The authors acknowledge (1) financial supports from Department of Nanobiomedical Science &#38; BK21 PLUS NBM Global Research Center for Regenerative Medicine in Dankook University, and from the Priority Research Centers Program (No. 2009-0093829) funded by NRF, Republic of Korea, (2) the facilities, and the scientific and technical assistance, of the Australian Centre for Microscopy and Microanalysis at the University of Sydney. HKC is grateful to the Australian Research Council for the financial supports through a Discovery Project grant (DP0985367&#38; DP120102778). WCh is grateful to the Australian Research Council for the financial supports through a linkage Project grant (LP120200489, LP110200316).

1. Preparing the Colloidal Probe and AFM Substrate
<ol>
	<li>To prepare colloidal probes, use a method developed previously by the authors.19
	<ol>
		<li>In brief, use a 45&#176; angle holder to affix a tipless cantilever at the specific angle of 45&#176; (Figure 1A).</li>
		<li>Prepare an epoxy slide by smearing a thin layer of epoxy onto a microscope slide. Use a clean spatula or a slow stream of nitrogen to ensure that the layer of epoxy added to the microscope slide is of minimal height.</li>
		<li>Affix the epoxy slide to a 40X optical zoom microscope lens using a custom designed holder (Figure 1B). Then use the cantilever to approach the epoxy slide and acquire a small amount of epoxy on the cantilever.</li>
		<li>Repeat these steps to also attach a single particle of interest at the apex of the cantilever (Figure 1C).</li>
	</ol>
	</li>
	<li>Prepare the AFM substrate by affixing colloidal particles onto an AFM coverslip using a thermoplastic mounting adhesive.
	<ol>
		<li>Heat a 35 mm round cover slip to 120 &#176;C, and apply a small amount of the adhesive to the coverslip. The high temperature is necessary to melt the thermoplastic adhesive for application.</li>
		<li>Then cool, the coverslip to 40 &#176;C before dusting the colloidal particles onto the glue. NOTE: At 40 &#176;C the glue is sufficiently set that the particles will not become embedded into the glue, but the glue is sticky enough to ensure that the particles will adhere to the substrate.</li>
		<li>Further cool the coverslip to RT and use a gentle stream of nitrogen to blow off any excess unattached particles.</li>
		<li>Wash the substrate several times with the liquid medium that will be used for colloidal probe measurements to ensure that all unattached particles are removed from the substrate. NOTE: This is important to reduce the effects of free flowing particles during the measurement, which can interact with the cantilever and introduce errors in the results.</li>
	</ol>
	</li>
</ol>
2. Mounting the Colloidal Probe, Aligning Laser, and Equilibrating System
<ol>
	<li>Mount the coverslip with the colloidal particles into the bottom half of a liquid cell, making sure that the O-ring is seated properly to prevent any leaking.</li>
	<li>Place a hydrophobic transparent sheet onto the microscope stage to guard against any liquid that may leak during the experiment, especially if only using the bottom half of a liquid cell for the measurement, and place the liquid cell onto the microscope stage. NOTE: For simplicity one can use only the bottom half of a liquid cell, given that the system can be equilibrated adequately; tip &#8211; the evaporation changes the condition of the measurement and impacts the results/reading.</li>
	<li>Attach the colloidal probe to the AFM scanning head and assemble onto the AFM. With the AFM instrument software on, use the knobs on the scanning head to bring the cantilever tip into focus. NOTE: All procedural steps and measurements were completed using an MFP-3D-Bio AFM with Asylum Research software.</li>
	<li>To maximize intensity, align the laser onto the tip of the cantilever using the appropriate adjustment knobs on the scanning head.</li>
	<li>Allow the system to equilibrate for 5-10 min or until the deflection value stabilizes. Use the deflection adjustment knob to bring the deflection to zero or slightly negative.</li>
	<li>After the system has equilibrated in air, use the AFM software (Thermal Panel in the Master Panel window) to thermally calculate the InvOLS (sensitivity) and spring constant of the colloidal probe. NOTE: This sensitivity will be temporarily used until the true sensitivity is measured at the completion of the measurement (see step 4).
