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.).

Preparação 1. Amostra <ol><li> Purificação de monómeros de temperatura-sensível <ol><li> Dissolve-se 20 g de N -isopropylacrylamide (INAP) em 100 ml de tolueno. </li><li> Filtra-se a solução sob vácuo para eliminar o pó. </li><li> Misturar-se o filtrado com 500 ml de éter de petróleo. </li><li> Colocar o recipiente de reacção num banho de gelo-água. </li><li> Agita-se a solução até que os monómeros são precipitados (tipicamente 30 minutos). </li><li> Filtra-se a solução sob sucção para se obter os monómeros precipitados. </li><li> Secam-se os monómeros sob pressão reduzida (100 Pa) durante a noite. </li></ol></li><li> Preparação da solução de polímero de temperatura-sensível <ol><li> Desgaseifica 20 mL de água desionizada durante 1,0 min utilizando uma bomba de diafragma. </li><li> Dissolve-se 780,8 mg do INAP purificado em 9,5 ml de água desgaseif içada e desionizada. </li><li> Colocar o recipiente de reacção num banho de gelo-água. </li><li> Proteger a reação deluz, cobrindo o aparelho com folha de alumínio. </li><li> Agita-se a solução suavemente durante 10 min enquanto se introduz uma corrente moderada de gás Ar suavemente através de uma ponta de pipeta ligada ao cilindro de gás com um tubo. </li><li> Adicionar 11,9 mL de N, N, N &#39;, N&#39; tetrametiletilenodiamina à solução por meio de uma micropipeta. </li><li> Agita-se a solução durante 1,0 min, enquanto a introdução de gás Ar, como mencionado no passo 1.2.5. </li><li> Enquanto se agitava a amostra, dissolver-se 4,0 mg de persulfato de amónio em 0,5 ml de água desgaseif içada e desionizada. </li><li> Misturar a solução de amostra (a partir do passo 1.2.7) e uma solução de persulfato de amónio (a partir do passo 1.2.8). </li><li> Agita-se a solução durante 30 segundos enquanto se introduz gás Ar, como mencionado no passo 1.2.5. </li><li> Cubra a solução com folha de alumínio e mantê-lo no frigorífico (4 ° C) durante a noite. </li></ol></li><li> Preparação de fixação do provete <ol><li> Colocar 60 mL de solução da amostra (a partir depasso 1.2.11) em um slide cavidade. </li><li> Cubra a solução com uma tampa de vidro circular. Não ser o cuidado de bolhas de ar armadilha. </li><li> Retire o excesso de solução usando uma micropipeta e laboratoriais toalhetes. </li><li> Selar a amostra com cola. Deixe secar cola à temperatura ambiente (tipicamente 6 h). </li><li> Prepare outro slide preenchido com látex de 0,1% em peso de poliestireno (100 nm de diâmetro das partículas) suspensão por seguir os passos 1.3.1-1.3.4. Esta corrediça é utilizada como um padrão. </li></ol></li></ol> 2. Partícula medição do tamanho com um Dynamic Light Scattering Microscópio <ol><li> Optimização do instrumento <ol><li> Colocar a lâmina suspensão de látex de poliestireno (a partir do passo 1.3.5) sobre a fase de microscópio invertido. Pelo lado da tampa de vidro deve estar voltada para baixo. </li><li> Coloque um amortecedor feixe em frente do detector (um fotodiodo de avalanche e um autocorrelador). </li><li> Aplicar um feixe de laser (laser de estado sólido, λ = 488 nm, 30 mW, contínuaOnda) para a amostra através de uma lente objetiva (10 ×). Uma porção da luz reflectida passa por um espelho lançamento do microscópio e é observada por uma câmara CCD montada na porta do lado do microscópio (Figura 1). </li><li> Ajuste a altura da lente objetiva para definir o ponto focal na suspensão da amostra, deslocando a altura da lente objetiva a partir da posição de baixa para alta. Durante este processo, a imagem reflectida é focada três vezes: na superfície do vidro de cobertura, na interface entre a cobertura de vidro e a amostra, e na interface entre a amostra e o vidro furo-corrediça. Defina o ponto focal entre o segundo e terceiro pontos. </li><li> Atenuar a intensidade da luz dispersa, alterando a potência do laser. </li><li> Introduzir a luz dispersa para o detector por remoção do amortecedor de feixe em frente do detector. Esta unidade mede a correlação temporal da intensidade de luz. </li><li> Definir uma pinhole (φ = 50 mm) between o microscópio e o detector para alcançar o efeito confocal. Ajustar a posição do orifício para maximizar a intensidade da luz no detector. </li><li> Medir o tempo de função de correlação de intensidade de luz dispersa durante 30 s por iniciar a operação do dispositivo de correlação através de um computador. A função de correlação medido é muitas vezes expressa como g de (2) (t) - 1, em que t é o tempo de correlação e quatro <img alt="equação 1" src="/files/ftp_upload/54885/54885eq1.jpg" /> . Aqui, I (t) é a intensidade da luz dispersa no tempo t e (•••) T tem uma média de tempo. O tempo de decaimento será de aproximadamente 0,1 ms. </li><li> Ajustar o ponto focal para se obter uma vasta gama para a amplitude iniciais da função de correlação de tempo (g (2) (t = 0) - 1). NOTA: A amplitude inicial é fortemente influenciada pela quantidade de luz refletida. Movendo a reboque ponto focalard a interface entre a cobertura de vidro e a amostra, aumenta a quantidade de luz refletida. Para dispersores de luz fortes, tais como látex de poliestireno, a amplitude inicial pode ser alterado a partir de 0 a 1. No entanto, é difícil definir a amplitude inicial perto de 1 de soluções de polímeros mais comuns porque a intensidade da luz reflectida é muito maior do que que parte da luz dispersa. </li><li> Aplicar a transformação inversa de Laplace (utilizando o programa CONTIN regularização constrangido 13,14) para a função de correlação obtida tempo para a aquisição da função de distribuição de tamanho. Nos casos em que a amplitude inicial é definida como inferior a 0,2, a função de distribuição do raio hidrodinâmico irá mostrar um pico acentuado a cerca de 100 nm, o que é o dobro do raio real (ver a discussão para detalhes). </li></ol></li><li> medição da amostra <ol><li> Ajustar a temperatura etapa até 25 ° C. </li><li> Coloque uma lâmina preparada com poli-INAP (PNIPA) solutde iões (passo 1.3.4) sobre a fase de microscópio. </li><li> Medir a função de correlação temporal da intensidade de luz dispersa através dos seguintes passos 2.1.4-2.1.8. Se a amplitude inicial é maior do que 0,2, ajustar o ponto focal para tornar a amplitude iniciais da função de correlação de tempo inferior a 0,2 seguindo o passo 2.1.9. Uma pequena amplitude inicial simplifica a análise. </li><li> Regule a temperatura do estágio de 35 ° C e aguarde até que a solução se torna turva. A temperatura crítica inferior de solução (TCIS) da solução PNIPA é de 32 ° C 15. </li><li> Medir a função de correlação de tempo seguindo os passos 2.1.4-2.1.8. Se possível, ajustar a posição do ponto focal para tornar a amplitude iniciais da função de correlação de tempo inferior a 0,2. Para soluções turvas, as suas amplitudes iniciais tendem a aumentar, desde que a intensidade da luz aumenta os dispersos, enquanto que a luz reflectida de permanece constante. </li><li> Aplicar o inverso Laplace transformatiem que as funções de correlação de tempo obtidas para obter as funções de distribuição de tamanho. Note-se que o tamanho real é metade do valor obtido em casos onde a amplitude inicial é inferior a 0,2. </li></ol></li></ol>

