanding of these devices to 3D will open up a significant range of interesting future phenomena and applications. Readers interested in further details of trajectory analysis are directed towards Reference 17 which describes common artifacts in propulsive systems and how to ensure accurate quantification of propulsion velocities.

Catalytic swimming devices are small scale, untethered colloids capable of autonomously generating motion in fluidic environments.1,2 These devices are attracting significant research interest as they have the potential to enable exciting new functions such as drug delivery,3 lab-on a chip transport4 and environmental remediation.5 One widely studied example are catalytic &#34;Janus&#34; swimmers.6 These particles get their name from having two distinct sides, or faces (Janus is a two faced Roman god). One side is catalytically active and able to perform a decomposition reaction, while the other is inert. In the presence of suitable dissolved fuel molecules, the resulting asymmetric chemical reaction creates gradients around the colloids which can produce motion via self-diffusiophoresis/electrophoresis.7
Characterizing the motion for these rapidly moving objects is challenging and many experimental observations to date have been limited to 2D. However, eventual applications are likely to exploit catalytic swimming devices ability to move throughout bulk solutions in 3D.8 To address this, here we describe a protocol that allows accurate 3D trajectories for swimming devices to be determined. This method is based on interpreting the ring structures produced by out of focus fluorescent colloids observed with a fixed focus objective,9 and is easy to apply using conventional unmodified microscopes. By clearly describing this method here, other researchers in this field will benefit by being able to access such 3D information. This will aid future insights into motion characteristics for swimming devices. Evidence of this potential is given by the recent report of swimming devices being directed by gravity,10,11 behavior which can most easily be visualized through the application of 3D tracking.11
This paper also clearly documents a method to manufacture catalytic Janus particle swimming devices, which will be of further benefit to standardize methods across the existing research groups investigating these devices, and additionally guide new researchers interested in making and investigating swimming devices.

<table><tbody><tr><td>Evaporator</td><td>Moorfield (UK)</td><td></td><td>Minilab 80 e-beam evaporator</td></tr><tr><td>Microscope</td><td>Nikon</td><td></td><td>Eclipse LV100</td></tr><tr><td>Fluorescence light source</td><td>Nikon</td><td></td><td>Nikon B2A filter cube</td></tr><tr><td>Objective</td><td>Nikon</td><td></td><td>20X, 0.45 NA</td></tr><tr><td>Cuvette</td><td>Hellma</td><td></td><td>fused quartz, 40 x 10 x 1 mm</td></tr><tr><td>Vortex mixer</td><td>IKA</td><td></td><td>Lab Dancer S2</td></tr><tr><td>Spin coater</td><td>Laurell Technologies Corp.</td><td></td><td>Model WS-400BZ-6NPP/Lite</td></tr><tr><td>Ultrasonic bath</td><td>Eumax</td><td></td><td>2 liter</td></tr><tr><td>Lens tissue</td><td>Whatman</td><td>2105 841</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Sigma-Aldrich</td><td>31642-1L</td><td>30 wt%</td></tr><tr><td>Platinum</td><td>Sigma-Aldrich</td><td>267171</td><td>0.25 mm, 99.99%</td></tr><tr><td>Colloids</td><td>Thermo Scientific</td><td></td><td>Fluoro-Max PS microspheres, d = 1.9&amp;nbsp;microns</td></tr><tr><td>Glass decontamination solution</td><td>Fisher Scientific</td><td>D/0025/15</td><td>Decon 90</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>E/0600DF/17</td><td>Absolute Ethanol</td></tr><tr><td>DI water</td><td>Elga</td><td></td><td>Purelab Option filtration system (15 MW)</td></tr><tr><td>Gellan gum</td><td>Sigma-Aldrich</td><td>P8169-100G</td><td>&quot;Phytagel&quot;</td></tr></tbody></table>

preparation and 3d tracking of catalytic swimming devices

We report a method to prepare catalytically active Janus colloids that "swim" in fluids and describe how to determine their 3D motion using fluorescence microscopy. One commonly deployed method for catalytically active colloids to produce enhanced motion is via an asymmetrical distribution of catalyst. Here this is achieved by spin coating a dispersed layer of fluorescent polymeric colloids onto a flat planar substrate, and then using directional platinum vapor deposition to half coat the exposed colloid surface, making a two faced "Janus" structure. The Janus colloids are then re-suspended from the planar substrate into an aqueous solution containing hydrogen peroxide. Hydrogen peroxide serves as a fuel for the platinum catalyst, which is decomposed into water and oxygen, but only on one side of the colloid. The asymmetry results in gradients that produce enhanced motion, or "swimming". A fluorescence microscope, together with a video camera is used to record the motion of individual colloids. The center of the fluorescent emission is found using image analysis to provide an x and y coordinate for each frame of the video. While keeping the microscope focal position fixed, the fluorescence emission from the colloid produces a characteristic concentric ring pattern which is subject to image analysis to determine the particles relative z position. In this way 3D trajectories for the swimming colloid are obtained, allowing swimming velocity to be accurately measured, and physical phenomena such as gravitaxis, which may bias the colloids motion to be detected.

