Name: use of an optical trap for study of host-pathogen interactions for dynamic live cell imaging
Uploaded: 2011-07-28T12:00:00
Description: Watch this Scientific Journal Video about Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging at JoVE.com

Эта работа была поддержана Massachusetts General Hospital отделение медицины фонды Внутренняя (СГН, MKM, MLC, JMV), Национальный институт биомедицинской визуализации и биоинженерии грант T32EB006348 (ЦИК), центр Массачусетского общего госпиталя для вычислительной и интегративной фонда развития биологии и AI062773 ( RJH), гранты AI062773, DK83756 и DK 043 351 (RJX), NSF 0643745 (MJL), NIH R21CA133576 (MJL) и Национального института аллергии и инфекционных заболеваний (NIAID) из Национального института здоровья (NIH) AI057999 (JMV ). Мы благодарим Николая С. Йодер за полезные обсуждения, и Чарльз войлок (RPI, Inc) для оказания технической помощи.

1. Условия культивирования патогенных для оптического захвата <ol><li> Расти А. fumigatus (B-5233/RGD12-8) на полутвердых сред, содержащих агар SBD (Сабуро декстрозы) при температуре 30 ° С в течение 3 дней. </li><li> Расти C. Albicans (SC5314) в YPD (дрожжи-пептон декстроза) культуральной жидкости, содержащей 10...

<ol>
	<li>Grakoui, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunological+synapse:+A+molecular+machine+controlling+T+cell+activation.">The immunological synapse: A molecular machine controlling T cell activation.</a> Science. 285, 221-227 (1999).</li><li>Monks, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+segregation+of+supramolecular+activation+clusters+in+T+cells.">Three-dimensional segregation of supramolecular activation clusters in T cells.</a> Nature. 395, 82-86 (1998).</li><li>Stuart, L. M., Ezekowitz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+and+comparative+innate+immunity:+Learning+on+the+fly.">Phagocytosis and comparative innate immunity: Learning on the fly.</a> Nat Rev Immunol. 8, 131-141 (2008).</li><li>Neuman, K. C., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping.">Optical trapping.</a> Rev Sci Instrum. 75, 2787-2809 (2004).</li><li>Ashkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping+and+manipulation+of+neutral+particles+using+lasers.">Optical trapping and manipulation of neutral particles using lasers.</a> Proc Natl Acad Sci USA. 94, 4853-4860 (1997).</li><li>Ashkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+and+trapping+of+particles+by+radiation+pressure.">Acceleration and trapping of particles by radiation pressure.</a> Phys Rev Lett. 24, 156-159 (1970).</li><li>Ashkin, A., Dziedzic, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping+and+manipulation+of+viruses+and+bacteria+Science.">Optical trapping and manipulation of viruses and bacteria Science.</a> Nature. 235, 1517-1520 (1987).</li><li>Khalil, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+M13+bacteriophage+tethering+and+stretching.">Single M13 bacteriophage tethering and stretching.</a> Proc Natl Acad Sci USA. 104, 4892-4897 (2007).</li><li>Khalil, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin's+cover-neck+bundle+folds+forward+to+generate+force.">Kinesin's cover-neck bundle folds forward to generate force.</a> Proc Natl Acad Sci USA.. 105, 19247-19252 (2008).</li><li>Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+tether+formation+from+outer+hair+cells+with+optical+tweezers.">Membrane tether formation from outer hair cells with optical tweezers.</a> Biophys J. , 1386-1395 (2002).</li><li>Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&#945;&#946;+T+cell+receptor+is+an+anisotropic+mechanosensor.">The &#945;&#946; T cell receptor is an anisotropic mechanosensor.</a> J Biol Chem. 284, 31028-31028 (2009).</li><li>Mohanty, S., Mohanty, K., Gupta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+interaction+of+RBC+with+optical+tweezers.">Dynamics of interaction of RBC with optical tweezers.</a> Opt. Express. 13, 4745-4751 (2005).</li><li>Tam, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+and+manipulation+of+pathogens+with+an+optical+trap+for+live+cell+imaging+of+intercellular+interactions.">Control and manipulation of pathogens with an optical trap for live cell imaging of intercellular interactions.</a> PLoS One. 5, e15215-e15215 (2010).</li><li>Lin, S. J., Schranz, J., Teutsch, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspergillosis+case-fatality+rate:+Systematic+review+of+the+literature.">Aspergillosis case-fatality rate: Systematic review of the literature.</a> Clin Infect Dis.. 32, 358-366 (2001).</li><li>Wey, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hospital-acquired+candidemia+-+the+attributable+mortality+and+excess+length+of+stay.">Hospital-acquired candidemia - the attributable mortality and excess length of stay.</a> Arch. Intern. Med. 148, 2642-2645 (1988).</li></ol>

В этой работе мы используем оптическую ловушку, чтобы захватить патогенов с размерами от 3 мкм - 5 мкм. Хотя возбудители интерес к нашей лаборатории как правило, эти размеры, оптическая система пинцет, описанный здесь, гибкими, чтобы ловушка большой диапазон размеров. Действительно оптич...

