This work has been financially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (No. 25248027 to M.S.).

1. Sample Preparation
<ol>
	<li>Purification of temperature-responsive monomers
	<ol>
		<li>Dissolve 20 g of N-isopropylacrylamide (NIPA) in 100 mL of toluene.</li>
		<li>Filter the solution under suction to eliminate dust.</li>
		<li>Mix the filtrate with 500 mL of petroleum ether.</li>
		<li>Place the reaction vessel in an ice-water bath.</li>
		<li>Stir the solution until the monomers are precipitated (typically 30 min).</li>
		<li>Filter the solution under suction to obtain the precipitated monomers.</li>
		<li>Dry the monomers under reduced pressure (100 Pa) overnight.</li>
	</ol>
	</li>
	<li>Preparation of the temperature-responsive polymer solution
	<ol>
		<li>Degas 20 mL of deionized water for 1.0 min using a diaphragm pump.</li>
		<li>Dissolve 780.8 mg of the purified NIPA in 9.5 mL of degassed and deionized water.</li>
		<li>Place the reaction vessel in an ice-water bath.</li>
		<li>Shield the reaction from light by covering the apparatus with aluminum foil.</li>
		<li>Stir the solution gently for 10 min while introducing a moderate flow of Ar gas gently via a pipette tip attached to the gas cylinder with a tube.</li>
		<li>Add 11.9 &#181;L of N,N,N&#39;,N&#39;-tetramethylethylenediamine to the solution via a micropipette.</li>
		<li>Stir the solution for 1.0 min while introducing Ar gas, as mentioned in step 1.2.5.</li>
		<li>While stirring the sample, dissolve 4.0 mg of ammonium persulfate in 0.5 mL of degassed and deionized water.</li>
		<li>Mix the sample solution (from step 1.2.7) and ammonium persulfate solution (from step 1.2.8).</li>
		<li>Stir the solution for 30 s while introducing Ar gas, as mentioned in step 1.2.5.</li>
		<li>Cover the solution with aluminum foil and keep it in a refrigerator (4 &#176;C) overnight.</li>
	</ol>
	</li>
	<li>Preparation of sample mounts
	<ol>
		<li>Place 60 &#181;L of the sample solution (from step 1.2.11) in a cavity slide.</li>
		<li>Cover the solution with a circular cover glass. Be careful not to trap air bubbles.</li>
		<li>Remove excess solution using a micropipette and laboratory wipes.</li>
		<li>Seal the sample with glue. Let the glue dry at room temperature (typically 6 h).</li>
		<li>Prepare another slide filled with 0.1 wt% polystyrene latex (100-nm particle diameter) suspension by following steps 1.3.1-1.3.4. This slide is used as a standard.</li>
	</ol>
	</li>
</ol>
2. Particle Size Measurement with a Dynamic Light Scattering Microscope
<ol>
	<li>Optimization of the instrument
	<ol>
		<li>Place the polystyrene latex suspension slide (from step 1.3.5) on the stage of the inverted microscope. The cover glass side should face downward.</li>
		<li>Place a beam damper in front of the detector (an avalanche photodiode and an autocorrelator).</li>
		<li>Apply a laser beam (solid-state laser, &#955; = 488 nm, 30 mW, continuous wave) to the sample through an objective lens (10&#215;). A portion of the reflected light goes through a launch mirror of the microscope and is observed by a CCD camera mounted at the side port of the microscope (Figure 1).</li>
		<li>Adjust the height of objective lens to set the focal point at the sample suspension by shifting the height of objective lens from the low-to-high position. During this procedure, the reflected image is focused three times: at the surface of the cover glass, at the interface between the cover glass and the sample, and at the interface between the sample and the hole-slide glass. Set the focal point between the second and third points.</li>
		<li>Attenuate the scattered light intensity by changing the laser power.</li>
		<li>Introduce the scattered light into the detector by removing the beam damper in front of the detector. This unit measures the time correlation of the light intensity.</li>
		<li>Set a pinhole (&#981; = 50 &#181;m) between the microscope and the detector to achieve the confocal effect. Adjust the position of the pinhole to maximize the light intensity at the detector.</li>
		<li>Measure the time correlation function of scattered light intensity for 30 s by initiating the operation of the correlator via a computer. The measured correlation function is often expressed as g(2) (t) - 1, where t is the correlation time4 and <img alt="Equation 1" src="/files/ftp_upload/54885/54885eq1.jpg" />. Here, I(t) is the scattered light intensity at the time t and (&#8226;&#8226;&#8226;)T is time averaging. The decay time will be approximately 0.1 ms.</li>
		<li>Adjust the focal point to obtain a wide range for the initial amplitude of the time correlation function (g(2) (t = 0) - 1). 
		NOTE: The initial amplitude is strongly affected by the amount of reflected light. By moving the focal point toward the interface between the cover glass and the sample, the amount of reflected light increases. For strong light scatterers, such as polystyrene latex, the initial amplitude can be altered from 0 to 1. However, it is difficult to set the initial amplitude close to 1 for more common polymer solutions because the intensity of the reflected light is much higher than that of the scattered light.</li>
		<li>Apply the inverse Laplace transformation (using the constrained regularization program CONTIN13,14) to the obtained time correlation function to acquire the size distribution function. In cases where the initial amplitude is set to less than 0.2, the distribution function of the hydrodynamic radius will show a sharp peak around 100 nm, which is twice the actual radius (see the discussion for details).</li>
	</ol>
	</li>
	<li>Sample measurement
	<ol>
		<li>Set the stage temperature to 25 &#176;C.</li>
		<li>Place a slide prepared with poly-NIPA (PNIPA) solution (step 1.3.4) on the stage of the microscope.</li>
		<li>Measure the time correlation function of the scattered light intensity by following steps 2.1.4-2.1.8. If the initial amplitude is larger than 0.2, adjust the focal point to make the initial amplitude of the time correlation function less than 0.2 by following step 2.1.9. A small initial amplitude simplifies the analysis.</li>
		<li>Set the stage temperature to 35 &#176;C and wait until the solution turns turbid. The lower critical solution temperature (LCST) of PNIPA solution is 32 &#176;C15.</li>
		<li>Measure the time correlation function by following steps 2.1.4-2.1.8. If feasible, adjust the position of the focal point to make the initial amplitude of the time correlation function less than 0.2. For turbid solutions, their initial amplitudes tend to increase, since the intensity of the scattered light increases while that of the reflected light remains constant.</li>
		<li>Apply the inverse Laplace transformation to the obtained time correlation functions to obtain the size distribution functions. Note that the actual size is half the obtained value in cases where the initial amplitude is less than 0.2.</li>
	</ol>
	</li>
</ol>

