The Deutsche Forschungsgemeinschaft (DFG) is acknowledged for financial support within the Sonderforschungsbereich SFB 985 "Functional Microgels and Microgel Systems".

1. Microgel Synthesis
NOTE: N-isopropylacrylamide (NIPAM) was recrystallized from n-hexane. Other reagents were used as received.
<ol>
	<li>Conventional Batch Synthesis of Poly(NIPAM) Matrix Microgels
	<ol>
		<li>Dissolve 1.8 g NIPAM and 24 mg N,N&#39;-bisacrylamide (BIS) in 245 ml filtered (0.2 &#181;m regenerated cellulose (RC) membrane filter) double distilled water in a 500 ml three-neck round bottom flask equipped with a reflux condenser, a stirrer and a rubber septum.</li>
		<li>Insert a thermometer and a 120 mm needle for the nitrogen input through the septum.</li>
		<li>Heat the solution to 60 &#176;C, while stirring. Deoxygenate the solution by purging with nitrogen for 40 min.</li>
		<li>Simultaneously prepare an initiator solution of 155 mg potassium persulfate (KPS) in 5 ml filtered double-distilled water and bubble the solution with nitrogen to remove oxygen.</li>
		<li>Transfer the complete 5 ml KPS solution in a nitrogen-washed syringe equipped with a 120 mm needle.</li>
		<li>Lift the nitrogen needle above the solution level in the three-neck flask and add the KPS solution rapidly through the rubber septum into the reactor.</li>
		<li>Let the polymerization proceed for 1 hr under nitrogen flow and slow stirring at 60 &#176;C.</li>
		<li>Use a Buchner funnel and filter paper to filter the hot reaction solution in order to discard big aggregates. Let the dispersion to cool down.</li>
		<li>Centrifuge and redisperse the dispersion three times for 40 min at 257,000 x g and finally redisperse the sediment in a minimum viable amount of double distilled water. Typically this is 2-4 ml.</li>
		<li>Lyophilize the dispersion for storing.</li>
	</ol>
	</li>
	<li>Non-stirred Synthesis of Fluorescently Labeled Poly(NIPAM) Microgels
	<ol>
		<li>Weigh 257.7 mg NIPAM, 3.5 mg BIS, and 1.5 mg methacryloxyethyl thiocarbamoyl rhodamine B (dye) in glass vessel and add 10 ml of filtered double distilled water.</li>
		<li>Ultrasonicate the dye&#8211;monomer solution for 15 min to dissolve the dye in water.</li>
		<li>Prepare the same solution without the dye into a separate glass vessel.</li>
		<li>Prepare various dilutions of the monomer solution with the dye using the monomer solution without the dye to obtain a concentration series with various dye concentrations. In this work, use dye in the concentration range of 0.02-0.1 mmol/L.</li>
		<li>Dissolve 8.4 mg KPS in 10 ml filtered double distilled water in order to get the initiator solution.</li>
		<li>Transfer 0.5 ml of the concentration series and 0.5 ml of the KPS solution to test tubes with 10 mm diameter to obtain the final reaction solutions and seal them with rubber septa.</li>
		<li>Preheat an oil bath in a double-walled glass vessel connected to a heating circulator to 63 &#176;C.</li>
		<li>Deoxygenize the reaction solutions by purging with nitrogen through 120 mm needles for 20 min.</li>
		<li>Insert the tubes into a floating platform and immerse the platform into the preheated oil bath. Set the temperature to 60 &#176;C. Initially higher temperature in the bath is necessary as the room temperature solutions cool down the bath. For high precision particle size tuning the temperature control during the initial reaction has to be rigorous, typically &#177;0.1 &#176;C.</li>
		<li>Let the reaction proceed for an appropriate time. Typically 1 hr is enough.</li>
		<li>Transfer the reaction tubes rapidly to an oven at 60 &#176;C and put one drop of the hot dispersion to 10 ml filtered double distilled water preheated over PNIPAM volume phase transition temperature (VPTT, 32 -34 &#176;C) 1, for DLS characterization in the collapsed state.</li>
		<li>Let the rest of the dispersions cool down to room temperature and transfer them into centrifuge tubes.</li>
		<li>Centrifuge the solution three times for 40 min at 257,000 x g and dilute the microgels finally in 2 ml filtered double distilled water for use as tracer particles.</li>
	</ol>
	</li>
</ol>
2. Light Scattering Characterization
<ol>
	<li>Hydrodynamic Radius Determination in Collapsed State by Dynamic Light Scattering
	<ol>
		<li>Wash cuvettes and glassware with acetone vapor.</li>