<table border="1"><tbody><tr><th> Название реагента </th><th> Компания </th><th> Номер в каталоге </th><th> Комментарии (необязательно) </th></tr><tr><td> A. fumigatus </td><td></td><td></td><td> Альбино напряжения, B-5233/RGD12-8, подарок от KJ Kwon-Chung, NIH </td></tr><tr><td> C. Albicans </td><td></td><td></td><td> SSY50-B мутант, подарок от Элефтериос Mylonakis, MGH; SC5314 напряжения, подарок от Джеральда Финк, Уайтхед Институт </td></tr><tr><td> Alexa Fluor 488 </td><td> Invitrogen </td><td> A20000 </td><td></td></tr><tr><td> Alexa Fluor 647 </td><td> Invitrogen </td><td> A20006 </td><td></td></tr><tr><td> диметилформамиде </td><td> Сигма </td><td> D4551 </td><td></td></tr><tr><td> Свежая кровь </td><td></td><td></td><td> Подарок от RJW Хит, MGH, HMS </td></tr><tr><td> Nikon инвертированного микроскопа </td><td> Nikon </td><td></td><td> Модель Ti-E </td></tr><tr><td> Перехват лазер, ChromaLase </td><td> Синий исследований Sky </td><td> CLAS-106-STF02-02 </td><td></td></tr><tr><td> Лазерного возбуждения флуоресценции </td><td> Когерентный </td><td></td><td> Модель Innova 70C </td></tr><tr><td> Макеты для улавливания компонентов </td><td> Thorlabs </td><td> MB1224, MB1218 </td><td></td></tr><tr><td> Оптический стол воздуха </td><td> Технические Manufacturing Corporation </td><td></td><td></td></tr><tr><td> Электронный затвор с педалью управления </td><td> Uniblitz </td><td></td><td> Приобретенные от Винсента Associates, Рочестер, штат Нью-Йорк </td></tr><tr><td> Одномодовое оптическое волокно </td><td> Оз оптики </td><td> PMJ-3S3S-1064-6 </td><td></td></tr><tr><td> Волоконно позиционером </td><td> Thorlabs </td><td> СУП-X-5-C </td><td></td></tr><tr><td> Волоконно коллиматор </td><td> Оз оптики </td><td> HPUCO-23-1064-P-25AC </td><td></td></tr><tr><td> Объективы для телескопов </td><td> Thorlabs </td><td> AC254-150-B </td><td> Фокусное расстояние 150 мм </td></tr><tr><td> Перевод этапов (х, у, г) </td><td> Ньюпорт </td><td> М-461-XYZ </td><td></td></tr><tr><td> ИК дихроичных зеркал </td><td> Цветность </td><td> ET750-SP-2p8 </td><td></td></tr><tr><td> Объектив (100X) </td><td> Nikon </td><td></td><td> NA = 1,49, нефть погружения, TIRF цель </td></tr><tr><td> Конфокальной головы </td><td> Yokogawa </td><td> ХСС-XI </td><td></td></tr><tr><td> Поляризатор </td><td> Nikon </td><td> MEN51941 </td><td></td></tr><tr><td> Призмы Волластона </td><td> Nikon </td><td> MBH76190 </td><td></td></tr><tr><td> EM-CCD камера </td><td> Hamamatsu </td><td> C9100-13 </td><td></td></tr><tr><td> ПЗС-камера (ORCA ER) </td><td> Hamamatsu </td><td> C4742-80-12AG </td><td></td></tr><tr><td> Фильтры колесо </td><td> Ludl </td><td> 99A353 </td><td></td></tr><tr><td> Фильтры колесо </td><td> Саттер </td><td> LB10-NWE </td><td></td></tr><tr><td> Патрон покровного стекла </td><td> Lab-Tek/Nunc </td><td> 155409 </td><td></td></tr><tr><td> Dynabeads </td><td> Invitrogen </td><td> 111-51D </td><td> Покрытие анти-CD3 </td></tr><tr><td> Дульбеко изменение Орла среды (DMEM) </td><td> Invitrogen / Gibco </td><td> 10313 </td><td></td></tr><tr><td> Пенициллин / стрептомицин </td><td> Invitrogen / Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> L-глютамин </td><td> Invitrogen / Gibco </td><td> 25030-081 </td><td></td></tr><tr><td> Эмбриональной телячьей сыворотки (Hyclone) </td><td> ThermoScientific </td><td> SH30071.03 </td><td></td></tr></tbody></table>

use of an optical trap for study of host-pathogen interactions for dynamic live cell imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

Watch this Scientific Journal Video about Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging at JoVE.com

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Метод описан в индивидуальном порядке выбирать, манипулировать, и изображение живых патогенов при оптической ловушки связаны с вращающимся диском микроскопом. Оптической ловушки обеспечивает пространственной и временной контроль организмов и помещает их рядом с клетками хозяина. Люминесцентной микроскопии захватывает динамических межклеточных взаимодействий с минимальным возмущением к клеткам.

Использование оптической ловушки для изучения хост-Возбудитель Взаимодействие для динамических изображений Живая сотовый

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Research

JoVE Journal

Immunology and Infection

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Использование EpiAirway модель Характеризуя Долгосрочные хост-возбудителя Взаимодействия

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Сравнительный подход к характеризации Пейзаж хозяин-патоген белок-белковых взаимодействий

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Study of Phagolysosome Biogenesis in Live Macrophages

Изучение фаголизосомы биогенеза в живых макрофагов

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Использование<em&gt; Shigella flexneri</em&gt; Для изучения аутофагии-Цитоскелет взаимодействий

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Оценка эффективности и токсичности РНК Ориентация ВИЧ-1 Производство для использования в генной или терапии наркотиками

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Изготовления поверхностей оптическое качество стекла для изучения макрофагов Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

В пробирке Формирование собак нейтрофилов внеклеточного ловушка: Динамические и количественный анализ по микроскопии флуоресцирования

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Оценка хост возбудитель ответы и эффективность вакцины в мышей

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Инфекция личинок зебры с спорами Аспергилла для анализа взаимодействия хост-патогенов

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

Без этикетки Количественные Протеомика Рабочий процесс для Discovery-Driven Хост-патоген взаимодействия

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...