<ol><li>Hiroi, T., Shibayama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+Light+Scattering+Microscope%3A+Accessing+Opaque+Samples+with+High+Spatial+Resolution%5BTitle%5D)+AND+%22Opt.+Express%22%5BJournal%5D)">Dynamic Light Scattering Microscope: Accessing Opaque Samples with High Spatial Resolution.</a> Opt. Express. 21, 20260-20267 (2013).</li>
<li>Barth, H. G., Flippen, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Particle+Size+Analysis%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">Particle Size Analysis.</a> Anal. Chem. 67, 257-272 (1995).</li>
<li>Liu, Y., Wang, Z., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+supramolecular+polymers%5BTitle%5D)+AND+%22Chem.+Soc.+Rev%22%5BJournal%5D)">Characterization of supramolecular polymers.</a> Chem. Soc. Rev. 41, 5922-5932 (2012).</li>
<li>Berne, B. J., Pecora, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBerne%2C+BJ+%22Dynamic+Light+Scattering+with+Applications+to+Chemistry%2C+Biology+and+Physics%22">Dynamic Light Scattering with Applications to Chemistry, Biology and Physics</a>. , Dover Publications, Inc. (2000).</li>
<li>Phillies, G. D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+demonstration+of+ruultiple-scattering+suppression+in+quasielastic-light-scattering+spectroscopy+by+homodyne+coincidence+techniques%5BTitle%5D)+AND+%22Phys.+Rev.+A%22%5BJournal%5D)">Experimental demonstration of ruultiple-scattering suppression in quasielastic-light-scattering spectroscopy by homodyne coincidence techniques.</a> Phys. Rev. A. 24, 1939-1943 (1981).</li>
<li>Phillies, G. D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Suppression+of+multiple+scattering+effects+in+quasielastic+light+scattering+by+homodyne+crosscorrelation+techniques%5BTitle%5D)+AND+%22J.+Chem.+Phys%22%5BJournal%5D)">Suppression of multiple scattering effects in quasielastic light scattering by homodyne crosscorrelation techniques.</a> J. Chem. Phys. 74, 260-262 (1981).</li>
<li>Ishii, K., Yoshida, R., Iwai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-scattering+spectroscopy+for+extremely+dense+colloidal+suspensions+by+use+of+a+low-coherence+interferometer%5BTitle%5D)+AND+%22Opt.+Lett%22%5BJournal%5D)">Single-scattering spectroscopy for extremely dense colloidal suspensions by use of a low-coherence interferometer.</a> Opt. Lett. 30, 555-557 (2005).</li>
<li>Xia, H., Ishi, K., Iwai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrodynamic+Radius+Sizing+of+Nanoparticles+in+Dense+Polydisperse+Media+by+Low-Coherence+Dynamic+Light+Scattering%5BTitle%5D)+AND+%22Jpn.+J.+Appl.+Phys%22%5BJournal%5D)">Hydrodynamic Radius Sizing of Nanoparticles in Dense Polydisperse Media by Low-Coherence Dynamic Light Scattering.</a> Jpn. J. Appl. Phys. 44, 6261-6264 (2005).</li>
<li>Maret, G., Wolf, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+light+scattering+from+disordered+media.+The+effect+of+brownian+motion+of+scatterers%5BTitle%5D)+AND+%22Z.+Phys.+B%22%5BJournal%5D)">Multiple light scattering from disordered media. The effect of brownian motion of scatterers.</a> Z. Phys. B. 65, 409-413 (1987).</li>
<li>Pine, D. J., Weitz, D. A., Chaikin, P. M., Herbolzheimer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diffusing+wave+spectroscopy%5BTitle%5D)+AND+%22Phys.+Rev.+Lett%22%5BJournal%5D)">Diffusing wave spectroscopy.</a> Phys. Rev. Lett. 60, 1134-1137 (1988).</li>
