This project was supported by grants NIH NIGMS 8P41 GM103540-28 and P50-GM076516

Preparação 1 Amostra <ol><li> Manter células CHOK1 em frascos de cultura de tecidos, utilizando meio DMEM suplementado com 10% de soro fetal bovino e 100 UI / ml de penicilina de 50 ug / ml de estreptomicina. Incubar as células em uma incubadora humidificada 5% CO 2 a 37 ° C. </li><li> Colheita e, em seguida, as células CHOK1 prato sobre um diâmetro de micro-reservatório 14 mm, com uma espessura de superfície de 0,16 mm. Células de sementeira para a densidade óptima para a imagiologia, cerca de 60-70% de confluência. </li><li> Incubar as células durante a noite a 37 ° C, 5% de CO 2. Lavam-se as células três vezes em HBSS (Solução Salina Tamponada de Hank), e incubar as células numa solução que contém 50 nM de Lyso Tracker DND26 verde e 150 nM de verde tubulina rastreador. Incubar as células durante 1 hora a 37 ° C. Passo opcional: Antes da incubação, realizar uma mancha adicional coloração com um corante de matriz mitocondrial (25 nM). </li><li> Lavam-se as células três vezes em HBSS para remover qualquer corante não ligado. Adicionar meio de crescimento DMEM fresco antes de imagção das células. </li></ol> 2. Microscópio Configurações O microscópio para o rastreamento de partículas descrito no protocolo de vídeo está montado na armação de um microscópio invertido comercialmente disponível (Figura 4). No entanto módulos comerciais para 3D rastreamento de partículas orbital já estão disponíveis. <ol><li> Use a Ti Coherent Chameleon-Ultra II: fonte de luz laser de excitação femtosegundo Sa, com um comprimento de onda de saída ajustável entre 690 nm-1040 nm. </li><li> Certifique-se de que o feixe de laser é alinhado na porta traseira do microscópio usando espelhos revestidos por IR. O feixe de laser é atenuado usando uma placa de meia onda rotativa seguida por um polarizador linear calcite. Atenuar o feixe de modo a que a potência média da amostra está entre 0,5 e 2 mW. NOTA: O feixe de laser é reflectido por um par de espelhos accionados galvanómetro-motores que permitem o controlo da posição e da trajectória do feixe focado no plano da amostra. Numaconfiguração típica do feixe laser colimado de sair dos espelhos galvanométricos ampliada (10x), que passa através de um expansor de feixe, antes de entrar na parte de trás da objectiva de microscópio, após reflexão no espelho dicróico de curta passagem. </li><li> Recolha a luz de fluorescência da amostra através da colocação de um objectivo água abertura numérica elevada (60X, NA 1.2) no caminho da luz. Escolher um cubo de filtro de fluorescência de acordo com os comprimentos de onda de emissão desejadas em uma ou duas configurações de canal. Empregam filtros de banda de emissão para seleccionar ainda mais a gama espectral da fluorescência emitida. </li><li> Colocar a amostra para um palco motorizado, e ajustar o movimento fino da objetiva do microscópio usando um piezo-controlador objetivo. </li><li> Dirigir a luz a partir do cubo de filtro em tubos fotomultiplicadores, onde o sinal é discriminados e enviadas para um I / O cartão de aquisição de dados digital. NOTA: Os utilizadores são formas de onda da saída analógica da placa de I / O e são provided para os scanners sistema eletrônico. A entrada de contagem de fótons dos fotomultiplicadores e do sinal de saída para os scanners são medidos e controlados através do cartão de I / O por do Laboratório de Fluorescência software Dynamics SimFCS. </li></ol> 3 Imagens <ol><li> Posicione as células no palco e se concentrar utilizando transmitida iluminação de luz. </li><li> Mudar para a imagem raster varredura para identificar as células que incorporaram o corante e visualizar os microtúbulos subjacentes. Identificar a área inicial onde o movimento vesícula está presente. </li><li> Uma vez que uma vesícula isolada é identificado, definir os seguintes parâmetros para o rastreamento orbital: <ol><li> Seleccione o raio da órbita para definir o tamanho da varredura circular de acordo com o tamanho da partícula a ser rastreado. Para um ponto emissor, definir o raio da órbita igual à cintura da Função propagação ponto (PSF) do feixe de excitação para maximizar a sensibilidade e de resposta. </li><li> Defina o axialdistanciar para definir a distância entre as duas órbitas que são realizados para localizar a posição de partículas ao longo da direcção axial. Defina a distância axial de 1,5-3 vezes a cintura PSF. </li><li> Define o tempo de permanência de acordo com o brilho das partículas para definir o tempo gasto em cada ponto da órbita, que também irá determinar a taxa de branqueamento foto-. Use o tempo de espera entre 10-100 ms. </li><li> Defina o número de pontos em cada órbita a 64 ou 128 para produzir períodos de órbita na ordem de 4-32 ms e fornecer uma alta resolução temporal para determinar a posição da partícula. </li></ol></li><li> Alterar o DC offset sinal enviado para os espelhos, a fim de centralizar o feixe sobre a partícula através da interface gráfica do usuário através de uma seleção cursor na imagem digitalizada-raster. </li><li> Começar a acompanhar desligando o modo de microscópio de varredura de geração de imagens para digitalização orbital. </li><li> Ative a coleta feedback e dados. </li></ol> 4. TrajectAnálise ory <ol><li> Usar o software para exibir a fluorescência recolhidos em cada ponto ao longo da órbita e, em cada ponto de tempo sob a forma de um «tapete intensidade &#39;. A intensidade coletadas ao longo do tapete fornece informações sobre a interação da partícula com outros objetos brilhantes. Usar o software para mostrar tanto a informação percurso (isto é, o deslocamento de DC ao longo do tempo de x, y e digitalizadores do -piezo z), bem como a intensidade de fluorescência recolhido a partir do tubo de fotomultiplicador ao longo do tempo sob a forma de séries de tempo. </li><li> Use a representação de séries temporais e as informações &#39;intensidade tapete &quot;para selecionar apenas uma região de interesse na trajetória. </li><li> Use o software para exibir uma projeção 2D da parte selecionada da trajetória da partícula. Selecione os controles apropriados para cor-código a trajetória de acordo com a intensidade de fluorescência de partículas. </li><li> Selecione a opção para exibir o partigo trajetória em 3D, e cor code-lo de acordo com a intensidade de fluorescência. Gire a trajetória em 3D usando os controles para visualizar as características do movimento lisossomo ao longo do microtúbulo. </li></ol>