<ol>
	<li>Hiroi, T., Shibayama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Light+Scattering+Microscope:+Accessing+Opaque+Samples+with+High+Spatial+Resolution.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+Size+Analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+supramolecular+polymers.">Characterization of supramolecular polymers.</a> Chem. Soc. Rev. 41, 5922-5932 (2012).</li><li>Berne, B. J., Pecora, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dynamic Light Scattering with Applications to Chemistry, Biology and Physics. , (2000).</li><li>Phillies, G. D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+demonstration+of+ruultiple-scattering+suppression+in+quasielastic-light-scattering+spectroscopy+by+homodyne+coincidence+techniques.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+multiple+scattering+effects+in+quasielastic+light+scattering+by+homodyne+crosscorrelation+techniques.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-scattering+spectroscopy+for+extremely+dense+colloidal+suspensions+by+use+of+a+low-coherence+interferometer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+Radius+Sizing+of+Nanoparticles+in+Dense+Polydisperse+Media+by+Low-Coherence+Dynamic+Light+Scattering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+light+scattering+from+disordered+media.+The+effect+of+brownian+motion+of+scatterers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusing+wave+spectroscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Dynamic+Microscopy:+ProbingWave+Vector+Dependent+Dynamics+with+a+Microscope.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+Concentrated,+Multiply+Scattering,+and+Actively+Driven+Fluorescent+Systems+with+Confocal+Differential+Dynamic+Microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+constrained+regularization+method+for+investing+data+represented+by+linear+algebraic+or+integral+equations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+dynamic+light+scattering+autocorrelation+functions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-angle+Neutron+Scattering+Study+on+Preparation+Temperature+Dependence+of+Thermosensitive+Gels.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Light+Scattering+by+Non-Ergodic+Media.">Dynamic Light Scattering by Non-Ergodic Media.</a> Physica A. 157, 705-741 (1989).</li><li>Chu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laser Light Scattering. 2nd Ed. , (1991).</li><li>Shibayama, M., Tanaka, T., Han, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-Angle+Neutron-Scattering+Study+on+Poly(N-Isopropyl+Acrylamide)+Gels+near+Their+Volume-Phase+Transition-Temperature.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+Kinetics+of+Volume+Phase+Transition+of+Gels.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transitions+of+Aggregation+States+for+Concentrated+Carbon+Nanotube+Dispersion.">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.