<li>Cerbino, R., Trappe, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+Dynamic+Microscopy%3A+ProbingWave+Vector+Dependent+Dynamics+with+a+Microscope%5BTitle%5D)+AND+%22Phys.+Rev.+Lett%22%5BJournal%5D)">Differential Dynamic Microscopy: ProbingWave Vector Dependent Dynamics with a Microscope.</a> Phys. Rev. Lett. 108, 188102(2012).</li>
<li>Lu, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterizing+Concentrated%2C+Multiply+Scattering%2C+and+Actively+Driven+Fluorescent+Systems+with+Confocal+Differential+Dynamic+Microscopy%5BTitle%5D)+AND+%22Phys.+Rev.+Lett%22%5BJournal%5D)">Characterizing Concentrated, Multiply Scattering, and Actively Driven Fluorescent Systems with Confocal Differential Dynamic Microscopy.</a> Phys. Rev. Lett. 108, 218103(2012).</li>
<li>Provencher, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+constrained+regularization+method+for+investing+data+represented+by+linear+algebraic+or+integral+equations%5BTitle%5D)+AND+%22Comp.+Phys.+Comm%22%5BJournal%5D)">A constrained regularization method for investing data represented by linear algebraic or integral equations.</a> Comp. Phys. Comm. 27, 213-227 (1982).</li>
<li>Provencher, S. W., Stepanek, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+analysis+of+dynamic+light+scattering+autocorrelation+functions%5BTitle%5D)+AND+%22Part.+Part.+Syst.+Charact%22%5BJournal%5D)">Global analysis of dynamic light scattering autocorrelation functions.</a> Part. Part. Syst. Charact. 13, 291(1996).</li>
<li>Takata, S., Norisuye, T., Shibayama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small-angle+Neutron+Scattering+Study+on+Preparation+Temperature+Dependence+of+Thermosensitive+Gels%5BTitle%5D)+AND+%22Macromolecules%22%5BJournal%5D)">Small-angle Neutron Scattering Study on Preparation Temperature Dependence of Thermosensitive Gels.</a> Macromolecules. 35, 4779-4784 (2002).</li>
<li>Pusey, P. N., van Megen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+Light+Scattering+by+Non-Ergodic+Media%5BTitle%5D)+AND+%22Physica+A%22%5BJournal%5D)">Dynamic Light Scattering by Non-Ergodic Media.</a> Physica A. 157, 705-741 (1989).</li>
<li>Chu, B. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aChu%2C+B+%22Laser+Light+Scattering.+2nd+Ed%22">Laser Light Scattering. 2nd Ed</a>. , Academic Press. (1991).</li>
<li>Shibayama, M., Tanaka, T., Han, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small-Angle+Neutron-Scattering+Study+on+Poly%28N-Isopropyl+Acrylamide%29+Gels+near+Their+Volume-Phase+Transition-Temperature%5BTitle%5D)+AND+%22J.+Chem.+Phys%22%5BJournal%5D)">Small-Angle Neutron-Scattering Study on Poly(N-Isopropyl Acrylamide) Gels near Their Volume-Phase Transition-Temperature.</a> J. Chem. Phys. 97, 6829-6841 (1992).</li>
<li>Tanaka, T., Sato, E., Hirokawa, Y., Hirotsu, S., Peetermans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Critical+Kinetics+of+Volume+Phase+Transition+of+Gels%5BTitle%5D)+AND+%22Phys.+Rev.+Lett%22%5BJournal%5D)">Critical Kinetics of Volume Phase Transition of Gels.</a> Phys. Rev. Lett. 55, 2455-2458 (1985).</li>
<li>Hiroi, T., Ata, S., Shibayama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transitions+of+Aggregation+States+for+Concentrated+Carbon+Nanotube+Dispersion%5BTitle%5D)+AND+%22J.+Phys.+Chem.+C%22%5BJournal%5D)">Transitions of Aggregation States for Concentrated Carbon Nanotube Dispersion.</a> J. Phys. Chem. C. 120, 5776-5782 (2016).</li>
</ol>