		<li>Heat 10 ml of filtered (e.g., 200 nm or smaller RC filter) double distilled water over PNIPAM VPTT.</li>
		<li>Transfer a drop of hot dispersion to the filtered water using a pre-heated needle (0.9 x 40 mm) and syringe (1 ml).</li>
		<li>Temper the DLS goniometer index match bath to 50 &#176;C and transfer the sample to the instrument without letting it cool down.</li>
		<li>Find the largest scattering angle where the scattered intensity is sufficient to acquire a correlogram by performing test measurements.
		<ol>
			<li>Insert sample cuvette (10 mm diameter glass tube with 1 ml of particle dispersion). Move the detector arm to small scattering angle (here 30&#176;).</li>
			<li>Check the beam profile for multiple scattering: no glow around the primary beam, no multiple scattering, etc. Check that the count range is suitable for measurement at the lowest scattering angle (approx. between 30 and 600 kHz; top right corner of the software window.)</li>
			<li>Move the goniometer arm to highest scattering angle (choose 120&#176; here). Check that the count rate is still high enough for the measurement (between 30 and 600 kHz). If the intensity is too low, move the arm to lower scattering angle.</li>
		</ol>
		</li>
		<li>Check the beam visually through the toluene bath glass at the lowest scattering angle, if glow around the incident beam is observed multiple scattering takes place. In this case, reduce the laser intensity or use a higher dilution.</li>
		<li>Acquire 20 correlograms between the minimum and maximum scattering angle (e.g., 30&#176; - 140&#176;) with minimum acquisition time of 60 sec. Increase the acquisition time for weak intensity large scattering angles if necessary.</li>
	</ol>
	</li>
	<li>Data Analysis 37
	<ol>
		<li>Calculate scattering vector magnitudes for the scattering angle according to <img alt="Eq 2" src="/files/ftp_upload/54419/54419eq2.jpg" />, where n is the refractive index of the dispersion, <img alt="Eq 3" src="/files/ftp_upload/54419/54419eq3.jpg" />the wavelength of the laser in vacuum and <img alt="Eq 4" src="/files/ftp_upload/54419/54419eq4.jpg" /> the scattering angle.</li>
		<li>In the case the measurement software provides the intensity correlation function <img alt="Eq 5" src="/files/ftp_upload/54419/54419eq5.jpg" />, transform it to electric field correlation function&#160;<img alt="Eq 6" src="/files/ftp_upload/54419/54419eq6.jpg" /> according to <img alt="Eq 7" src="/files/ftp_upload/54419/54419eq7.jpg" />. Parameter <img alt="Eq 8" src="/files/ftp_upload/54419/54419eq8.jpg" /> is an uninteresting instrumental parameter related to the degree of spatial coherence of the scattered light over the detector area.</li>
		<li>Perform cumulant analysis on correlograms, i.e., fit second order polynomial to the logarithm of each electric field correlation function <img alt="Eq 9" src="/files/ftp_upload/54419/54419eq9.jpg" /> by linear least squares. <img alt="Eq 8" src="/files/ftp_upload/54419/54419eq8.jpg" /> appears as the intercept of the fit and its exact value is unimportant in respect to the data analysis. Restrict the fit to a meaningful lag time &#964; value, e.g., so that the correlation amplitude is 10 - 20 % of the maximum amplitude. The coefficient of the first order term is the mean decay rate of the correlation function, <img alt="Eq 10" src="/files/ftp_upload/54419/54419eq10.jpg" />.</li>
		<li>Find the most likely value for the mean diffusion coefficient&#160;<img alt="Eq 11" src="/files/ftp_upload/54419/54419eq11.jpg" /> of the particles by linear least squares fit on <img alt="Eq 12" src="/files/ftp_upload/54419/54419eq12.jpg" />. If <img alt="Eq 10" src="/files/ftp_upload/54419/54419eq10.jpg" /> against <img alt="Eq 13" src="/files/ftp_upload/54419/54419eq13.jpg" /> does not appear linear and go through the origin within the error, the particle size distribution is broad and hydrodynamic radius will be poorly defined.&#160;</li>
		<li>Calculate the mean hydrodynamic radius from the Stokes-Einstein relation <img alt="Eq 14" src="/files/ftp_upload/54419/54419eq14.jpg" />, where <img alt="Eq 15" src="/files/ftp_upload/54419/54419eq15.jpg" /> is the Boltzmann coefficient, <img alt="Eq 16" src="/files/ftp_upload/54419/54419eq16.jpg" /> the absolute temperature and <img alt="Eq 17" src="/files/ftp_upload/54419/54419eq17.jpg" /> the viscosity of the dispersion at <img alt="Eq 16" src="/files/ftp_upload/54419/54419eq16.jpg" />. Propagate the standard deviation of <img alt="Eq 11" src="/files/ftp_upload/54419/54419eq11.jpg" /> to <img alt="Eq 18" src="/files/ftp_upload/54419/54419eq18.jpg" />.&#160;&#160;&#160;</li>