A amplitude inicial da função de correlação de tempo depende muito do ponto focal, como mostrado na Figura 2 (a). Isto aparentemente contraria o facto de que a solução é homogénea (excepto para a camada fina na interface) 8. Esta variação na amplitude inicial é atribuída a uma variação na quantidade de luz reflectida. Teoria heterodyne parcial 16 prevê que a amplitude inicial, A, a intensidade da luz dispersa, eu s, e a intensidade da luz refletida, I r, satisfazer a seguinte equação 1 <img alt="equação 2" src="/files/ftp_upload/54885/54885eq2.jpg" /> Esta equação mostra que a maior I r torna-se, o mais pequeno se torna um. Portanto, A é reduzida, definindo a posição focal próximo da interface. A aparente de difusão constante D Um can ser obtido ajustando a função de correlação de tempo no caso de soluções monodispersas: <img alt="equação 3" src="/files/ftp_upload/54885/54885eq3.jpg" /> Onde <img alt="equação 4" src="/files/ftp_upload/54885/54885eq4.jpg" /> . Aqui, n é o índice de refracção do solvente (água, 1,33), θ é o ângulo dispersa (180 °), e λ é o comprimento de onda da luz (514,5 nm). Uma vez que aplicada geometria retroespalhamento, o valor de q é fixo. No entanto, este ponto é resolvido pelo uso de diferentes comprimentos de onda de luz. Por favor note que qualquer tipo de fonte de laser de onda contínua está disponível para construir o microscópio DLS. Graças ao pequeno volume irradiado, o factor de coerência 17 está estimada em mais de 0,99 e é insignificante. Para soluções polidispersos, a função de distribuição de D A é obtido pela transformação inversa de Laplace. th heterodyne parciaiseory também prevê que D A não é a mesma que a constante de difusão D efectiva. Estas duas constantes de difusão satisfazer a seguinte equação: <img alt="equação 5" src="/files/ftp_upload/54885/54885eq5.jpg" /> A constante de difusão D é convertido no raio Rh hidrodinâmico utilizando a equação de Stokes-Einstein 4. Quando A = 1, esta relação se torna D A = D. Neste caso, o processo de conversão de dados é o mesmo que o descrito para a dispersão dinâmica de luz comum. A linha vermelha mostrada na Figura 2 (b) corresponde a este caso. Em contraste, esta relação torna-se D A = 0,5 D ao limite de A → 0. Por conseguinte, o tamanho é estimada para ser duas vezes tão grande como o tamanho real quando A é pequeno (na prática, inferior a 0,2), como mostrado pela A linha azul da Figura 2 (b) </strong&gt;. Se se sabe que A é significativamente pequena, o eixo horizontal pode ser deslocado, como mostrado na Figura 2 (c). Em princípio, podemos converter D A em D para qualquer valor de. Na prática, no entanto, é melhor para definir a amplitude inicial menor do que 0,2, uma vez que a aproximação simples D A ~ 0,5 D é válido. As características proeminentes da técnica de dispersão de microscópio de luz dinâmica foram demonstrados usando uma solução PNIPA. A conformação do PNIPA abaixo e acima da TCIS tem sido extensivamente estudada usando pequeno ângulo de dispersão de neutrões 15,18. Em contraste, a dispersão dinâmica de luz não tem sido utilizada para a análise de PNIPA acima da TCIS devido à sua turbidez 19. Este problema é resolvido pelo microscópio de luz de dispersão dinâmica, como se mostra nas Figuras 3 (a) e (b). O tamanho destes agregados é várias &amp;# 181; m, o que não pode ser obtida por qualquer pequeno ângulo de raios X de dispersão / neutrões ou técnicas de dispersão de luz convencionais. medições resolvida no tempo usando este sistema dar informações sobre o processo de agregação durante a mudança de temperatura. A desvantagem do microscópio de dispersão de luz dinâmica é também ilustrada na Figura 3. Para o resultado abaixo da TCIS, a função de correlação de tempo é fortemente afetada pela pequena quantidade de poeira presente (as linhas pretas na Figura 3). Por exemplo, a função de correlação de tempo não se decompor completamente, mesmo com tempos de correlação da ordem de 1,0 s. Isto é porque o volume irradiado com este aparelho (aproximadamente 1.0 um) é significativamente menor do que irradiada com o aparelho de dispersão de luz dinâmica habitual (cerca de 100 um). Nos casos em que a intensidade de luz dispersa é fraco, o sinal está obscurecido pelo ruído, tal como a causada por Squantidades de shopping de poeira na solução. Portanto, os três picos mostrados na Figura 3 (b) pode não ter importância quantitativa embora a ordem geral do tamanho é significativa. Note-se que um espalhador tal fraco pode ser medida por um aparelho de dispersão de luz dinâmica convencional. Nós demonstramos que a dinâmica microscópio de dispersão da luz permite-nos medir amostras tanto transparentes e turvos com a mesma configuração. Uma vez que o comprimento do percurso óptico nas amostras é curto, esta técnica pode ser aplicada a amostras de absorção de luz fortes, tais como suspensões de nanotubos de carbono 20. Além disso, devido à sua alta resolução espacial, esta técnica pode ser aplicada a células biológicas. Para a sua aplicação para a biologia, este método também pode ser combinada com outras técnicas de imagiologia, tais como imagiologia de fluorescência e Raman. Assim, acreditamos que a dinâmica microscópio de dispersão da luz é uma ferramenta poderosa para uma ampla gama de campos de pesquisa.