The authors have nothing to disclose.

The initial amplitude of the time correlation function heavily depends on the focal point, as shown in Figure 2(a). This seemingly contradicts the fact that the solution is homogeneous (except for the thin layer at the interface)8. This variation in the initial amplitude is attributed to a variation in the amount of reflected light. Partial heterodyne theory16 predicts that the initial amplitude, A, the scattered light Intensity, Is, and the reflected light intensity, Ir, satisfy the following equation1
<img alt="Equation 2" src="/files/ftp_upload/54885/54885eq2.jpg" />
This equation shows that the larger Ir becomes, the smaller A becomes. Therefore, A is reduced by setting the focal position close to the interface. The apparent diffusion constant DA can be obtained by fitting the time correlation function in the case of monodisperse solutions:
<img alt="Equation 3" src="/files/ftp_upload/54885/54885eq3.jpg" />
where <img alt="Equation 4" src="/files/ftp_upload/54885/54885eq4.jpg" />. Here, n is the refractive index of the solvent (water, 1.33), &#952; is the scattered angle (180&#176;), and&#160;&#955; is the wavelength of light (514.5 nm). Since we applied backscattering geometry, the value of q is fixed. However, this point is solved by using different wavelengths of light. Please note that any kind of continuous-wave laser source is available to construct the DLS microscope. Thanks to the small irradiated volume, the coherence factor17 is estimated to be more than 0.99 and is negligible. For polydisperse solutions, the distribution function of DA is obtained by the inverse Laplace transformation. Partial heterodyne theory also predicts that DA is not the same as the actual diffusion constant D. These two diffusion constants satisfy the following equation:
<img alt="Equation 5" src="/files/ftp_upload/54885/54885eq5.jpg" />
The diffusion constant D is converted into the hydrodynamic radius Rh using the Einstein-Stokes equation4. When A = 1, this relationship becomes DA = D. In this case, the data conversion process is the same as that for the common dynamic light scattering. The red line shown in Figure 2(b) corresponds to this case. In contrast, this relationship becomes DA = 0.5D at the limit of A &#8594; 0. Therefore, the size is estimated to be twice as large as the actual size when A is small (practically, less than 0.2), as shown by the blue line of Figure 2(b). If we know that A is significantly small, the horizontal axis can be shifted, as shown in Figure 2(c). In principle, we can convert DA into D for any value of A. In practice, however, it is better to set the initial amplitude smaller than 0.2, since the simple approximation DA &#8764; 0.5D holds true.
The prominent features of the dynamic light scattering microscope technique were demonstrated using a PNIPA solution. The conformation of PNIPA below and above the LCST has been extensively studied using small-angle neutron scattering15,18. In contrast, dynamic light scattering has not been utilized for the analysis of PNIPA above the LCST because of its turbidity19. This problem is solved by the dynamic light scattering microscope, as shown in Figures 3(a) and (b). The size of these aggregates is several &#181;m, which cannot be obtained by either small-angle X-ray/neutron scattering or conventional light scattering techniques. Time-resolved measurements using this system give information on the aggregation process during the temperature change.
The drawback of the dynamic light scattering microscope is also illustrated in Figure 3. For the result below the LCST, the time correlation function is strongly affected by the very small amount of dust present (the black lines in Figure 3). For example, the time correlation function does not decay completely, even with correlation times in the order of 1.0 s. This is because the volume irradiated with this apparatus (approximately 1.0 &#181;m) is significantly smaller than that irradiated with the usual dynamic light scattering apparatus (approximately 100 &#181;m). In cases where the intensity of scattered light is weak, the signal is obscured by the noise, such as that caused by small amounts of dust in the solution. Therefore, the three peaks shown in Figure 3(b) may not have quantitative importance although the general order of the size is meaningful. Note that such a weak scatterer can be measured by a conventional dynamic light scattering apparatus.
We have demonstrated that the dynamic light scattering microscope enables us to measure both transparent and turbid samples with the same setup. Since the optical path length in the samples is short, this technique can be applied to strong light-absorbing samples, such as carbon nanotube suspensions20. In addition, due to its high spatial resolution, this technique can be applied to biological cells. For its application to biology, this method can also be combined with other imaging techniques, such as fluorescence and Raman imaging. Thus, we believe that the dynamic light scattering microscope is a powerful tool for a wide range of research fields.