	</ol>
	</li>
	<li>Particle Structure Determination by Static Light Scattering
	<ol>
		<li>Wash cuvettes and glassware with acetone vapor. Use 20 mm diameter or larger cuvettes to minimize the cylindrical lens effect.</li>
		<li>Filter (200 nm RC filter or smaller) approximately 20 ml of double distilled water to a glass vial and transfer a drop of purified dispersion to the vial. Wash the filter with 10 ml water before using it for sample preparation to remove impurities remaining from the manufacturing process.</li>
		<li>Check sample against any ambient light source. If blue hue is observed, the sample is likely to be too concentrated. Dilute accordingly.</li>
		<li>Prepare background water sample by flushing the cuvette multiple times with filtered water and then fill up to appropriate sample volume, depending on the cuvette and the laser position in the instrument. The laser must pass through the sample without being refracted from the meniscus.</li>
		<li>Calibrate the instrument using a toluene sample.</li>
		<li>Measure water scattering (background) throughout the available angular range.</li>
		<li>Measure the scattering intensity from the sample throughout the available angular range preferably at several wavelengths. The scattering pattern normalized to the forward scattering intensity is known as the form factor.</li>
		<li>If particle structure is known, use the appropriate model expression to calculate global fit on the datasets measured at different wavelengths.</li>
		<li>For unknown particle structure use regularized direct (such as FitIt! 33) or a more general indirect inverse Fourier transform 21,22 routine in conjunction with deconvolution of the pair distance distribution function (only for spherical particles) 23,24 for approximate classification of particle type.</li>
		<li>In case the fitting or inversion routine provides an estimate of the particle radius distribution function, calculate the polydispersity index (standard deviation of the distribution divided by its mean).</li>
	</ol>
	</li>
</ol>
3. Particle Tracking by Wide-field Fluorescence Microscopy
NOTE: Tracer and matrix particles of 465 &#177; 7 nm and 405 &#177; 7 nm hydrodynamic radii at 20 &#176;C, respectively, were used for particle tracking.
<ol>
	<li>Sample Preparation
	<ol>
		<li>Prepare concentrated matrix microgel dispersion by redispersing known amount of lyophilized unlabeled microgel to known amount of double distilled water. Add a small volume of labeled tracer particles.</li>
		<li>Confirm the appropriate tracer microgel concentration in the microscope. The optimal concentration is a compromise between simultaneous acquisitions of maximum number of tracks, while having the tracer concentration low enough so that the probability that the tracer particle tracks cross during the acquisition is negligible.</li>
		<li>Prepare concentrated dispersions by evaporating water in an oven. Determine weight concentration by comparing the weight of the dispersion to the original weight of the sample before evaporation.</li>
	</ol>
	</li>
	<li>Data Acquisition and Analysis
	<ol>
		<li>Use an appropriate objective lens of the desired magnification and aperture for excitation of the tracers and simultaneous fluorescence collection from the sample. In this work, use a 100X/1.3&#160;NA oil immersion objective lens.</li>
		<li>Place the moisture chamber onto a xyz-piezo table, which fits into a commercial microscope.</li>
		<li>To prevent the sample from drying, place a plasma cleaned cover slip into the moisture chamber and pipette 10 &#181;l of poly(NIPAM) dispersion of the desired concentration onto the slip.</li>
		<li>Depending on the excitation and emission spectra of the fluorescent dye, use a suitable laser for excitation and adjust the laser power appropriately. The intensity should be sufficiently low to avoid fast photobleaching of the dyes, but at the same time strong enough for accurate single particle positioning (see below). In this work, use a 561 nm diode-pumped solid-state laser and keep the laser power constant at 16 mW (ca. 0.5 kW cm-2 at the sample) for all the measurements.</li>