	<ol>
		<li>Select either &#8220;Cal Spring Constant&#8221; or &#8220;Cal InvOLS&#8221; and then click on &#8220;Capture Thermal Data&#8221;.</li>
		<li>Once a prominent peak is apparent, stop capturing data, and click to zoom over the main peak.</li>
		<li>Click on &#8220;Initialize Fit&#8221; followed by &#8220;Fit Thermal Data,&#8221; to obtain the automatically calculated spring constant or InvOLS values.</li>
	</ol>
	</li>
	<li>Slowly add 2 ml of the liquid medium to the liquid cell using a syringe and ensure that no bubbles are present around the cantilever. Re-align the laser, since the refractive index of the medium has now changed, and once again equilibrate the system allowing the deflection value to stabilize before adjusting the deflection back to zero. NOTE: If a large temperature difference exists between the environment and liquid, equilibration will take longer.</li>
</ol>
3. Imaging and Data Acquisition
<ol>
	<li>Set the initial scan size to 20 &#181;m, scan rate to 1 Hz, scan angle to 90&#176;, set point to 0.2 V and obtain a scan of the sample. Adjust the gain as needed to obtain overlapped trace and retrace curves.</li>
	<li>Once a particle of interest is found, immediately zoom onto that particle to limit extended probe interactions with the substrate prior to obtaining force volume measurements.</li>
	<li>Once zoomed in, acquire a sufficient image of a single particle or portion of a single particle. Then switch to the Force Panel in the software. Bring the red position bar to the highest position, set the force distance to 5 &#181;m, scan rate to 0.1 Hz, trigger channel to none and conduct a single force measurement. Make sure the probe does not contact the substrate.</li>
	<li>From the single measurement graph obtained, calculate the virtual deflection line by right clicking on the graph window, and selecting the &#8220;Calculate Virtual Def Line&#8221; option. This will automatically calculate the virtual deflection and update the value as needed within the software.</li>
	<li>Change the trigger channel to deflection and set the trigger point to 20 nm. Set the force distance to 1 &#181;m and adjust the scan velocity as desired depending on the measured forces of interest.</li>
	<li>Manually adjust the value for the deflection Inverse Optical Lever Sensitivity (InvOLS) in the Review Force Panel after conducting 2-3 consecutive preliminary single force measurements.
	<ol>
		<li>Conduct a single force measurement, then click on the &#8220;Review&#8221; button on the Force Panel which opens up a Master Force Panel.</li>
		<li>Highlight the most recently completed force measurement. Under the &#8220;Axis&#8221; heading ensure that only &#8220;DeflV&#8221; is checked. Change the &#8220;X-Axis&#8221; input field to &#8220;Sep&#8221; using the dropdown menu and click on &#8220;make graph.&#8221;</li>
		<li>Click on the &#8220;Parm&#8221; tab on the Master Force Panel and adjust the value of &#8220;InvOLS&#8221; until the contact region of the graph is completely vertical. Then populate this value in the &#8220;Defl InvOLS&#8221; field located under the Cal&#160;sub-tab in the Force tab located on the main Master Panel window.</li>
		<li>Repeat this 2-3 times to ensure that the InvOLS value does not change significantly.</li>
	</ol>
	</li>
	<li>Now that all parameters have been set up, ensure that the liquid medium level is still sufficient and that the deflection is still stable. NOTE: At this time, single force curves or force maps can be obtained. If dwell force measurements are desired, the dwell options can be accessed in the Force Panel.</li>
</ol>
4. Post-tuning of Sensitivity for Analysis
<ol>
	<li>After the completion of measurement acquisition, measure the true sensitivity of the colloidal probe. To do this, conduct a force measurement using a relatively large deflection/force with the colloidal probe in the same liquid medium against an &#8220;infinitely&#8221; hard surface such as mica. NOTE: Sensitivity was obtained after completion of the experiments because the large deflection/force may damage colloidal probes prepared with porous or fragile colloids.</li>
	<li>The slope of the contact region is used by the software to automatically calculate the sensitivity (Figure 2). Use this true value of sensitivity during data analysis of all the curves obtained using that particular colloidal probe.</li>
</ol>

