Summary

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

Published: July 28, 2011
doi:

Summary

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.

Abstract

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.

Protocol

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

Discussion

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…

Declarações

The authors have nothing to disclose.

Acknowledgements

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.

Materials

Name of the reagent Company Catalogue number Comments (optional)
A. fumigatus     Albino strain, B-5233/RGD12-8, gift from K.J. Kwon-Chung, NIH
C. albicans     SSY50-B mutant, gift from Eleftherios Mylonakis, MGH; SC5314 strain, gift from Gerald Fink, Whitehead Institute
Alexa Fluor 488 Invitrogen A20000  
Alexa Fluor 647 Invitrogen A20006  
dimethylformamide Sigma D4551  
Fresh blood     Gift from R.J.W. Heath, MGH, HMS
Nikon inverted microscope Nikon   Model Ti-E
Trapping laser, ChromaLase Blue Sky Research CLAS-106-STF02-02  
Fluorescence excitation laser Coherent   Model Innova 70C
Breadboards for trapping components Thorlabs MB1224, MB1218  
Optical air table Technical Manufacturing Corporation    
Electronic shutter with pedal control Uniblitz   Purchased from Vincent Associates, Rochester, NY
Singlemode optical fiber Oz Optics PMJ-3S3S-1064-6  
Fiber positioner Thorlabs PAF-X-5-C  
Fiber collimator Oz Optics HPUCO-23-1064-P-25AC  
Lenses for telescope Thorlabs AC254-150-B Focal length of 150 mm
Translation stages (x, y, z) Newport M-461-XYZ  
IR dichroic mirror Chroma ET750-sp-2p8  
Objective lens (100X) Nikon   NA = 1.49, oil immersion, TIRF objective
Confocal head Yokogawa CSU-XI  
Polarizer Nikon MEN51941  
Wollaston prism Nikon MBH76190  
EM-CCD camera Hamamatsu C9100-13  
CCD camera (ORCA ER) Hamamatsu C4742-80-12AG  
Filter wheel Ludl 99A353  
Filter wheel Sutter LB10-NWE  
Chambered coverglass Lab-Tek/Nunc 155409  
Dynabeads Invitrogen 111-51D Coated with anti-CD3
Dulbecco’s modified Eagle’s medium (DMEM) Invitrogen/Gibco 10313  
Penicillin/streptomycin Invitrogen/Gibco 15140-122  
L-glutamine Invitrogen/Gibco 25030-081  
Fetal Bovine Serum (HyClone) ThermoScientific SH30071.03  

Referências

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Tam, J. M., Castro, C. E., Heath, R. J. W., Mansour, M. K., Cardenas, M. L., Xavier, R. J., Lang, M. J., Vyas, J. M. Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging. J. Vis. Exp. (53), e3123, doi:10.3791/3123 (2011).

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