		<li>To obtain homogeneous sample illumination, use the critical illumination setup described here. For this, couple the laser into a multimode fiber (NA 0.22 &#177; 0.02, 0.6 mm core diameter), shake the fiber using a vortexer in order to temporally average out laser speckles, and project the fiber end into the sample plane.</li>
		<li>Calibrate the z distance from the back reflection of the cover slip and focus several micrometers into the sample by moving the objective slightly up and fix the z-position using a z-compensator. This avoids any interface effects with the coverslip.</li>
		<li>Adjust the detector parameters, such as exposure time, to the strength of the fluorescence signal. In this case, use an EMCCD camera with exposure time of 0.1 sec, electron multiplying mode and gain of 50.</li>
		<li>Acquire several movies with the appropriate number of frames to obtain adequate lag time to calculate the mean square displacement of the microgels in different regions of the sample. In this work, use acquisition frame numbers of 500 or 1,000 frames.</li>
		<li>Analyze the data by positioning the particles in each frame using Gaussian fitting 25 and use an appropriate particle tracking algorithm 26 to obtain the mean square displacement. 27 Calculate mean values and standard deviation by averaging over all the tracks in all the movies. Calculate the long lag time diffusion coefficients by linear regression from <img alt="Eq 19" src="/files/ftp_upload/54419/54419eq19.jpg" />, where&#160;<img alt="Eq 20" src="/files/ftp_upload/54419/54419eq20.jpg" /> is the mean square displacement, D the mean diffusion coefficient and &#964; the lag time.</li>
		<li>Estimate the anomaly parameter &#947; from the anomalous diffusion equation <img alt="Eq 21" src="/files/ftp_upload/54419/54419eq21.jpg" /> by transforming the data to logarithmic scale, yielding <img alt="Eq 22" src="/files/ftp_upload/54419/54419eq22.jpg" />. The anomaly parameter <img alt="Eq 23" src="/files/ftp_upload/54419/54419eq23.jpg" /> is given by the derivative of the plot. The derivative can be estimated by the finite differences of the data points, or fitting the data points by polynomial functions and differentiating analytically. Determine the sufficient degree of the polynomial fit functions by plotting the fit residuals and residual norm for increasing polynomial order.</li>
		<li>Repeat the same procedure for different concentrations of the microgel matrices.</li>
	</ol>
	</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdiffusion+and+the+cage+effect+studied+near+the+colloidal+glass+transition.">Subdiffusion and the cage effect studied near the colloidal glass transition.</a> Chem. Phys. 284 (1-2), 361-367 (2002).</li><li>Ernst, D., K&#246;hler, J., Weiss, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+type+of+anomalous+diffusion+with+single-particle+tracking.">Probing the type of anomalous diffusion with single-particle tracking.</a> Phys. Chem. Chem. Phys. 16 (17), 7686-7691 (2014).</li><li>. FitIt! (Version 1.1.4) Available from: <a target="_blank" href="https://www.github.com/ovirtanen/fitit">https://www.github.com/ovirtanen/fitit</a> (2015)</li><li>Provencher, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Constrained+Regularization+Method+For+Inverting+Data+Represented+By+A+Linear+Algebraic+or+Integral+Equations.">A Constrained Regularization Method For Inverting Data Represented By A Linear Algebraic or Integral Equations.</a> Comput. Phys. Commun. 27 (3), 213-227 (1982).</li><li>Holtzer, L., Meckel, T., Schmidt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanometric+three-dimensional+tracking+of+individual+quantum+dots+in+cells.">Nanometric three-dimensional tracking of individual quantum dots in cells.</a> Appl. Phys. Lett. 90 (5), 053902-053904 (2007).</li><li>Diezmann, A. V., Lee, M. Y., Lew, M. D., Moerner, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correcting+field-dependent+aberrations+with+nanoscale+accuracy+in+three-dimensional+single-molecule+localization+microscopy.">Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy.</a> Optica. 2 (11), 985-989 (2015).</li><li>Lindner, P., Zemb, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neutrons, X-rays and Light: Scattering Methods Applied to Soft Condensed Matter. , (2002).</li></ol>