Particle size is one of the most fundamental properties of colloidal and polymer solutions. Numerous techniques are used to measure the particle size. Particle sizes of 1.0 &#181;m or larger can be measured directly using an optical microscope. For smaller particles, alternative techniques, such as laser diffraction, electron microscopy, or atomic force microscopy, are used2,3. Dynamic light scattering is a commonly-used technique for the measurement of particle size distributions in solutions4. The results obtained using this technique are not derived from images of the particles but from the characteristic time of the fluctuations in scattered light intensity. These fluctuations originate from Brownian motion, which is characterized by a diffusion constant. The size distribution is obtained from the distribution of diffusion constants using the Einstein-Stokes equation. Due to its simplicity, dynamic light scattering is widely used for the routine assessment of solutions, such as paints and food colloids.
Pretreatment is required for most of the techniques used for the particle size measurement of solution samples. In the case of electron microscopy and atomic force microscopy, the sample must be analyzed under vacuum conditions. Therefore, it is difficult to observe the samples in their native forms. Furthermore, for laser diffraction and dynamic light scattering, only diluted samples that are free from multiple scattering and light absorption can be measured. To overcome this difficulty, several new techniques have been proposed for the measurement of dynamic light scattering from undiluted, concentrated solutions, such as cross-correlation spectroscopy5,6, low-coherence dynamic light scattering7,8, diffusing-wave spectroscopy9,10, and differential dynamic microscopy11,12.
We have developed a new apparatus called a dynamic light scattering microscope1. This apparatus enables us to measure turbid samples without dilution by means of a confocal optical system in which multiple scattering is eliminated using a pinhole. However, the measurement procedure and data analysis are slightly more complicated than those of commercially-available instruments. This video explains the measurement procedure and data analysis in detail using the analysis of the temperature-responsive polymer, poly(N-isopropylacrylamide), as an example.

<table><tbody><tr><td>&lt;em&gt;N&lt;/em&gt;-isopropylacrylamide, 98%</td><td>Tokyo Chemical Industry Co., Ltd.</td><td>I0401</td><td></td></tr><tr><td>toluene, 99%</td><td>Wako Pure Chemical Industries, Ltd.</td><td>201-01876</td><td></td></tr><tr><td>petroleum ether, distillation temperature 30 ~ 60 &amp;deg;C</td><td>Wako Pure Chemical Industries, Ltd.</td><td>169-22565</td><td></td></tr><tr><td>&lt;em&gt;N,N,N&#039;,N&#039;&lt;/em&gt;-tetramethylethylenediamine, 99%</td><td>Sigma</td><td>T9281</td><td></td></tr><tr><td>ammonium persulfate, 98%</td><td>Sigma</td><td>248614</td><td></td></tr><tr><td>polystyrene latex suspension, 1 wt%</td><td>Duke Scientific Corporation</td><td>3500A</td><td></td></tr><tr><td>argon</td><td>Koike Sanso Kogyo Co., Ltd.</td><td></td><td>purity &gt; 99.999 vol.%</td></tr><tr><td>cavity slide</td><td>Matsunami Glass Ind.,Ltd.</td><td>83-0336</td><td></td></tr><tr><td>inverted microscope</td><td>Nikon Instech Co., Ltd.</td><td>ECLIPSE Ti-U</td><td></td></tr><tr><td>Thermo Plate</td><td>Tokai Hit CO.,Ltd</td><td>TP-108R-C</td><td></td></tr><tr><td>Solid-state laser</td><td>Coherent</td><td>OBIS 488LX</td><td></td></tr><tr><td>avalanche photodiode</td><td>ALV-GmbH</td><td>ALV-High Q.E. Avalanche Photo Diode</td><td></td></tr><tr><td>correlator</td><td>ALV-GmbH</td><td>ALV-5000/EPP</td><td></td></tr></tbody></table>