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	<li>So, P. T., Dong, C. Y., Masters, B. R., Berland, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+photon+excitation+fluorescence+microscopy.">Two photon excitation fluorescence microscopy.</a> Annu Rev Biomed Eng. 2, 399-429 (2000).</li><li>Dupont, A., Lamb, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+three+dimensional+single+particle+tracking.">Nanoscale three dimensional single particle tracking.</a> Nanoscale. 3, 4532-4541 (2011).</li><li>Estrada, L. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+motors+at+the+intersection+of+trafficking+and+transport.">Microtubule motors at the intersection of trafficking and transport.</a> Trends Cell Biol. 16, 530-537 (2006).</li><li>Cardarelli, F., Lanzano, L., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+directed+molecular+motion+in+the+nuclear+pore+complex+of+live+cells.">Capturing directed molecular motion in the nuclear pore complex of live cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 9863-9868 (2012).</li></ol>

The authors have nothing to disclose.

Apesar dos avanços enormes de técnicas de microscopia de fluorescência e instrumentações, nos últimos anos, alcançando trajetórias de partículas fluorescentes em três dimensões com uma resolução temporal alta manteve-se um desafio no campo. Se alta resolução temporal foi alcançado partículas de rastreio em duas dimensões, a extensão no sentido axial tipicamente traz uma drástica redução na resposta de frequência do sistema 10. Neste protocolo de vídeo, foca...

Um grande número de abordagens têm sido desenvolvidas até agora para controlar partículas fluorescentes em três dimensões utilizando um microscópio. A maioria das abordagens se baseiam na utilização de câmeras rápidas, ideal para acompanhar em duas dimensões, normalmente combinados com modificações personalizadas da óptica de emissão do microscópio para alcançar rastreamento na direção axial. Microscópios de varredura a laser (ou confocal ou dois fótons) convencionalmente pode acompanhar uma partícula fluorescente através da realização de uma seqüência de tempo de z-stacks, embora este processo é normalmente demorado, e produz resolução de tempo razoável (10 Hz) somente se a partícula ser rastreado é mantida no centro de uma pequena região de imagem raster por um mecanismo de feedback activo 2. A ideia de bloqueio em que a partícula é a base do método de controle orbital. Em vez de um raster digitalizar uma órbita circular é realizada em torno da partícula fluorescente. A intensidade da fluorescência ao longoa órbita localiza com precisão a posição de partícula 4. A posição localizada da partícula pode então ser utilizado para accionar os scanners de microscópio e centro-re a órbita na posição de partícula. Os scanners galvanométricos do microscópio são accionados por uma tensão analógica. O componente AC desta voltagem permite realizar uma órbita com o feixe de laser focado, ou seja, um seno e co-seno de onda aplicada aos X e Y digitalização espelhos irá permitir a realização de uma órbita circular. O desvio do sinal de corrente contínua facilita a mudança de posição do centro da órbita. Depois de um período de órbita, é determinada e a forma de onda para o sinal de corrente alternada está configurada, o sistema de realimentação tem de actualizar apenas o componente DC do sinal. Um software capaz de ler o sinal de fluorescência recolhido dos detectores é necessário para controlar os leitores e detectores, para calcular a posição da partícula e actualizar o CC foradefinido. Para uma reacção de sucesso imagens de uma cuidadosa escolha dos parâmetros da órbita (tamanho e de sincronização) é necessária e estes parâmetros têm de ser ajustadas com base nas características das partículas fluorescentes que é necessário para controlar. Os fundamentos físicos e matemáticos da técnica foram descritos no passado de 4,5 para ambas as aplicações de 2D e 3D. Neste protocolo, descrevemos resumidamente os principais componentes da configuração de hardware, e em mais detalhe a escolha dos parâmetros e a preparação da amostra necessária para uma experiência típica, permitindo-nos a seguir o deslocamento de um lisossoma em alta resolução temporal dentro de uma célula viva.

<table border="0" cellpadding="0" cellspacing="0" width="525">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:197px;">Name</td>
			<td style="width:160px;">Company</td>
			<td style="width:168px;">Model</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysotracker DND 26</td>
			<td>Life technologies</td>
			<td>L-7526</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tubuline tracker Green</td>
			<td>Life technologies</td>
			<td>T34075</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mitotracker RED</td>
			<td>Life technologies</td>
			<td>M7512</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coherent Chamelon- ultra II TI</td>
			<td>Coherent</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcite polarize</td>
			<td>Melles Griot Glan&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Galvanometer-motor mirror</td>
			<td>Cambridge technologies</td>
			<td>M 6350</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dichroic mirror</td>
			<td>Chroma technologies</td>
			<td>700 DCSPXR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Motorized stage</td>
			<td>ASI</td>
			<td>MS2000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Piezo&#160;</td>
			<td>PI</td>
			<td>P721-LLQ</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>H7422P-40</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Data acquisition card</td>
			<td>IO tech</td>
			<td>PCI 1128-4000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging software</td>
			<td>LFD</td>
			<td>Global for images-SimFCS</td>
		</tr>
	</tbody>
</table>

3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles

O objetivo deste protocolo de vídeo é discutir como executar e analisar uma fluorescente orbital experimento de rastreamento de partículas tridimensional usando uma versão modificada do microscópio de dois fótons 1. Ao contrário de abordagens convencionais (digitalização raster ou campo grande baseado em uma pilha de quadros), o monitoramento orbital 3D permite localizar e seguir com uma alta espacial (precisão nm 10) e resolução temporal (frequência de resposta de 50 Hz) o deslocamento 3D uma partícula fluorescente passar comprimento escalas de centenas de microns 2. O método baseia-se num algoritmo de realimentação que controla o hardware de um de dois fotões microscópio de varrimento laser, a fim de realizar uma órbita circular em torno do objecto a ser rastreado: o mecanismo de realimentação vai manter o objecto fluorescente no centro através do controlo da deslocação da o feixe de varrimento 3-5. Para demonstrar as vantagens desta técnica, seguimos uma organela em movimento rápido, o lisossoma, dentro de alcélula iving 6,7. As células foram semeadas de acordo com protocolos convencionais, e corados com um corante comercialmente lisossoma. Discutimos brevemente a configuração de hardware e de forma mais detalhada o software de controle, para realizar um experimento de monitoramento orbital 3D dentro de células vivas. Discutimos detalhadamente os parâmetros necessários, a fim de controlar o microscópio de varredura e permitir o movimento do feixe em uma órbita fechada em torno da partícula. Conclui-se, demonstrando como esse método pode ser efetivamente usado para rastrear o movimento rápido de um lisossoma marcado ao longo dos microtúbulos em 3D dentro de uma célula viva. Lisossomas podem mover-se com velocidades na gama de 0,4-0,5 um / seg, exibindo tipicamente um movimento dirigido ao longo da rede de microtúbulos 8.

De acordo com este protocolo de rastreio rápido 3D única partícula pode ser realizada dentro de células vivas utilizando um microscópio de dois fotões modificado para controlar o deslocamento de lisossomos marcados com fluorescência. O experimento realizado consiste em acompanhamento de um lisossoma isolado em movimento no interior da célula após o processo de maturação endosome 9. Os lisossomos foram coradas utilizando um corante fluorescente verde e animado em 930 nm exploram dois fótons de exci...

Watch this Scientific Journal Video about 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles at JoVE.com

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3D Orbital Rastreamento em um microscópio de dois fotões Modificação: Uma Aplicação para o rastreamento de vesículas intracelulares

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

3d-orbital-tracking-modified-two-photon-microscope-an-application-to

Research

JoVE Journal

Bioengineering

10.1K visualizações.  University of California, Irvine. In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope. 

Vídeo: 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

Rastreamento de Sinalização Hipóxico dentro agregados celulares Encapsulated

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

Bioengenharia

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

Medição Longitudinal de Matriz Extracelular Rigidez em 3D tumorais modelos com o uso de rastreamento de partículas Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Longo prazo intravital Imunofluorescência imagem do tecido componentes da matriz com epifluorescência e microscopia de dois fótons

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Em Situ Mapeamento das propriedades mecânicas de biofilmes por de rastreamento de partículas Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Plataforma microfluídico com multiplexada detecção electrónica do Ordenamento do Rastreamento de Partículas

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

Montagem e Rastreamento do Desenvolvimento de Comunidade Microbiana em uma Plataforma Microwell Array

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Um protocolo para rastreamento em tempo real 3D única partícula

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Um Orbital agitando a cultura de células de mamíferos em vasos em forma do produzir agregados uniformes

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Segmentando o crescimento de células endoteliais em placas de 6-bem em um agitador orbital para estudos mecanobiológicos

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.