The authors have nothing to disclose.

Addition of small amounts of functional comonomer can have a significant effect on the particle size and structure of the PNIPAM derived microgels. Simultaneous small-scale test tube polymerization is a good method to account for such changes, and helps to rapidly find the right reactant compositions for target particle size for upscaling the reaction as needed. The mass of the particles is approximately exponentially dependent on the ...

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels 1,2 have attracted continuous interest over the past two decades due to their potential in various smart applications. Demonstrated use cases include switchable emulsion stabilizers 3-8, microlenses 9, cell culture substrates for easy cell harvesting 10,11, and smart carriers for low molecular weight compounds and other biomedical uses 12. From a fundamental research point of view these particles have been proven to be useful for investigating subjects such as colloidal interactions 13-15 and polymer-solvent interactions 16-18.
Successful use of PNIPAM microgels and their derivatives in any given application typically requires knowledge on the mean particle size and width of the particle size distribution. For the correct interpretation of the experimental results involving PNIPAM microgels, the particle structure, which can be affected by functional comonomers, has to be known. Dynamic and static light scattering (DLS and SLS, respectively) are uniquely suited for acquiring this information because these methods are fast and relatively easy to use; and they probe the particle properties non-invasively in their native environment (dispersion). DLS and SLS also collect data from vast number of particles avoiding the bias arising from small sample sizes, typical for microscopy methods. Therefore, the first aim of this work is to introduce good practice regarding light scattering for practitioners new to colloidal characterization.
Typically, precipitation polymerization is carried out in laboratory scale and finding the right reaction conditions for specific particle properties can be laborious and require many repetitions of the synthesis. In contrast to large batch synthesis, non-stirred precipitation polymerization 19,20 is a rapid procedure in which batches of different reactant composition can be polymerized simultaneously yielding particles of narrow size distribution. Simultaneous polymerization minimizes experimental variation and large output means that right reaction conditions can be found fast for upscaling the reaction. Hence, our second aim is to demonstrate the usefulness of non-stirred precipitation polymerization in prototyping and in applications that do not require a large amount of product.
Different aspects of synthesis and characterization come together in the example of application of fluorescent labeled PNIPAM microgels in colloidal interaction research. Here we use highly accurate single particle tracking to investigate the diffusion of labeled tracer microgels in dispersion of unlabeled matrix microgels over a wide matrix concentration range and resolve the cage effect in concentrated colloidal dispersion. Wide-field fluorescence microscopy is well suited for this purpose as it can characterize the specific behavior of a few tracer molecules among a large number of potentially different matrix species. This is in contrast to techniques such as DLS, SLS and rheology, which measure the ensemble average properties of systems and therefore cannot resolve behavior of small number of probe particles in a large system. Furthermore, in this specific example conventional light scattering methods cannot be utilized also due to high particle concentration, which leads to strong multiple scattering invalidating any standard analysis. Use of automated data processing and statistical methods enable analysis of overall system behavior also for single particle tracking, when averaged over large sample sizes.

