1. Microgel Synthesis
NOTE: N-isopropylacrylamide (NIPAM) was recrystallized from n-hexane. Other reagents were used as received.
2. Light Scattering Characterization
3. Particle Tracking by Wide-field Fluorescence Microscopy
NOTE: Tracer and matrix particles of 465 ± 7 nm and 405 ± 7 nm hydrodynamic radii at 20 °C, respectively, were used for particle tracking.
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 the mean final particle volume in the collapsed state with increasing dye concentration, shown in Figure 1. The increase in volume can be attributed to the hydrophobic comonomer dye, which promotes microgel nuclei aggregation at early reaction times, decreasing the particle concentration and increasing the final particle volume.
Results from a successful DLS measurements are shown in Figure 2. For the six smallest final particle volume batches linear dependence of the mean decay rate Γ2 on q2 and zero y-intercept within the error indicate that the particle size distributions for these batches are relatively narrow and a well-defined estimate for the mean diffusion coefficient can be obtained from the slope of the linear fit. Figure 3 shows a more complicated result from the two largest volume batches, where Γ2 deviates from the linear behavior in the intermediate q range. The non-linearity originates from the form factor (angular scattering pattern) minimum which coincides with these q values. 28 The phenomenon in question can be observed for particles with dimensions comparable to the wavelength of the incident laser radiation and even a moderate particle size distribution width. Determination of diffusion coefficient in this q range results in strongly biased results and therefore poses a notable risk of mischaracterization. In the specific case of Figure 3, Γ2 reflects the mean behavior again at high q, where all particle size fractions contribute more evenly to the total scattered intensity. A straightforward way to obtain a reasonable estimate for the mean diffusion coefficient is to exclude the intermediate Γ2 values from the linear fit. If the form factor of the particles are known, a more elaborate fitting method can be used 28.
Determining the hydrodynamic volume in the collapsed state without letting the samples cool down below the PNIPAM VPTT ensures that the non-gelled sol fraction has not detached from the particles. Therefore the volume in the collapsed state reflects the mass and the number of the particles during the polymerization, which is important if the fundamental properties of the precipitation polymerization are being investigated 20. Volume in the collapsed state also provides a good quantity for comparing different reaction parameters, because it is independent of the swelling properties and the fraction of non-gelled polymer in the particles regulated by the amount of cross-linker in the monomer mixture. Smaller size and higher scattering contrast in the collapsed state also facilitate the DLS characterization.
Static light scattering data measured at two wavelengths of 642 nm and 404 nm for the matrix and tracer particles are shown in Figure 4. Visual inspection of the angular scattering patterns reveals that the particles are well-defined: Multiple distinguished oscillations typical for spherical particles throughout the q indicates narrow polydispersity, in this case 7% and 6% for the tracer and matrix microgels, respectively. Smooth behavior at low q indicates that the samples are sufficiently diluted and no significant particle aggregation is present. The increase in scattered intensity at extreme q can be attributed to the scattering due to the back reflected beam from the inner cuvette wall. Inversion of the form factors of the matrix particles confirm typical microgel structure 29 with dense core and radially decaying density profile resulting from cross-linker copolymerization kinetics 30 (see inset). The dashed line shows the form factor of the reference hard sphere with the same mean radius of gyration as the matrix particles. The experimental form factor decays faster with q than the hard sphere form factor, which is typical for particles with fuzzy surface. In contrast, tracer particles exhibit unconventional microgel structure. This can be also seen from the reference hard sphere form factor, which shows that the experimental form factor initially does not decay faster than the reference. This result shows that the incorporating dye molecules to microgels can affect their structure, which must be accounted for in interpretation of the results.