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 border="0" cellpadding="0" cellspacing="0" width="591">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="76">
			<td height="76" style="height:76px;width:216px;">N-isopropylacrylamide, 98%</td>
			<td style="width:89px;">Tokyo Chemical Industry Co., Ltd.</td>
			<td style="width:119px;">I0401</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:216px;">toluene, 99%</td>
			<td style="width:89px;">Wako Pure Chemical Industries, Ltd.</td>
			<td style="width:119px;">201-01876</td>
			<td></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:216px;">petroleum ether, distillation temperature 30 ~ 60 &#176;C</td>
			<td style="width:89px;">Wako Pure Chemical Industries, Ltd.</td>
			<td style="width:119px;">169-22565</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">N,N,N&#39;,N&#39;-tetramethylethylenediamine, 99%</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:119px;">T9281</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">ammonium persulfate, 98%</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:119px;">248614</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">polystyrene latex suspension, 1 wt%</td>
			<td style="width:89px;">Duke Scientific Corporation</td>
			<td style="width:119px;">3500A</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">argon</td>
			<td style="width:89px;">Koike Sanso Kogyo Co., Ltd.</td>
			<td style="width:119px;"></td>
			<td>purity &#62; 99.999 vol.%</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;"></td>
			<td style="width:89px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">cavity slide</td>
			<td style="width:89px;">Matsunami Glass Ind.,Ltd.</td>
			<td style="width:119px;">83-0336</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">inverted microscope</td>
			<td style="width:89px;">Nikon Instech Co., Ltd.</td>
			<td style="width:119px;">ECLIPSE Ti-U</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Thermo Plate</td>
			<td style="width:89px;">Tokai Hit CO.,Ltd</td>
			<td style="width:119px;">TP-108R-C</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Ar-Kr ion laser</td>
			<td style="width:89px;">Spectra-Physics</td>
			<td style="width:119px;">Stabilite 2018</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">avalanche photodiode</td>
			<td style="width:89px;">ALV-GmbH</td>
			<td style="width:119px;">ALV-High Q.E. Avalanche Photo Diode</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">correlator</td>
			<td style="width:89px;">ALV-GmbH</td>
			<td style="width:119px;">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.