<table><tbody><tr><td>Acetone</td><td>VWR Chemicals</td><td>KRAF13455</td><td></td></tr><tr><td>Bisacrylamid</td><td>AppliChem</td><td>A3636</td><td></td></tr><tr><td>n-Hexane</td><td>Merck</td><td>104374</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Isopropylacrylamide</td><td>Fisher Scientific</td><td>AC412785000</td><td>recrystallized from n-hexane</td></tr><tr><td>Methacryloxyethyl thiocarbamoyl rhodamine B</td><td>Polysciences</td><td>23591</td><td></td></tr><tr><td>Potassium peroxodisulfate</td><td>Merck</td><td>105091</td><td></td></tr><tr><td>Silicone oil 47 V 350</td><td>VWR Chemicals</td><td>83851</td><td></td></tr><tr><td>Toluene</td><td>Sigma Aldrich</td><td>244511</td><td></td></tr><tr><td>F12 Refrigerated/heating circulator</td><td>Julabo</td><td>9116612</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>IX83</td><td></td></tr><tr><td>XY(Z) Piezo System</td><td>Physik Instrumente</td><td>P-545.3R7</td><td></td></tr><tr><td>100X Oil immersion objective</td><td>Olympus</td><td>UPLSAPO</td><td></td></tr><tr><td>QuadLine Beamsplitter</td><td>AHF Analysentechnik</td><td>F68-556T</td><td></td></tr><tr><td>Cobolt Jive 150 laser</td><td>Cobolt</td><td>0561-04-01-0150-300</td><td></td></tr><tr><td>Multimode Fiber</td><td>Thorlabs</td><td>UM22-600</td><td></td></tr><tr><td>iXON Ultra 897 EMCCD camera</td><td>Andor</td><td>DU-897U-CS0-BV</td><td></td></tr><tr><td>Laser goniometer</td><td>SLS Systemtechnik</td><td>Mark III</td><td></td></tr><tr><td>CF40 Cryo-compact circulator</td><td>Julabo</td><td>9400340</td><td></td></tr><tr><td>Laser goniometer system&amp;nbsp;</td><td>ALV GmbH</td><td>ALV / CGS-8F</td><td></td></tr><tr><td>Multi-tau corretator</td><td>ALV GmbH</td><td>ALV-7004</td><td></td></tr><tr><td>Light scattering electronics</td><td>ALV GmbH</td><td>ALV / LSE 5004</td><td></td></tr><tr><td>Photon counting module</td><td>PerkinElmer</td><td>SPCM-CD2969</td><td>2 units in pseudo cross-correlation mode</td></tr><tr><td>633 nm HeNe Laser</td><td>JDS Uniphase</td><td>1145P</td><td></td></tr><tr><td>F32 Refrigerated/heating circulator</td><td>Julabo</td><td>9312632</td><td></td></tr></tbody></table>

controlled synthesis and fluorescence tracking of highly uniform poly(n-isopropylacrylamide) microgels

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.

The number of PNIPAM microgel particles in the batch, and thus the final particle volume, is determined early in the reaction during the nucleation phase 20 Hydrophobic co-monomer dye methacryloxyethyl thiocarbamoyl rhodamine B influences the nucleation by reducing the particle number density in the batch. The decrease in particle concentration for two different initial NIPAM concentrations can be seen as increase in...

Watch this Scientific Journal Video about Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels at JoVE.com

Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this ...