Figure 1 shows a typical dispersion of colloids on a clean glass slide prior to depositing platinum. Figure 2 shows a typical back-scattered SEM image for a half platinum coated Janus swimmer, under this imaging mode the platinum coated region produces bright contrast. The desired hemispherical platinum layer is apparent. Figure 3 displays the appearance of a typical fluorescent Janus swimmer under optimum illumination conditions fixed in gellan gum. The swimmer appears as a symmetrical ring feature, and it is the radius of the ring that can be used to determine the z-position of the colloid relative to the focus position. Figure 4 shows representative cross-sections for the radial brightness intensity distribution that is used in combination with image analysis algorithms to accurately locate the center and apparent radius of the colloid. Figure 5 contains a calibration curve obtained using a fixed colloidal sample and a calibrated microscope z-translation stage to relate apparent colloidal size and distance from focus position. This curve is fitted to a cubic function, which is used to convert apparent radius into z-coordinate. Finally, Figure 6 displays a typical x,y,z trajectory for a fluorescent Janus particle swimmer.
<img alt="Figure 1" src="/files/ftp_upload/54247/54247fig1.jpg" /> 
Figure 1. Optical image of 1.9 &#181;m diameter Polystyrene microspheres. Microspheres are dispersed on a cleaned glass slide before Platinum deposition. Scale bar represents 40 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54247/54247fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54247/54247fig2.jpg" /> 
Figure 2. SEM backscatter image of a 1.9 &#181;m diameter Polystyrene microspheres. Microspheres are shown after platinum deposition. Scale bar represents 2 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54247/54247fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54247/54247fig3.jpg" /> 
Figure 3. Calibration images of a 4.8 &#181;m diameter fluorescent polystyrene sphere fixed in gellan gum recorded using a 20X objective (0.4 N.A.). The distances below each image indicate the distance of the focal plane of the objective above the sphere. As the image is defocused from 0 &#181;m to 200 &#181;m the in focus image of a bright disc changes to a bright ring, the radius of which is dependent upon magnification, the sphere size and its distance from the focal plane. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54247/54247fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54247/54247fig4.jpg" /> 
Figure 4. X, y, z particle tracking procedure. A set of self-written algorithms is used to first locate the (x, y) center of the bright ring by extracting a series of vertical and horizontal lines and finding the mean mid-point between the bright peaks (a). The ring radius is then calculated from the peak intensity of a spline fitted to the average pixel grey-values radiating out from the ring center (b). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54247/54247fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54247/54247fig5.jpg" /> 
Figure 5. Z-coordinate calibration chart for Janus spheres obtained by measuring the bright ring radius of spheres fixed in gellan gum (see Figures 3 and 4). The calibration chart is used by our algorithms to convert the measured ring radius to a z-coordinate. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54247/54247fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54247/54247fig6.jpg" /> 
Figure 6. A trajectory of a typical fluorescent Janus sphere swimming device. A sequence of images of the moving swimming device was recorded over a period of 30 sec at a frame rate of 33 Hz. The (x, y, z) coordinates of the trajectory were obtained by locating the bright ring center (Figure 4 (a)) and comparing the measured ring radius to the calibration chart for each image in the sequence (Figures 4 (b) and 5). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54247/54247fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Preparation and 3D Tracking of Catalytic Swimming Devices

A method to prepare catalytically active Janus colloids that can "swim" in fluids and determine their 3D trajectories is presented.

We report a method to prepare catalytically active Janus colloids that "swim" in fluids and describe how to determine their 3D motion using fluorescence ...

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7.5K Views.  University of Sheffield. A method to prepare catalytically active Janus colloids that can "swim" in fluids and determine their 3D trajectories is presented.

Video: Preparation and 3D Tracking of Catalytic Swimming Devices

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

Developmental Biology

A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen consumption is typically used as the criterion measurement in this regard, two metabolic breakpoints that reflect changes in the dynamics of lactate production/consumption as the work rate is increased are perhaps more relevant for endurance athletes from a functional standpoint. Exercise economy, which represents the rate of oxygen consumption relative to performance of submaximal work, is also an important parameter to measure for endurance-athlete assessment. Ramp incremental tests comprising a gradual but rapid increase in work rate until the limit of exercise tolerance is reached are useful for determining these parameters. This type of test is typically performed on a cycle ergometer or treadmill because there is a need for precision with respect to work-rate incrementation. However, athletes should be tested while performing the mode of exercise required for their sport. Consequently, swimmers are typically assessed during free-swimming incremental tests where such precision is difficult to achieve. We have recently suggested that stationary swimming against a load that is progressively increased (incremental tethered swimming) can serve as a &#34;swim ergometer&#34; by allowing sufficient precision to accommodate a gradual but rapid loading pattern that reveals the aforementioned metabolic breakpoints and exercise economy. However, the degree to which the peak rate of oxygen consumption achieved during such a protocol approximates the maximal rate that is measured during free swimming remains to be determined. In the present article, we explain how this rapidly incremented tethered-swimming protocol can be employed to assess the cardiorespiratory capacity of a swimmer. Specifically, we explain how assessment of a short-distance competitive swimmer using this protocol revealed that his rate of oxygen uptake was 30.3 and 34.8 mL&#8729;min-1&#8729;kg-1BM at his gas-exchange threshold and respiratory compensation point, respectively.