Funções tempo de correlação de intensidade da luz dispersa para uma suspensão de látex de poliestireno (raio de partícula: 50 nm) foram medidos em diferentes pontos focais, como mostrado na Figura 2 (a). Estas funções de correlação foram convertidos para as funções de distribuição do raio hidrodinâmico pela transformação inversa de Laplace (consulte a Figura 2 (b) e (c)). Utilizando o mesmo procedimento, as funções de correlação de tempo e funções de distribuição do raio hidrodinâmico da solução PNIPA foram obtidos a 25 ° C e 35 ° C, respectivamente. As Figuras 3 (a) e (b) mostram as funções de correlação de tempo da intensidade da luz dispersa e as funções de distribuição de tamanho correspondente da solução abaixo PNIPA (25 ° C) e acima (35 ° C) a TCIS. As funções de distribuição de tamanhos foram obtidos pela transformação inversa de Laplace seguido pelacorreção do heterodyne parcial. O raio hidrodinâmico médio abaixo da TCIS é de várias dezenas de nanómetros, o que é típico para soluções de polímero. Em contraste, o raio hidrodinâmico acima da TCIS é de cerca de 1,0 um. Este resultado é consistente com o facto que a solução é turva acima da TCIS. As linhas vermelhas e azuis na Figura 3 representam a distribuição do tamanho de soluções PNIPA obtidos imediatamente após 20 min e após a solução se tornar turva, respectivamente. A Figura 3 (b) indica claramente o crescimento da agregação. <img alt="figura 1" src="/files/ftp_upload/54885/54885fig1.jpg" /> Figura 1. Esquema do microscópio de dispersão de luz dinâmica. Pinhole (PH), divisor de feixe (BS), polarizador (Pol) e fotodiodo avalanche (APD). <a href="http://ecsource.jove.com/files/ftp_upload/54885/54885fig1large.jpg" target="_blank">Por favor clique aqui para ver</a>uma versão maior desta figura. <img alt="Figura 2" src="/files/ftp_upload/54885/54885fig2.jpg" /> Figura 2. Os resultados representativos de uma suspensão de látex de poliestireno. (A) funções de correlação temporal da intensidade da luz dispersa para a suspensão de látex de poliestireno. O raio nominal é de 50 nm, e a concentração é de 0,1% em peso. Dois conjuntos de dados foram obtidos a partir de diferentes pontos de dispersão. (B), (c) funções de distribuição de tamanho correspondente para a suspensão de látex de poliestireno, obtidos por transformação inversa de Laplace de (a). A linha vermelha corresponde à função de correlação de tempo cuja amplitude inicial é de cerca de 1,0, e a linha azul corresponde ao que com uma amplitude inicial que é de aproximadamente 0,2. O eixo horizontal foi calculado sem (b) e com (c), considerando o efeito de heterodyning parcial (PHD), quando A &lt;&lt;<html> ; 1. <a href="http://ecsource.jove.com/files/ftp_upload/54885/54885fig2large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54885/54885fig3.jpg" /> Figura 3. Os resultados representativos para uma solução PNIPA. (A) funções de correlação temporal de intensidade de luz dispersa para a solução PNIPA. (B) funções de distribuição de tamanho correspondente para a solução PNIPA obtido pela transformação inversa de Laplace de (a). O eixo horizontal foi calculado considerando o efeito de heterodyning parcial para cada conjunto de dados. A linha preta representa os dados obtidos a 25 ° C. A linha vermelha representa os dados obtidos imediatamente após a solução se tornar turva (35 ° C). A linha azul representa os dados obtidos após uma medição de 20 min da linha vermelha./54885/54885fig3large.jpg &quot;Target =&quot; _ blank &quot;&gt; Clique aqui para ver uma versão maior desta figura.

Watch this Scientific Journal Video about Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy at JoVE.com

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

Um protocolo para a medição directa da distribuição de tamanho de partículas em soluções concentradas usando microscopia de luz espalhamento dinâmico é apresentado.

Medição do Tamanho de Partículas Distribuição em turvas Solutions pela Dynamic dispersão de luz Microscopia

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

measurement-particle-size-distribution-turbid-solutions-dynamic-light

Universidad de Cartagena UDEC

Research

Education

JoVE Core

JoVE Journal

Electrical Engineering

Chemistry

Unit 1

The Laplace Transform

SCIENTISTS IN ACTION

14.2K visualizações.  University of Tokyo.  Um protocolo para a medição directa da distribuição de tamanho de partículas em soluções concentradas usando microscopia de luz espalhamento dinâmico é apresentado. 