<ol>
	<li>Sindel, U., Zimmermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+interaction+forces+between+individual+powder+particles+using+an+atomic+force+microscope.">Measurement of interaction forces between individual powder particles using an atomic force microscope.</a> Powder Technology. 117, 247-254 (2001).</li><li>Ducker, W. A., Senden, T. J., Pashley, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+colloidal+forces+using+an+atomic+force+microscope.">Direct measurement of colloidal forces using an atomic force microscope.</a> Nature. 353, 239-241 (1991).</li><li>Israelachvili, J. N., Adams, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+forces+between+two+mica+surfaces+in+aqueous+electrolyte+solutions+in+the+range+0&#8211;100+nm.">Measurement of forces between two mica surfaces in aqueous electrolyte solutions in the range 0&#8211;100 nm.</a> Journal of the Chemical Society, Faraday Transactions. 1, 975-1001 (1978).</li><li>Upadhyay, D., 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=Magnetised+thermo+responsive+lipid+vehicles+for+targeted+and+controlled+lung+drug+delivery.">Magnetised thermo responsive lipid vehicles for targeted and controlled lung drug delivery.</a> Pharmaceutical Research. 29, 2456-2467 (2012).</li><li>Chrzanowski, W., 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=Biointerface:+protein+enhanced+stem+cells+binding+to+implant+surface.">Biointerface: protein enhanced stem cells binding to implant surface.</a> Journal of Materials Science: Materials in Medicine. 23, 2203-2215 (2012).</li><li>Chrzanowski, W., 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=Nanomechanical+evaluation+of+nickel&#8211;titanium+surface+properties+after+alkali+and+electrochemical+treatments.">Nanomechanical evaluation of nickel&#8211;titanium surface properties after alkali and electrochemical treatments.</a> Journal of The Royal Society Interface. 5, 1009-1022 (2008).</li><li>Tran, C. T., Kondyurin, A., Chrzanowski, W., Bilek, M. M., McKenzie, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+pH+on+yeast+immobilization+on+polystyrene+surfaces+modified+by+energetic+ion+bombardment.">Influence of pH on yeast immobilization on polystyrene surfaces modified by energetic ion bombardment.</a> Colloids and Surfaces B: Biointerfaces. 104, 145-152 (2013).</li><li>Page, K., 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=Study+of+the+adhesion+of+Staphylococcus+aureus+to+coated+glass+substrates.">Study of the adhesion of Staphylococcus aureus to coated glass substrates.</a> Journal of materials science. 46, 6355-6363 (2011).</li><li>Binnig, G., Quate, C. F., Gerber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscope.">Atomic force microscope.</a> Physical Review Letters. 56, 930-933 (1103).</li><li>Butt, H. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+electrostatic,+van+der+Waals,+and+hydration+forces+in+electrolyte+solutions+with+an+atomic+force+microscope.">Measuring electrostatic, van der Waals, and hydration forces in electrolyte solutions with an atomic force microscope.</a> Biophysical Journal. 60, 1438-1444 (1991).</li><li>Johnson, K., Kendall, K., Roberts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+energy+and+the+contact+of+elastic+solids.">Surface energy and the contact of elastic solids.</a> Proceedings of the Royal Society of London. A. Mathematical and Physical Sciences. 324, 301-313 (1971).</li><li>Deraguin, B., Landau, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+the+stability+of+strongly+charged+lyophobic+sols+and+of+the+adhesion+of+strongly+charged+particles+in+solution+of+electrolytes.">Theory of the stability of strongly charged lyophobic sols and of the adhesion of strongly charged particles in solution of electrolytes.</a> Acta Physicochim: USSR. 14, 633-662 (1941).</li><li>Derjaguin, B., Muller, V., Toporov, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+contact+deformations+on+the+adhesion+of+particles.">Effect of contact deformations on the adhesion of particles.</a> Journal of Colloid and Interface Science. 53, 314-326 (1975).</li><li>Verwey, E. J. W., Overbeek, J. T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+the+stability+of+lyophobic+colloids.">Theory of the stability of lyophobic colloids.</a> DoverPublications.com, doi:10.1021/j150453a001. , (1999).</li><li>Kappl, M., Butt, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+colloidal+probe+technique+and+its+application+to+adhesion+force+measurements.">The colloidal probe technique and its application to adhesion force measurements.</a> Particle &amp; Particle Systems Characterization. 19, 129-143 (2002).</li><li>Tran, C. T., Kondyurin, A., Chrzanowski, W., Bilek, M. M., McKenzie, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+pH+on+yeast+immobilization+on+polystyrene+surfaces+modified+by+energetic+ion+bombardment.">Influence of pH on yeast immobilization on polystyrene surfaces modified by energetic ion bombardment.</a> Colloids and Surfaces B: Biointerfaces. , (2012).</li><li>Sa, D. J., de Juan Pardo, E. M., de Las Rivas Astiz, R., Sen, S., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+indentational+elasticity+measurements+of+hydrogel+extracellular+matrix+substrates.">High-throughput indentational elasticity measurements of hydrogel extracellular matrix substrates.</a> Applied Physics Letters. 95, 063701-063701 (2009).</li><li>Zauscher, S., Klingenberg, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friction+between+cellulose+surfaces+measured+with+colloidal+probe+microscopy.">Friction between cellulose surfaces measured with colloidal probe microscopy.</a> Colloids and Surfaces A: Physicochemical and Engineering Aspects. 178, 213-229 (2001).</li><li>Sa, D., Chan, H. -. K., Chrzanowski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+of+Micro-+and+Nano-particles+on+Tipless+Cantilevers+for+Colloidal+Probe+Microscopy.">Attachment of Micro- and Nano-particles on Tipless Cantilevers for Colloidal Probe Microscopy.</a> International Journal of Colloid and Interface. , (2014).</li></ol>