The high uniformity of the synthesized particles is of high interest for studies of their diffusion at volume fractions around the glass transition temperature in order to accurately determine the evolution behavior in this regime 13, and compare it to hard particles 31. Therefore, a low fraction of labeled microgels were mixed with non-labeled microgel of comparable size. Excitation and emission spectra of microgel-incorporated dye molecules along with the excitation wavelength and filter configuration used in the emission path are presented in Figure 5. Absorbance and emission maxima of methacryloxyethyl thiocarbamoyl rhodamine B are close to excitation wavelength and fluorescence collection range, respectively, enabling high collection efficiency in the particle tracking setup. The time evolution of mean square displacement for tracer microgels in various non-labeled microgel matrix concentrations are shown in Figure 6 and Figure 7 in linear and logarithmic scale, respectively. At low microgel matrix concentrations the tracer particles diffuse rapidly. Even though they are only visible for a limited number of frames before moving out of the focus plane, a reasonably good estimation of their mean square displacements is possible. The linear increase of the mean square displacement with time indicates normal diffusion behavior for all lag times measured. However, for microgel concentrations close to the colloidal glass transition, i.e., 29-36 mg/ml, the temporal evolution of the mean square displacements becomes non-linear (see Figure 7). The behavior resembles the one of colloidal micrometer-sized PMMA particles as described by Weeks and Weitz 31 and can be related to the cage effect. As shown schematically in Figure 10, a labeled microgel in a dense matrix can diffuse rather freely within the cage. For that reason, the mean square displacement increases linearly in the first few milliseconds. However, since particles are trapped in transient cages formed by their neighbors, a collective rearrangement of the surrounding microgels is necessary for microgels to move further. This cage effect expresses itself in a rather shallow slope in the second range of Figure 7, and can be also confirmed by inspecting the particle tracks in Figure 9. At short lag times the particles jiggle in their cages, from which they escape just to get trapped again. At long lag times, the linear diffusion behavior is recovered. Cage effects can be analyzed using anomalous diffusion models where the temporal evolution of the (two-dimensionally detected) mean square displacement is expressed by a power law in time: or in its logarithmic form with the anomaly parameter 32. For normal diffusion, the anomaly parameter equals 1, subdiffusion is represented by values below 1. Figure 8 presents the temporal evolution of the anomaly parameter directly determined from the slope in the log-log-plot Figure 7. For the lower concentrations of microgels in our study, the anomaly parameter basically equals to 1. For lag times in the range of several seconds, the factor deviates from 1 towards lower values. This behavior is an artifact due to fact that the axial (z-)observation range in wide-field microscopy is limited to only a few micrometers. The narrow z-range biases the analysis for fast diffusion at long time intervals for rapidly diffusing tracers at low matrix concentrations. When increasing the microgel concentration, we find that the minimum of the anomaly parameter becomes much more pronounced and the transition to normal diffusion () appears later. This is a clear indication of the cage effect appearing for dense microgels systems when approaching their glass transition regime.
Figure 1: Single particle volume in collapsed state with initial dye concentration in the batch. Two different initial NIPAM concentrations were used, 57.5 mmol dm-3 (black circles) and 28.8 mmol dm-3 (grey rectangles). 1 mol-% of cross-linker was used. Initial KPS concentration was the same in all the batches at 1.56 mmol dm-3. Error bars denote the standard deviation. Please click here to view a larger version of this figure.
Figure 2: Decay rate with the square of the scattering vector magnitude for the four smallest volume microgel batches. Linear dependence of on q2 and zero intercept indicate narrow particle size distribution and indicates that well-defined estimate of the mean diffusion coefficient can be calculated from the slope of the linear fit. NIPAM concentrations were 57.5 mmol dm-3 (red squares and orange inverted triangles) and 28.8 mmol dm-3 (rest of the symbols). Dye concentrations were 0.044 mmol dm-3 (red squares), 0.022 mmol dm-3 (orange inverted triangles), 0.088 mmol dm-3 (green triangles), 0.066 mmol dm-3 (cyan rhombuses), 0.044 mmol dm-3 (dark blue triangles), and 0.022 mmol dm-3 (pink circles). Please click here to view a larger version of this figure.
Figure 3: Decay rate with the square of the scattering vector magnitude for the two largest volume batches. Non-linear behavior of Γ2 with q2 in the central q range is caused by the changes in the intensity weighting of signal by different size fractions in the vicinity of the form factor minimum. NIPAM concentration in the both batches were 57.5 mmol dm-3, the dye concentrations were 0.088 mmol dm-3 (black circles) and 0.066 mmol dm-3 (red triangles). Faded symbols were excluded from the linear fit. Please click here to view a larger version of this figure.
Figure 4: Form factors of the labeled tracer and unlabeled matrix particles. For both particles the form factor was measured at two wavelengths, 642 nm (light blue and red data points) and 404 nm (green and dark blue data points). Solid lines are global fits to the 642 nm and 404 nm datasets. Dashed lines show form factors of hard sphere reference particles with the same radii of gyration as matrix and tracer particles (orange and green dashed lines, respectively.) Insets show normalized particle density profiles from the core to the surface calculated, e.g., FitIt! Please click here to view a larger version of this figure.