measurement of particle size distribution in turbid solutions by dynamic light scattering microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

Time correlation functions of scattered light intensity for a polystyrene latex suspension (particle radius: 50 nm) were measured at different focal points, as shown in Figure 2(a). These correlation functions were converted into the distribution functions of the hydrodynamic radius by the inverse Laplace transformation (refer to Figure 2(b) and (c)). Using the same procedure, the time correlation functions and distribution functions of the hydrodynamic radius of the PNIPA solution were obtained at 25 &#176;C and 35 &#176;C, respectively. Figures 3(a) and (b) show the time correlation functions of the scattered light intensity and the corresponding size distribution functions of the PNIPA solution below (25 &#176;C) and above (35 &#176;C) the LCST. The size distribution functions were obtained by the inverse Laplace transformation followed by the correction of the partial heterodyne. The average hydrodynamic radius below the LCST is several tens of nanometers, which is typical for polymer solutions. In contrast, the hydrodynamic radius above the LCST is about 1.0 &#181;m. This result is consistent with the fact that the solution is turbid above the LCST. The red and blue lines in Figure 3 represent the size distribution of PNIPA solutions obtained immediately after and 20 min after the solution became turbid, respectively. Figure 3(b) clearly indicates the growth of the aggregation.
<img alt="Figure 1" src="/files/ftp_upload/54885/54885fig1.jpg" /> 
Figure 1. Schematics of the dynamic light scattering microscope. Pinhole (PH), beam splitter (BS), polarizer (Pol), and avalanche photodiode (APD). <a href="http://cloudflare.jove.com/files/ftp_upload/54885/54885fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54885/54885fig2.jpg" /> 
Figure 2. Representative results for a polystyrene latex suspension. (a) Time correlation functions of the scattered light intensity for the polystyrene latex suspension. The nominal radius is 50 nm and the concentration is 0.1 wt%. Two datasets were obtained from different scattering points. (b), (c) Corresponding size distribution functions for the polystyrene latex suspension obtained by the inverse Laplace transformation of (a). The red line corresponds to the time correlation function whose initial amplitude is approximately 1.0, and the blue line corresponds to that with an initial amplitude that is approximately 0.2. The horizontal axis was calculated without (b) and with (c) considering the effect of partial heterodyning (PHD) when A &#60;&#60; 1. <a href="http://cloudflare.jove.com/files/ftp_upload/54885/54885fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54885/54885fig3.jpg" /> 
Figure 3. Representative results for a PNIPA solution. (a) Time correlation functions of scattered light intensity for the PNIPA solution. (b) Corresponding size distribution functions for the PNIPA solution obtained by the inverse Laplace transformation of (a). The horizontal axis was calculated considering the effect of partial heterodyning for each data set. The black line represents the data obtained at 25 &#176;C. The red line represents the data obtained just after the solution turned turbid (35 &#176;C). The blue line represents the data obtained after a 20-min measurement of the red line. <a href="http://cloudflare.jove.com/files/ftp_upload/54885/54885fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

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14.3K Views.  University of Tokyo. A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

Video: Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

Bioengineering

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

Environment

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.

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.