The authors declare that they have no competing financial interests.

Several sources of system instability present during liquid colloidal probe nanoscopy can easily be mitigated through proper equilibration procedures. Instabilities as discussed previously result in erroneous results and force curves that are more difficult to analyze objectively. If all sources of instability have been tended and graphs similar to that shown in Figure 4 are still present, another measurement parameter may be the reason. Other measurement parameters that are important to consider during ...

Atomic force microscopy (AFM) is a technique that enables qualitative and quantitative imaging and probing of a material surface.4-6 Traditionally, AFM is used for the evaluation of surface topography, morphology and structure of multi-phasic materials. AFM has the capability to quantitatively evaluate nano-scale interactions, such as charge, attraction, repulsion and adhesion forces between a specific probe and substrate in both air and liquid mediums.7,8 The AFM originally developed by Binning, Quate and Gerber9 uses a probe of known/determined sensitivity and spring constant to approach and/or scan a sample. Due to the physical interactions between the probe and the sample, the cantilever is deflected during contact or proximity and depending on the mode of operation, this deflection can be translated to acquire the topography of the sample or measure forces present between the probe and sample. Modifications to the AFM technique, such as colloidal probe nanoscopy,10 have allowed scientist to directly evaluate the nano-force interactions between two materials present in a colloidal system of interest.
In colloidal probe nanoscopy, a spherical particle of choice is attached to the apex of a cantilever, replacing the traditional conical and pyramidal tips. A spherical particle is ideal to allow comparison with theoretical models such as the Johnson, Kendal, Roberts (JKR)11 and Derjaguin, Landau, Vervwey, Overbeek (DLVO)12-14 theories and to minimize the influence of surface roughness on the measurement.15 These theories are used to define the contact mechanics and inter-particle forces expected within a colloidal system. The DLVO theory combines the attractive van der Waal forces and repulsive electrostatic forces (due to electrical double layers) to quantitatively explain the aggregation behavior of aqueous colloidal systems, while the JKR theory incorporates the effect of contact pressure and adhesion to model elastic contact between two components. Once an appropriate probe is produced, it is used to approach any other material/particle to evaluate the forces between the two components. Using a standard manufactured tip one will be able to measure interactive forces between that tip and a material of choice, but the benefit of using a custom made colloidal probe allows the measurement of forces present between materials present within the studied system. Measurable interactions include: adhesive, attractive, repulsive, charge, and even electrostatic forces present between the particles.16 Additionally, the colloidal probe technique can be used to explore tangential forces present between particles and material elasticity.17,18
The ability to conduct measurements in various media is one of the major advantages of colloidal probe nanoscopy. Ambient conditions, liquid media, or humidity-controlled conditions can all be used to mimic environmental conditions of the system studied. The ability to conduct measurements in a liquid environment enables the study of colloidal systems in an environment that it naturally occurs; thus, being able to quantitatively acquire data that is directly translatable to the system in its natural state. For example, particle interactions present within metered dose inhalers (MDI) can be studied using a model liquid propellant with similar properties to the propellant used in MDIs. The same interactions measured in air would not be representative of the system existent in the inhaler. Furthermore, the liquid medium can be modified to evaluate the effect of moisture ingress, a secondary surfactant, or temperature on the particle interactions in an MDI. The ability to control temperature can be used to mimic certain steps in the manufacturing of colloidal systems to evaluate how temperature either in the manufacturing of or storage of colloidal systems may have an impact on particle interactions.
Measurements that can be obtained using colloidal probes include; Topography scanning, individual force-distance curves, force-distance adhesion maps, and dwell force-distance measurements. Key parameters that are measured using the colloidal probe nanoscopy method presented in this paper include the snap-in, max load, and separation energy values. Snap-in is a measurement of the attractive forces, max load the value of the maximum adhesion force, and the separation energy conveys the energy required to withdraw the particle from contact. These values can be measured through instantaneous or dwell force measurements. Two different types of dwell measurements include deflection and indentation. The length and type of dwell measurement can be specifically chosen to mimic specific interactions that are present within a system of interest. An example is using deflection dwell - which holds the samples in contact at a desired deflection value &#8211; to evaluate the adhesive bonds that develop in aggregates formed in dispersions. The adhesive bonds formed can be measured as a function of time and can provide insight into the forces required to redisperse the aggregates after prolonged storage. The plethora of data that can be obtained using this method is a testament to the versatility of the method.