Figure 5: Excitation and emission spectra of fluorescence labeled microgel particles. Blue line denotes the excitation and red line emission spectrum. Solid vertical line is the excitation wavelength. Shaded area denotes fluorescence collection wavelength range. Please click here to view a larger version of this figure.
Figure 6: Mean square displacement with lag time for the tracer particles. Unlabeled matrix microgel concentrations were 15.56 mg/ml (left), 22.05 mg/ml, 28.28 mg/ml, 28.67 mg/ml, 30.32 mg/ml, 31.13 mg/ml and 35.35 mg/ml. Points and error bars denote experimental values and standard deviation, respectively. Solid lines are linear fits to the data points. Inset shows a wide-field fluorescence micrograph of tracer microgels at 35.35 mg/ml matrix concentration. Please click here to view a larger version of this figure.
Figure 7: Mean square displacement with lag time for the tracer particles in logarithmic scale. Unlabeled matrix microgel concentrations were 15.56 mg/ml (left), 22.05 mg/ml, 28.28 mg/ml, 28.67 mg/ml, 30.32 mg/ml, 31.13 mg/ml and 35.35 mg/ml. Points and error bars denote experimental values and standard deviation, respectively. Solid lines are polynomial fits to the data points. Please click here to view a larger version of this figure.
Figure 8: Anomaly parameters with lag time for tracer particles. Unlabeled matrix microgel concentrations were 15.56 mg/ml (left), 22.05 mg/ml, 28.28 mg/ml, 28.67 mg/ml, 30.32 mg/ml, 31.13 mg/ml and 35.35 mg/ml. Points represent derivatives estimated by finite differences and solid lines analytically calculated derivatives from the polynomial fits in Figure 7. Please click here to view a larger version of this figure.
Figure 9: Particle tracks for 12 tracer microgels in dispersion with 35.35 mg/ml matrix concentration. Clustering of tracks to distinctive blobs results from the tracer's particles being trapped in transient cages formed by their unlabeled neighbors. Please click here to view a larger version of this figure.
Figure 10: Schematic illustration of tracer microgel diffusion in concentrated unlabeled matrix microgel dispersion. Red trajectory denotes rapid diffusion of the tracers within the transient cages (blue dashed line) formed by the neighboring particles. Blue trajectory denotes long lag time diffusion enabled by the collective rearrangement of the transient cages. Please click here to view a larger version of this figure.
Figure 11: Long lag time diffusion coefficients with unlabeled matrix microgel concentration. At low matrix concentration the diffusion of the tracer microgels is not affected by the matrix particles. With increasing matrix microgel concentration the long time diffusion slows down orders of magnitude because diffusion requires collective rearrangement of the transient cages, where the tracers are trapped. Please click here to view a larger version of this figure.
Acetone | VWR Chemicals | KRAF13455 | |
Bisacrylamid | AppliChem | A3636 | |
n-Hexane | Merck | 104374 | |
N-Isopropylacrylamide | Fisher Scientific | AC412785000 | recrystallized from n-hexane |
Methacryloxyethyl thiocarbamoyl rhodamine B | Polysciences | 23591 | |
Potassium peroxodisulfate | Merck | 105091 | |
Silicone oil 47 V 350 | VWR Chemicals | 83851 | |
Toluene | Sigma Aldrich | 244511 | |
F12 Refrigerated/heating circulator | Julabo | 9116612 | |
Microscope | Olympus | IX83 | |
XY(Z) Piezo System | Physik Instrumente | P-545.3R7 | |
100x Oil immersion objective | Olympus | UPLSAPO | |
QuadLine Beamsplitter | AHF Analysentechnik | F68-556T | |
Cobolt Jive 150 laser | Cobolt | 0561-04-01-0150-300 | |
Multimode Fiber | Thorlabs | UM22-600 | |
iXON Ultra 897 EMCCD camera | Andor | DU-897U-CS0-BV | |
Laser goniometer | SLS Systemtechnik | Mark III | |
CF40 Cryo-compact circulator | Julabo | 9400340 | |
Laser goniometer system | ALV GmbH | ALV / CGS-8F | |
Multi-tau corretator | ALV GmbH | ALV-7004 | |
Light scattering electronics | ALV GmbH | ALV / LSE 5004 | |
Photon counting module | PerkinElmer | SPCM-CD2969 | 2 units in pseudo cross-correlation mode |
633 nm HeNe Laser | JDS Uniphase | 1145P | |
F32 Refrigerated/heating circulator | Julabo | 9312632 |
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.
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.
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.