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

Podsumowanie

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.

Streszczenie

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.

Protokół

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 at 4000 rpm for 1 minute.
5. Aspirate supernatant, leaving pellet undisturbed.
6. Repeat (2.4) and (2.5) two more times.
7. Resuspend in 500μL of PBS.

3. Labeling of pathogens with fluorescent dye

1. Dissolve 1 mg of dye of interest (e.g. Alexa Fluor 488, Alexa Fluor 647) in 100 μL dimethylformamide (DMF) (concentration of 10 mg/mL).
2. Add 3 μL of dye mixture to reaction tubes containing washed pathogens.
3. Rotate or shake sample at 37°C for 1 hour.
4. Wash sample with PBS 3X by centrifuging at 4000 rpm for 1 minute.
5. Resuspend in 300 μL of PBS.

4. Harvesting T-cells from whole blood

1. Obtain whole blood (fresh).
2. Warm blood, PBS + 2% fetal bovine serum (FBS), and histopaque to room temperature.
3. Add RosetteSep Human CD4+ T Cell Enrichment Cocktail at 50 μL/mL of whole blood.
4. Rotate sample and incubate for 20 minutes at room temperature.
5. Dilute sample with an equal volume of PBS + 2% FBS and mix gently.
6. Layer diluted sample on top of histopaque, minimizing mixing
7. Centrifuge for 20 minutes at 1200 x g at room temperature with the brake off.
8. Remove the enriched cells.
9. Wash the enriched cells 2x with PBS + 2% FBS solution.
10. Lyse red blood cells for 2 minutes with red blood cell lysing buffer
11. Add 10 mL of PBS + 2% FBS and centrifuge lysed red blood cells at 1500 rpm for 5 minutes
12. Aspirate supernatant, careful not to disturb the pellet
13. Resuspend in IMDM media containing 10% fetal bovine serum

5. Preparation of RAW 264.7 macrophages into chamber slides

1. Prepare DMEM (Dulbecco's modified Eagle's medium) to contain 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine.
2. Warm media, trypsin, and PBS in warm bath to 37°C.
3. Wash plate 2x with PBS
4. Aspirate PBS between each wash.
5. Add 5 mL trypsin to plate to cover surface (for a 10 cm tissue culture plate).
6. Incubate for 5 min at 37°C.
7. Gently knock side of plate to detach cells from plate surface. Be careful not to splash trypsin outside of the plate.
8. Add 5 mL or equivalent amount of media to trypsin.
9. