Name: measurement of particle size distribution in turbid solutions by dynamic light scattering microscopy
Uploaded: 2017-01-09T12:30:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy at JoVE.com

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>

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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><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.

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="https://ecsource-jove-com.bdigitaluss.remotexs.co/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="https://ecsource-jove-com.bdigitaluss.remotexs.co/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.

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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 ...

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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

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.

Método de extrusão constante pressão controlada para a Preparação de vesículas lipídicas nanométricas

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

Bioengenharia

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.

Dispersão de nanomateriais em meios aquosos: para otimização de protocolo

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

Ambiente

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.

Síntese controlada e Fluorescência de rastreamento de Poly altamente uniforme (<em&gt; N</em&gt; -isopropylacrylamide) microgéis

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

Química

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 ...

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. ...

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.

Montagem e Caracterização de Micelis Complexos polieletrolitos

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.

Método de triagem para micropartículas em transfusões de plaquetas de rotina

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

Medicina

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.

Quantificando a Dinâmica do Citoesqueleto usando microscopia dinâmica diferencial

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.

Um método inovador para exossomo Quantificação e Size Measurement

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

Biologia

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.

Análise dinâmica de espalhamento de luz para a determinação do tamanho de partículas de complexos ferro-carboidrato

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