As opposed to measurement during free swimming, which presents inherent challenges and limitations, determination of important parameters of cardiorespiratory function for swimmers can be made using a more feasible and easier to administer tethered-swimming rapidly incremented protocol with gas exchange and ventilatory data collection.

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen ...

Biochemistry

3D Kinematic Gait Analysis for Preclinical Studies in Rodents

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of complex algorithms and mathematical modeling that accompany 3D data collection and analysis procedures. This work provides a simple, user-friendly, step-by-step detailed methodology for 3D kinematic gait analysis during treadmill locomotion in healthy and neurotraumatic rats using a six-camera motion capture system. Also provided are details on 1) calibration of the system in an experimental set-up customized for quadrupedal locomotion, 2) data collection for treadmill locomotion in adult rats using markers positioned on all four limbs, 3) options available for video tracking and processing, and 4) basic 3D kinematic data generation and visualization and quantification of data using the built-in data collection software. Finally, it is suggested that the utility of this motion capture system be expanded to studying a variety of motor behaviors before and after neurotrauma.

Presented here is a protocol to collect and analyze three-dimensional kinematics of quadrupedal locomotion in rodents for preclinical studies.

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of ...

Behavior

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Neuroscience

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living components. Due to their inherent advantages, biorobots are gaining interest as alternatives to traditional fully artificial robots. Various studies have focused on harnessing the power of biological actuators, but only recently studies have quantitatively characterized the performance of biorobots and studied their geometry to enhance functionality and efficiency. Here, we demonstrate the development of a self-stabilizing swimming biorobot that can maintain its pitch, depth, and roll without external intervention. The design and fabrication of the PDMS scaffold for the biological actuator and biorobot followed by the functionalization with fibronectin is described in this first part. In the second part of this two-part article, we detail the incorporation of cardiomyocytes and characterize the biological actuator and biorobot function. Both incorporate a base and tail (cantilever) which produce fin-based propulsion. The tail is constructed with soft lithography techniques using PDMS and laser engraving. After incorporating the tail with the device base, it is functionalized with a cell adhesive protein and seeded confluently with cardiomyocytes. The base of the biological actuator consists of a solid PDMS block with a central glass bead (acts as a weight). The base of the biorobot consists of two composite PDMS materials, Ni-PDMS and microballoon-PDMS (MB-PDMS). The nickel powder (in Ni-PDMS) allows magnetic control of the biorobot during cells seeding and stability during locomotion. Microballoons (in MB-PDMS) decrease the density of MB-PDMS, and enable the biorobot to float and swim steadily. The use of these two materials with different mass densities, enabled precise control over the weight distribution to ensure a positive restoration force at any angle of the biorobot. This technique produces a magnetically controlled self-stabilizing swimming biorobot.

In this two-part study, a biological actuator was developed using highly flexible polydimethylsiloxane (PDMS) cantilevers and living muscle cells (cardiomyocytes), and characterized. The biological actuator was incorporated with a base made of modified PDMS materials to build a self-stabilizing, swimming biorobot.

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living ...

Bioengineering

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Biology

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes&#8212;many not associated with the oral apparatus (OA)&#8212;that are differentially regulated in phases throughout the regeneration process. It was hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new OA will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression. However, the morphological complexity of S. coeruleus necessitated the development of an assay to capture the statistics and timescale. A custom script was used to track cells in short videos, and statistics were compiled over a large population (N ~100). Upon loss of the OA, S. coeruleus initially loses the ability for directed motion; then starting at ~4 h, it exhibits a significant drop in speed until ~8 h. This assay provides a useful tool for the screening of motility phenotypes and can be adapted for the investigation of other organisms.

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

We present a protocol for the characterization of motility and behavior of a population of hundred micron- to millimeter-sized cells using brightfield microscopy and cell tracking. This assay reveals that Stentor coeruleus transitions through four behaviorally distinct phases when regenerating a lost oral apparatus.

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds ...

Preparation and 3D Tracking of Catalytic Swimming Devices

Summary

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Chapters in this video

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers

3D Kinematic Gait Analysis for Preclinical Studies in Rodents

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

A Protocol for Real-time 3D Single Particle Tracking

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking