This work was supported by Massachusetts General Hospital Department of Medicine Internal Funds (J.M.T., M.K.M, M.L.C., J.M.V.), National Institute of Biomedical Imaging and Bioengineering grant T32EB006348 (C.E.C.), Massachusetts General Hospital's Center for Computational and Integrative Biology development fund and AI062773 (R.J.H.), grants AI062773, DK83756, and DK 043351 (R.J.X.), NSF 0643745 (M.J.L.), NIH R21CA133576 (M.J.L.), and National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) AI057999 (J.M.V.). We thank Nicholas C. Yoder for helpful discussions, and Charles Felts (RPI, Inc.) for technical assistance.

1. Culture conditions of pathogens for optical trapping
<ol>
	<li>Grow A. fumigatus (B-5233/RGD12-8) on a semi-solid agar media containing SBD (Sabouraud dextrose) at 30&deg;C for 3 days. </li>
	<li>Grow C. albicans (SC5314) in YPD (Yeast-Peptone Dextrose) liquid culture containing 100 &mu;g/mL ampicillin overnight in a shaker incubator at 30&deg;C.</li>
</ol>
2. Preparation of pathogens for fluorescent labeling
<ol>
	<li>Harvest desired amount of pathogens and transfer to a ...

<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>

No conflicts of interest declared.

In this work we use an optical trap to capture pathogens with dimensions between 3 &mu;m - 5 &mu;m. Although pathogens of interest to our lab typically have these dimensions, the optical tweezer system described here is flexible to trap a large range of sizes. Indeed optical traps have been used to capture particles ranging from single atoms to cells approximately 10 &mu;m in diameter. Additionally, this optical trapping system was able to capture particles of various shapes: spherical, elliptical, and extremely elon...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>A. fumigatus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Albino strain, B-5233/RGD12-8, gift from K.J. Kwon-Chung, NIH</td>
		</tr>
		<tr>
			<td>C. albicans</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>SSY50-B mutant, gift from Eleftherios Mylonakis, MGH; SC5314 strain, gift from Gerald Fink, Whitehead Institute</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A20000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>A20006</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>dimethylformamide</td>
			<td>Sigma</td>
			<td>D4551</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fresh blood</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Gift from R.J.W. Heath, MGH, HMS</td>
		</tr>
		<tr>
			<td>Nikon inverted microscope</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>Model Ti-E </td>
		</tr>
		<tr>
			<td>Trapping laser, ChromaLase</td>
			<td>Blue Sky Research</td>
			<td>CLAS-106-STF02-02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence excitation laser</td>
			<td>Coherent</td>
			<td>&nbsp;</td>
			<td>Model Innova 70C </td>
		</tr>
		<tr>
			<td>Breadboards for trapping components</td>
			<td>Thorlabs</td>
			<td>MB1224, MB1218</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Optical air table</td>
			<td>Technical Manufacturing Corporation</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electronic shutter with pedal control</td>
			<td>Uniblitz</td>
			<td>&nbsp;</td>
			<td>Purchased from Vincent Associates, Rochester, NY</td>
		</tr>
		<tr>
			<td>Singlemode optical fiber</td>
			<td>Oz Optics</td>
			<td>PMJ-3S3S-1064-6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fiber positioner</td>
			<td>Thorlabs</td>
			<td>PAF-X-5-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fiber collimator</td>
			<td>Oz Optics</td>
			<td>HPUCO-23-1064-P-25AC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lenses for telescope</td>
			<td>Thorlabs</td>
			<td>AC254-150-B</td>
			<td>Focal length of 150 mm</td>
		</tr>
		<tr>
			<td>Translation stages (x, y, z)</td>
			<td>Newport</td>
			<td>M-461-XYZ</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IR dichroic mirror</td>
			<td>Chroma</td>
			<td>ET750-sp-2p8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Objective lens (100X)</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>NA = 1.49, oil immersion, TIRF objective</td>
		</tr>
		<tr>
			<td>Confocal head</td>
			<td>Yokogawa</td>
			<td>CSU-XI</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polarizer</td>
			<td>Nikon</td>
			<td>MEN51941</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Wollaston prism</td>
			<td>Nikon</td>
			<td>MBH76190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EM-CCD camera</td>
			<td>Hamamatsu</td>
			<td>C9100-13</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CCD camera (ORCA ER)</td>
			<td>Hamamatsu</td>
			<td>C4742-80-12AG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter wheel</td>
			<td>Ludl</td>
			<td>99A353</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter wheel</td>
			<td>Sutter</td>
			<td>LB10-NWE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chambered coverglass</td>
			<td>Lab-Tek/Nunc</td>
			<td>155409</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dynabeads</td>
			<td>Invitrogen</td>
			<td>111-51D</td>
			<td>Coated with anti-CD3</td>
		</tr>
		<tr>
			<td>Dulbecco's modified Eagle's medium (DMEM)</td>
			<td>Invitrogen/Gibco</td>
			<td>10313</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen/Gibco</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
			<tr>
			<td>L-glutamine</td>
			<td>Invitrogen/Gibco</td>
			<td>25030-081</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (HyClone)</td>
			<td>ThermoScientific</td>
			<td>SH30071.03</td>
			<td>&nbsp;</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

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

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

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Research

JoVE Journal

Immunology and Infection

11.9K visualizações.  Massachusetts General Hospital, Harvard Medical School. Um método é descrito para selecionar individualmente, manipular e patógenos imagem ao vivo usando uma armadilha óptica acoplada a um microscópio disco giratório. A armadilha óptica fornece controle espacial e temporal dos organismos e os coloca ao lado células hospedeiras. Microscopia de fluorescência captura dinâmica interações intercelulares com perturbação mínima para as células.