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

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

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

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

O exame quantitativo e qualitativo dos Interações partícula-partícula coloidal Usando Probe Nanoscopia

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

Química

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

Dinâmica eletroquímico Medição de íons cloreto

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

Dissolução dinâmico Nuclear Polarização Instrumentação para em tempo real enzimática Reaction Taxa de Medições por RMN

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

Fabricação e teste dos termómetros fotônicos

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

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.

DNA-magnético partícula análise por dispersão de luz dinâmica e eletroforético de vinculação

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

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

Chips microfluídicos por difração de raio x de cristal In Situ e In Situ Difusão dinâmica da luz de cristalografia de Serial

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid based on volatile solar and wind sources. The pressing demand of developing high-performance electrochemical energy storage solutions, i.e., batteries, relies on both fundamental understanding and practical developments from both the academy and industry. The formidable challenge of developing successful battery technology stems from the different requirements for different energy-storage applications. Energy density, power, stability, safety, and cost parameters all have to be balanced in batteries to meet the requirements of different applications. Therefore, multiple battery technologies based on different materials and mechanisms need to be developed and optimized. Incisive tools that could directly probe the chemical reactions in various battery materials are becoming critical to advance the field beyond its conventional trial-and-error approach. Here, we present detailed protocols for soft X-ray absorption spectroscopy (sXAS), soft X-ray emission spectroscopy (sXES), and resonant inelastic X-ray scattering (RIXS) experiments, which are inherently elemental-sensitive probes of the transition-metal 3d and anion 2p states in battery compounds. We provide the details on the experimental techniques and demonstrations revealing the key chemical states in battery materials through these soft X-ray spectroscopy techniques.

Here, we present a protocol for typical experiments of soft X-ray absorption spectroscopy (sXAS) and resonant inelastic X-ray scattering (RIXS) with applications in battery material studies.

Elemental-sensíveis à deteção da química em baterias através de espectroscopia de absorção de raios-x moles e espalhamento inelástico de raios-x ressonante

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

Imagem de fotoelétron de ânions ilustrado por 310 Nm destacamento de F−

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

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.

Aplicação da tensão na análise dinâmica do tamanho da partícula de dispersão de luz

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

Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures

Neutron crystallography is a structural technique that allows determination of hydrogen atom positions within biological macromolecules, yielding mechanistically important information about protonation and hydration states while not inducing radiation damage. X-ray diffraction, in contrast, provides only limited information on the position of light atoms and the X-ray beam&#160;rapidly induces radiation damage of photosensitive&#160;cofactors and metal centers. Presented here is the workflow employed for the IMAGINE and MaNDi beamlines at Oak Ridge National Laboratory (ORNL) to obtain a neutron diffraction structure once a protein crystal of suitable size (&#62; 0.1 mm3) has been grown. We demonstrate mounting of hydrogenated protein&#160;crystals in quartz capillaries for neutron diffraction data collection. Also presented is the vapor exchange process of the mounted crystals with D2O-containing buffer to ensure replacement of hydrogen atoms at exchangeable sites with deuterium. The incorporation of deuterium reduces the background arising from the incoherent scattering of hydrogen atoms and prevents density cancellation caused by their negative coherent scattering length. Sample alignment and room temperature data collection strategies are illustrated using quasi-Laue data collection at IMAGINE at the High Flux Isotope Reactor (HFIR). Furthermore, crystal mounting and rapid freezing in liquid nitrogen for cryo-data collection to trap labile reaction intermediates is demonstrated at the MaNDi time-of-flight instrument at the Spallation Neutron Source (SNS). Preparation of the model coordinate and diffraction data files and visualization of the neutron scattering length density (SLD) maps will also be addressed. Structure refinement against neutron data-only or against joint X-ray/neutron data to obtain an all-atom structure of the protein of interest will finally be discussed. The process of determining a neutron structure will be demonstrated using crystals of the lytic polysaccharide monooxygenase Neurospora crassa LPMO9D, a copper-containing metalloprotein involved in the degradation of recalcitrant polysaccharides via oxidative cleavage of the glycosidic bond.

Neutron protein crystallography is a structural technique that permits the localization of hydrogen atoms, thereby providing important mechanistic details of protein function. We present here the workflow for mounting a protein crystal, neutron diffraction data collection, structure refinement and analysis of the neutron scattering length density maps.

Medição do Tamanho de Partículas Distribuição em turvas Solutions pela Dynamic dispersão de luz Microscopia

Resumo

Explore mais vídeos

Capítulos neste vídeo