गतिशील लाइव सेल इमेजिंग के लिए मेजबान पैथोजन सहभागिता के अध्ययन के लिए एक ऑप्टिकल जाल का प्रयोग करें
Summary
एक विधि के लिए व्यक्तिगत का चयन करें, हेरफेर वर्णित है, और छवि रहते रोगज़नक़ों एक कताई डिस्क खुर्दबीन एक ऑप्टिकल मिलकर जाल का उपयोग कर. ऑप्टिकल जाल स्थानिक और लौकिक जीवों के नियंत्रण प्रदान करता है और उन्हें मेजबान कोशिकाओं के लिए आसन्न स्थानों. प्रतिदीप्ति माइक्रोस्कोपी कोशिकाओं को कम से कम गड़बड़ी के साथ गतिशील कहनेवाला बातचीत कब्जा है.
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
Discussion
5 सुक्ष्ममापी – इस काम में हम एक ऑप्टिकल 3 सुक्ष्ममापी बीच आयामों के साथ जाल रोगज़नक़ों कब्जा का उपयोग. हालांकि हमारी प्रयोगशाला में ब्याज की रोगज़नक़ों आम तौर पर इन आयाम है, ऑप्टिकल चिमटी से नोचना यहाँ ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
इस काम मैसाचुसेट्स चिकित्सा आंतरिक फंड के जनरल अस्पताल विभाग (JMT, MKM, विधान परिषद के सदस्य, JMV), नेशनल इंस्टीट्यूट ऑफ बायोमेडिकल इमेजिंग के और बायोइन्जिनियरिंग T32EB006348 अनुदान (सीईसी), कम्प्यूटेशनल और एकीकृत जीवविज्ञान विकास निधि के लिए मैसाचुसेट्स जनरल ने अस्पताल केंद्र और AI062773 द्वारा समर्थित था ( RJH), AI062773 अनुदान, DK83756, और 043351 डी.के. (RJX), NSF 0643745 (MJL), R21CA133576 NIH (MJL), और एलर्जी और संक्रामक रोगों के राष्ट्रीय संस्थान (NIAID) के राष्ट्रीय स्वास्थ्य संस्थान के (NIH) के AI057999 (JMV ). हम निकोलस सी. Yoder उपयोगी विचार – विमर्श के लिए धन्यवाद, और तकनीकी सहायता के लिए चार्ल्स Felts (आरपीआई इंक).
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 |
References
- Grakoui, A. The immunological synapse: A molecular machine controlling T cell activation. Science. 285, 221-227 (1999).
- Monks, C. R. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature. 395, 82-86 (1998).
- Stuart, L. M., Ezekowitz, R. A. Phagocytosis and comparative innate immunity: Learning on the fly. Nat Rev Immunol. 8, 131-141 (2008).
- Neuman, K. C., Block, S. M. Optical trapping. Rev Sci Instrum. 75, 2787-2809 (2004).
- Ashkin, A. Optical trapping and manipulation of neutral particles using lasers. Proc Natl Acad Sci USA. 94, 4853-4860 (1997).
- Ashkin, A. Acceleration and trapping of particles by radiation pressure. Phys Rev Lett. 24, 156-159 (1970).
- Ashkin, A., Dziedzic, J. Optical trapping and manipulation of viruses and bacteria Science. Nature. 235, 1517-1520 (1987).
- Khalil, A. S. Single M13 bacteriophage tethering and stretching. Proc Natl Acad Sci USA. 104, 4892-4897 (2007).
- Khalil, A. S. Kinesin’s cover-neck bundle folds forward to generate force. Proc Natl Acad Sci USA.. 105, 19247-19252 (2008).
- Li, Z. Membrane tether formation from outer hair cells with optical tweezers. Biophys J. , 1386-1395 (2002).
- Kim, S. The αβ T cell receptor is an anisotropic mechanosensor. J Biol Chem. 284, 31028-31028 (2009).
- Mohanty, S., Mohanty, K., Gupta, P. Dynamics of interaction of RBC with optical tweezers. Opt. Express. 13, 4745-4751 (2005).
- Tam, J. Control and manipulation of pathogens with an optical trap for live cell imaging of intercellular interactions. PLoS One. 5, e15215-e15215 (2010).
- Lin, S. J., Schranz, J., Teutsch, S. M. Aspergillosis case-fatality rate: Systematic review of the literature. Clin Infect Dis.. 32, 358-366 (2001).
- Wey, S. B. Hospital-acquired candidemia – the attributable mortality and excess length of stay. Arch. Intern. Med. 148, 2642-2645 (1988).