<table><tbody><tr><td>Double-Bubble Epoxy</td><td>Hardman</td><td>4004</td><td></td></tr><tr><td>Veeco Tipless Probes</td><td>Veeco</td><td>NP-O10&amp;nbsp;</td><td></td></tr><tr><td>Porous Particles</td><td>Pearl Therapeutics</td><td></td><td></td></tr><tr><td>Atomic Force Microscope (MFP)</td><td>Asylum&amp;nbsp;</td><td>MFP-3D</td><td></td></tr><tr><td>SPIP Scanning Probe Image Processor Software</td><td>NanoScience&amp;nbsp; Instruments</td><td></td><td></td></tr><tr><td>35 mm Coverslips</td><td>Asylum</td><td>504.003</td><td></td></tr><tr><td>Tempfix</td><td>Ted Pella. Inc.</td><td>16030</td><td></td></tr></tbody></table>

quantitative and qualitative examination of particle-particle interactions using colloidal probe nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Liquid colloidal systems are used for several pharmaceutical drug delivery systems. For inhalation drug delivery, a common colloidal system is the suspension pressurized metered dose inhaler (pMDI). Particle interactions present within the pMDI play a vital role in formulation physical stability, storage, and drug delivery uniformity. In this manuscript, inter-particle forces between porous lipid-based particles (~2 &#181;m optical mean particle diameter) in a model propellant (2H,3H-perfluoropentane) were evaluated at R...

Watch this Scientific Journal Video about Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy at JoVE.com

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

quantitative-qualitative-examination-particle-particle-interactions

Research

JoVE Journal

Chemistry

11.0K Views.  University of Sydney. Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems. 

Video: Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures

The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed assembly1-3, drug delivery4, improved hydrocarbon recovery5-7, and flowable electrodes for energy storage8. Suspensions containing fluorescent colloids and non-adsorbing polymers are appealing model systems, as the ratio of the polymer radius of gyration to the particle radius&#160;and concentration of polymer control the range and strength of the interparticle attraction, respectively. By tuning the polymer properties and the volume fraction of the colloids, colloid fluids, fluids of clusters, gels, crystals, and glasses can be obtained9.&#160;Confocal microscopy, a variant of fluorescence microscopy, allows an optically transparent and fluorescent sample to be imaged with high spatial and temporal resolution in three dimensions. In this technique, a small pinhole or slit blocks the emitted fluorescent light from regions of the sample that are outside the focal volume of the microscope optical system. As a result, only a thin section of the sample in the focal plane is imaged. This technique is particularly well suited to probe the structure and dynamics in dense colloidal suspensions at the single-particle scale: the particles are large enough to be resolved using visible light and diffuse slowly enough to be captured at typical scan speeds of commercial confocal systems10. Improvements in scan speeds and analysis algorithms have also enabled quantitative confocal imaging of flowing suspensions11-16,37.&#160;In this paper, we demonstrate confocal microscopy experiments to probe the confined phase behavior and flow properties of colloid-polymer mixtures. We first prepare colloid-polymer mixtures that are density- and refractive-index matched. Next, we report a standard protocol for imaging quiescent dense colloid-polymer mixtures under varying confinement in thin wedge-shaped cells. Finally, we demonstrate a protocol for imaging colloid-polymer mixtures during microchannel flow.