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

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Application of Voltage in Dynamic Light Scattering Particle Size Analysis

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. Modern instrumentation permits measurement of particle size as a function of time and/or temperature, but currently there is no simple method for performing DLS particle size distribution measurements in the presence of applied voltage. The ability to perform such measurements would be useful in the development of electroactive, stimuli-responsive polymers for applications such as sensing, soft robotics, and energy storage. Here, a technique using applied voltage coupled with DLS and a temperature ramp to observe changes in aggregation and particle size in thermoresponsive polymers with and without electroactive monomers is presented. The changes in aggregation behavior observed in these experiments were only possible through the combined application of voltage and temperature control. To obtain these results, a potentiostat was connected to a modified cuvette in order to apply voltage to a solution. Changes in polymer particle size were monitored using DLS in the presence of constant voltage. Simultaneously, current data were produced, which could be compared with particle size data, to understand the relationship between current and particle behavior. The polymer poly(N-isopropylacrylamide) (pNIPAM) served as a test polymer for this technique, as pNIPAM's response to temperature is well-studied. Changes in the lower-critical solution temperature (LCST) aggregation behavior of pNIPAM and poly(N-isopropylacrylamide)-block-poly(ferrocenylmethyl methacrylate), an electrochemically active block-copolymer, in the presence of applied voltage are observed. Understanding the mechanisms behind such changes will be important when trying to achieve reversible polymer structures in the presence of applied voltage.

Here, a protocol to apply voltage to solution during dynamic light scattering particle size measurements with the intent to explore the effect of voltage and temperature changes on polymer aggregation is presented.

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. ...

Assembly and Characterization of Polyelectrolyte Complex Micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Medicine

Quantifying Cytoskeleton Dynamics Using Differential Dynamic Microscopy

Cells can crawl, self-heal, and tune their stiffness due to their remarkably dynamic cytoskeleton. As such, reconstituting networks of cytoskeletal biopolymers may lead to a host of active and adaptable materials. However, engineering such materials with precisely tuned properties requires measuring how the dynamics depend on the network composition and synthesis methods. Quantifying such dynamics is challenged by variations across the time, space, and formulation space of composite networks. The protocol here describes how the Fourier analysis technique, differential dynamic microscopy (DDM), can quantify the dynamics of biopolymer networks and is particularly well suited for studies of cytoskeleton networks. DDM works on time sequences of images acquired using a range of microscopy modalities, including laser-scanning confocal, widefield fluorescence, and brightfield imaging. From such image sequences, one can extract characteristic decorrelation times of density fluctuations across a span of wave vectors. A user-friendly, open-source Python package to perform DDM analysis is also developed. With this package, one can measure the dynamics of labeled cytoskeleton components or of embedded tracer particles, as demonstrated here with data of intermediate filament (vimentin) networks and active actin-microtubule networks. Users with no prior programming or image processing experience will be able to perform DDM using this software package and associated documentation.

Differential dynamic microscopy (DDM) combines features of dynamic light scattering and microscopy. Here, the process of using DDM to characterize reconstituted cytoskeleton networks by quantifying the subdiffusive and caged dynamics of particles in vimentin networks and the ballistic motion of active myosin-driven actin-microtubule composites is presented.

Cells can crawl, self-heal, and tune their stiffness due to their remarkably dynamic cytoskeleton. As such, reconstituting networks of cytoskeletal ...

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 ...

Biology

Dynamic Light Scattering Analysis for the Determination of the Particle Size of Iron-Carbohydrate Complexes

Intravenously administered iron-carbohydrate nanoparticle complexes are widely used to treat iron deficiency. This class includes several structurally heterogeneous nanoparticle complexes, which exhibit varying sensitivity to the conditions required for the methodologies available to physicochemically characterize these agents. Currently, the critical quality attributes of iron-carbohydrate complexes have not been fully established. Dynamic light scattering (DLS) has emerged as a fundamental method to determine intact particle size and distribution. However, challenges still remain regarding the standardization of methodologies across laboratories, specific modifications required for individual iron-carbohydrate products, and how the size distribution can be best described. Importantly, the diluent and serial dilutions used must be standardized. The wide variance in approaches for sample preparation and data reporting limit the use of DLS for the comparison of iron-carbohydrate agents. Herein, we detail a robust and easily reproducible protocol to measure the size and size distribution of the iron-carbohydrate complex, iron sucrose, using the Z-average and polydispersity index.

Dynamic light scattering (DLS) has emerged as a suitable assay for evaluating the particle size and distribution of intravenously administered iron-carbohydrate complexes. However, the protocols lack standardization and need to be modified for each iron-carbohydrate complex analyzed. The present protocol describes the application and special considerations for the analysis of iron sucrose.

Intravenously administered iron-carbohydrate nanoparticle complexes are widely used to treat iron deficiency. This class includes several structurally ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

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