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Research

JoVE Journal

Chemistry

10.2K Views.  RWTH Aachen University.  Niet-geroerde precipitatie polymerisatie geeft een snelle, reproduceerbare prototyping aanpak van de synthese van stimuli-gevoelige poly (N -isopropylacrylamide) microgels smalle grootteverdeling. In dit protocol synthese, lichtverstrooiing karakterisering en enkel deeltje fluorescentie volgen van deze microgels in een wide-field microscopie setup worden gedemonstreerd. 

Video: Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

Synthesis of Poly(N-isopropylacrylamide) Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this unique property, Janus microparticles have been recognized as a new class of materials, thereby attracting a great deal of attention from various research fields. The versatility of these microparticles has been exemplified through their uses as building blocks for self-assembly, electrically responsive actuators, emulsifiers for painting and cosmetics, and carriers for drug delivery. This study introduces a detailed protocol that explicitly describes a synthetic method for designing novel Janus microhydrogels composed of a single base material, poly(N-isopropylacrylamide) (PNIPAAm). Janus microdroplets are firstly generated via a hydrodynamic focusing microfluidic device (HFMD) based on the separation of a supersaturated aqueous NIPAAm monomer solution and subsequently polymerized through exposure to UV irradiation. The resulting Janus microhydrogels were found to be entirely composed of the same base material, featured an easily identifiable compartmentalized morphology, and exhibited anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability. We believe that the proposed method introduces a novel hydrogel platform with the potential for advanced synthesis of multi-functional Janus microhydrogels.

Synthesis of PolyN-isopropylacrylamide Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

We present a protocol to synthesize Janus microhydrogels composed entirely of the same base material, poly(N-isopropylacrylamide) (PNIPAAm), with a clearly compartmentalized structure base on the phase separation of a supersaturated NIPAAm monomer solution. The synthesized Janus microhydrogels show unique properties such as anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability.

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this ...

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and efficient synthesis of well-controlled fluorescent statistical glycopolymers using reversible addition-fragmentation chain-transfer (RAFT)-based polymerization is demonstrated. The synthesis starts with the preparation of &#946;-galactose-containing glycomonomer 2-lactobionamidoethyl methacrylamide obtained by reaction of lactobionolactone and N-(2-aminoethyl) methacrylamide (AEMA). 2-Gluconamidoethyl methacrylamide (GAEMA) is used as a structural analog lacking a terminal &#946;-galactoside. The following RAFT-mediated copolymerization reaction involves three different monomers: N-(2-hydroxyethyl) acrylamide as spacer, AEMA as target for further fluorescence labeling, and the glycomonomers. Tolerant of aqueous systems, the RAFT agent used in the reaction is (4-cyanopentanoic acid)-4-dithiobenzoate. Low dispersities (&#8804;1.32), predictable copolymer compositions, and high reproducibility of the polymerizations were observed among the products. Fluorescent polymers are obtained by modifying the glycopolymers with carboxyfluorescein succinimidyl ester targeting the primary amine functional groups on AEMA. Lectin-binding specificities of the resulting glycopolymers are verified by testing with corresponding agarose beads coated with specific glycoepitope recognizing lectins. Because of the ease of the synthesis, the tight control of the product compositions and the good reproducibility of the reaction, this protocol can be translated towards preparation of other RAFT-based glycopolymers with specific structures and compositions, as desired.

An efficient, three-step synthesis of RAFT-based fluorescent glycopolymers, consisting of glycomonomer preparation, copolymerization, and post-modification, is demonstrated. This protocol can be used to prepare RAFT-based statistical glycopolymers with desired structures.

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

Bioengineering

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...