Confocal microscopy is used to image quiescent and flowing colloid-polymer mixtures, which are studied as model systems for attractive suspensions. Image analysis algorithms are used to calculate structural and dynamic metrics for the colloidal particles that measure changes due to geometric confinement.

The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed ...

Implementation of a Reference Interferometer for Nanodetection

A thermally and mechanically stabilized fiber interferometer suited for examining ultra-high quality factor microcavities is fashioned. After assessing its free spectral range (FSR), the module is put in parallel with a fiber taper-microcavity system and then calibrated through isolating and eliminating random shifts in the laser frequency (i.e. laser jitter noise). To realize the taper-microcavity junction and to maximize the optical power that is transferred to the resonator, a single-mode optical fiber waveguide is pulled. Solutions containing polystyrene nanobeads are then prepared and flown to the microcavity in order to demonstrate the system&#8217;s ability to sense binding to the surface of the microcavity. Data is post-processed via adaptive curve fitting, which allows for high-resolution measurements of the quality factor as well as the plotting of time-dependent parameters, such as resonant wavelength and split frequency shifts. By carefully inspecting steps in the time-domain response and shifting in the frequency-domain response, this instrument can quantify discrete binding events.

A reference interferometer technique, which is designed to remove undesirable laser jitter noise for nanodetection, is utilized for probing an ultra-high quality factor microcavity. Instructions for assembly, setup, and data acquisition are provided, alongside the measurement process for specifying the cavity quality factor.

A thermally and mechanically stabilized fiber interferometer suited for examining ultra-high quality factor microcavities is fashioned. After assessing ...

Engineering

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 &#181;g.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a microfluidic chamber assembled in the specimen holder for Transmission Electron Microscopy (TEM) and STEM. The microfluidic system consists of two silicon microchips supporting thin Silicon Nitride (SiN) membrane windows. This article describes the basic steps of sample loading and data acquisition. Most important of all is to ensure that the liquid compartment is correctly assembled, thus providing a thin liquid layer and a vacuum seal. This protocol also includes a number of tests necessary to perform during sample loading in order to ensure correct assembly. Once the sample is loaded in the electron microscope, the liquid thickness needs to be measured. Incorrect assembly may result in a too-thick liquid, while a too-thin liquid may indicate the absence of liquid, such as when a bubble is formed. Finally, the protocol explains how images are taken and how dynamic processes can be studied. A sample containing AuNPs is imaged both in pure water and in saline.

This protocol describes the operation of a liquid flow specimen holder for scanning transmission electron microscopy of AuNPs in water, as used for the observation of nanoscale dynamic processes.

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a ...

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few can observe immobilized particles. Moreover, a limited number of studies have experimentally measured the exertable forces on the particles. The designs can be classified as the protruding nanodisk type or the suppressed nanohole type. For the latter, microscopic observation is extremely challenging. In this paper, a new plasmonic tweezer system is introduced to monitor particles, both in directions parallel and orthogonal to the symmetric axis of a plasmonic nanohole structure. This feature enables us to observe the movement of each particle near the rim of the nanohole. Furthermore, we can quantitatively estimate the maximal trapping forces using a new fluidic channel.

A microchip fabrication process that incorporates plasmonic tweezers is presented here. The microchip enables the imaging of a trapped particle to measure maximal trapping forces.

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few ...

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

Biochemistry

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. The instrument is optimized for the exploration of the wide momentum transfer Q range between 1x10-4 and 0.5 &#197;-1 by combining classical pinhole, focusing (with lenses), and time-of-flight (with chopper) methods, while simultaneously providing high-neutron intensities with an adjustable resolution. Because of its ability to adjust the intensity and the resolution within wide limits during the experiment, combined with the possibility to equip specific sample environments and ancillary devices, the KWS-2 shows a high versatility in addressing the broad range of structural and morphological studies in the field. Equilibrium structures can be studied in static measurements, while dynamic and kinetic processes can be investigated over time scales between minutes to tens of milliseconds with time-resolved approaches. Typical systems that are investigated with the KWS-2 cover the range from complex, hierarchical systems that exhibit multiple structural levels (e.g., gels, networks, or macro-aggregates) to small and poorly-scattering systems (e.g., single polymers or proteins in solution). The recent upgrade of the detection system, which enables the detection of count rates in the MHz range, opens new opportunities to study even very small biological morphologies in buffer solution with weak scattering signals close to the buffer scattering level at high Q.
In this paper, we provide a protocol to investigate samples with characteristic size levels spanning a wide length scale and exhibiting ordering in the mesoscale structure using KWS-2. We present in detail how to use the multiple working modes that are offered by the instrument and the level of performance that is achieved.

Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to &#181;m that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. ...

Bioengineering

Nanoscale Characterization of Liquid-Solid Interfaces by Coupling Cryo-Focused Ion Beam Milling with Scanning Electron Microscopy and Spectroscopy

Physical and chemical processes at solid-liquid interfaces play a crucial role in many natural and technological phenomena, including catalysis, solar energy and fuel generation, and electrochemical energy storage. Nanoscale characterization of such interfaces has recently been achieved using cryogenic electron microscopy, thereby providing a new path to advancing our fundamental understanding of interface processes.
This contribution provides a practical guide to mapping the structure and chemistry of solid-liquid interfaces in materials and devices using an integrated cryogenic electron microscopy approach. In this approach, we pair cryogenic sample preparation which allows stabilization of solid-liquid interfaces with cryogenic focused ion beam (cryo-FIB) milling to create cross-sections through these complex buried structures. Cryogenic scanning electron microscopy (cryo-SEM) techniques performed in a dual-beam FIB/SEM enable direct imaging as well as chemical mapping at the nanoscale. We discuss practical challenges, strategies to overcome them, as well as protocols for obtaining optimal results. While we focus in our discussion on interfaces in energy storage devices, the methods outlined are broadly applicable to a range of fields where solid-liquid interface play a key role.

Cryogenic Focused Ion Beam (FIB) and Scanning Electron Microscopy (SEM) techniques can provide key insights into the chemistry and morphology of intact solid-liquid interfaces. Methods for preparing high quality Energy Dispersive X-ray (EDX) spectroscopic maps of such interfaces are detailed, with a focus on energy storage devices.

Physical and chemical processes at solid-liquid interfaces play a crucial role in many natural and technological phenomena, including catalysis, solar ...

Co-localizing Kelvin Probe Force Microscopy with Other Microscopies and Spectroscopies: Selected Applications in Corrosion Characterization of Alloys

Kelvin probe force microscopy (KPFM), sometimes referred to as surface potential microscopy, is the nanoscale version of the venerable scanning Kelvin probe, both of which measure the Volta potential difference (VPD) between an oscillating probe tip and a sample surface by applying a nulling voltage equal in magnitude but opposite in sign to the tip-sample potential difference. By scanning a conductive KPFM probe over a sample surface, nanoscale variations in surface topography and potential can be mapped, identifying likely anodic and cathodic regions, as well as quantifying the inherent material driving force for galvanic corrosion.
Subsequent co-localization of KPFM Volta potential maps with advanced scanning electron microscopy (SEM) techniques, including back scattered electron (BSE) images, energy dispersive spectroscopy (EDS) elemental composition maps, and electron backscattered diffraction (EBSD) inverse pole figures can provide further insight into structure-property-performance relationships. Here, the results of several studies co-localizing KPFM with SEM on a wide variety of alloys of technological interest are presented, demonstrating the utility of combining these techniques at the nanoscale to elucidate corrosion initiation and propagation.
Important points to consider and potential pitfalls to avoid in such investigations are also highlighted: in particular, probe calibration and the potential confounding effects on the measured VPDs of the testing environment and sample surface, including ambient humidity (i.e., adsorbed water), surface reactions/oxidation, and polishing debris or other contaminants. Additionally, an example is provided of co-localizing a third technique, scanning confocal Raman microscopy, to demonstrate the general applicability and utility of the co-localization method to provide further structural insight beyond that afforded by electron microscopy-based techniques.

Kelvin probe force microscopy (KPFM) measures surface topography and differences in surface potential, while scanning electron microscopy (SEM) and associated spectroscopies can elucidate surface morphology, composition, crystallinity, and crystallographic orientation. Accordingly, the co-localization of SEM with KPFM can provide insight into the effects of nanoscale composition and surface structure on corrosion.

Kelvin probe force microscopy (KPFM), sometimes referred to as surface potential microscopy, is the nanoscale version of the venerable scanning Kelvin ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

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