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

Summary

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.

Abstract

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system^{1, 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 1970^{5, 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 phenomena^8-13.

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 individuals^{15, 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. Culture conditions of pathogens for optical trapping

1. Grow A. fumigatus (B-5233/RGD12-8) on a semi-solid agar media containing SBD (Sabouraud dextrose) at 30°C for 3 days.
2. Grow C. albicans (SC5314) in YPD (Yeast-Peptone Dextrose) liquid culture containing 100 μg/mL ampicillin overnight in a shaker incubator at 30°C.

2. Preparation of pathogens for fluorescent labeling

1. Harvest desired amount of pathogens and transfer to a 1.5 mL reaction tube.
2. Add 300 μL of phosphate buffered saline (PBS) to reaction tube.
3. Sonicate mixture for 30 seconds.
4. Centrifuge a....

Discussion

In this work we use an optical trap to capture pathogens with dimensions between 3 μm - 5 μm. 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 μm in diameter. Additionally, this optical trapping system was able to capture particles of various shapes: spherical, elliptical, and extremely elon.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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-Aldrich	D4551
Fresh blood			Gift from R.J.W. Heath, MGH, HMS
Nikon inverted microscope	Nikon Instruments		Model Ti-E
Trapping laser, ChromaLase	Blue Sky Research	CLAS-106-STF02-02
Fluorescence excitation laser	Coherent Inc.		Model Innova 70C
Breadboards for trapping components	Thorlabs Inc.	MB1224, MB1218
Optical air table	Technical Manufacturing Corp.
Electronic shutter with pedal control	Uniblitz		Purchased from Vincent Associates, Rochester, NY
Singlemode optical fiber	Oz Optics	PMJ-3S3S-1064-6
Fiber positioner	Thorlabs Inc.	PAF-X-5-C
Fiber collimator	Oz Optics	HPUCO-23-1064-P-25AC
Lenses for telescope	Thorlabs Inc.	AC254-150-B	Focal length of 150 mm
Translation stages (x, y, z)	Newport Corp.	M-461-XYZ
IR dichroic mirror	Chroma Technology Corp.	ET750-sp-2p8
Objective lens (100X)	Nikon Instruments		NA = 1.49, oil immersion, TIRF objective
Confocal head	Yokogawa	CSU-XI
Polarizer	Nikon Instruments	MEN51941
Wollaston prism	Nikon Instruments	MBH76190
EM-CCD camera	Hamamatsu Corp.	C9100-13
CCD camera (ORCA ER)	Hamamatsu Corp.	C4742-80-12AG
Filter wheel	Ludl	99A353
Filter wheel	Sutter Instrument Co.	LB10-NWE
Chambered coverglass	Lab-Tek	155409
Dynabeads	Invitrogen	111-51D	Coated with anti-CD3
Dulbecco’s modified Eagle’s medium (DMEM)	Invitrogen	10313
Penicillin/streptomycin	Invitrogen	15140-122
L-glutamine	Invitrogen	25030-081
Fetal Bovine Serum (HyClone)	Thermo Fisher Scientific, Inc.	SH30071.03