Synthesis of Stimuli-responsive Nanogels using Aqueous One-step Crosslinking and Co-nanopolymerization

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to their versatile bottom-up synthesis and biocompatibility. While various methods have been employed for nanogel synthesis to date, very few have achieved it without the use of harsh organic solvents or high temperatures that can damage the integrity of the biological payload. In contrast, the methodology presented here accomplishes the synthesis of sub-100 nm sized, protein-loaded nanogels using mild reaction conditions. Here, we present a method for the non-covalent encapsulation of protein-based payloads within nano-gels that were synthesized using an aqueous-based, single-step, crosslinking copolymerization technique. In this technique, we initially electrostatically bind a protein-based payload to a cationic quaternary ammonium monomer and simultaneously cross-link and co-polymerize it using ammonium persulfate and N,N,N&#39;,N&#39;-tetramethylethylenediamine to form nanogels that entrap the protein payload. The size and polydispersity index of the nanogels is determined using dynamic light scattering (DLS), while the surface morphology is assessed by transmission electron microscopy (TEM). The mass of protein entrapped within nanogels is determined by calculating the encapsulation efficiency. Furthermore, the controlled-release ability of the nanogels via the gradual degradation of redox-responsive structural elements is also assessed in bioreduction assays. We provide examples of nanoparticle optimization data to demonstrate all caveats of nanogel synthesis and characterization using this technique. In general, uniformly sized nanogels were obtained with an average size of 57 nm and a polydispersity index value of 0.093. A high encapsulation efficiency of 76% was achieved. Furthermore, the nanogels exhibited controlled release of up to 86% of the encapsulated protein by gradual degradation of novel redox-responsive components in the presence of glutathione over 48 h.

Nanogels are an excellent and versatile nanoparticle platform for the delivery of biologics. Stimuli-responsive poly(ethylene) glycol-based polymeric nano-gels, capable of encapsulating protein-based payloads, were synthesized using a one-step cross-linking co-nanopolymerization strategy in aqueous conditions. The optimal fabrication and characterization of these novel nanoparticles are presented here.

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold

The microporous annealed particle (MAP) scaffold platform is a subclass of granular hydrogels. It is composed of an injectable slurry of microgels that can form a structurally stable scaffold with cell-scale porosity in situ following a secondary light-based chemical crosslinking step (i.e.,&#160;annealing). MAP scaffold has shown success in a variety of regenerative medicine applications, including dermal wound healing, vocal fold augmentation, and stem cell delivery. This paper describes the methods for synthesis and characterization of poly(ethylene glycol) (PEG) microgels as the building blocks to form a MAP scaffold. These methods include the synthesis of a custom annealing macromer (MethMAL), determination of microgel precursor gelation kinetics, microfluidic device fabrication, microfluidic generation of microgels, microgel purification, and basic scaffold characterization, including microgel sizing and scaffold annealing. Specifically, the high-throughput microfluidic methods described herein can produce large volumes of microgels that can be used to generate MAP scaffolds for any desired application, especially in the field of regenerative medicine.

This protocol describes a set of methods for synthesizing the microgel building blocks for microporous annealed particle scaffold, which can be used for a variety of regenerative medicine applications.

The microporous annealed particle (MAP) scaffold platform is a subclass of granular hydrogels. It is composed of an injectable slurry of microgels that ...

Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo tissue repair. In these granular scaffolds, the innate porosity generated by the void space between microgels enables cell infiltration and migration. Controlling the void fraction and particle fraction is critical for MAP scaffold design, as porosity is a bioactive cue for cells. Spherical microgels can be generated on a microfluidic device for controlled size and shape and subsequently freeze-dried using methods that prevent fracturing of the polymer network. Upon rehydration, the lyophilized microgels lead to controlled particle fractions in MAP scaffolds. The implementation of these methods for microgel lyophilization has led to reproducible studies showing the effect of particle fraction on macromolecule diffusion and cell spreading. The following protocol will cover the fabrication, lyophilization, and rehydration of microgels for controlling particle fraction in MAP scaffolds, as well as annealing the microgels through bio-orthogonal crosslinking for 3D cell culture in vitro.

Minimizing the variability in the particle fraction within granular scaffolds facilitates reproducible experimentation. This work describes methods for generating granular scaffolds with controlled particle fractions for in vitro tissue engineering applications.

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo ...

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

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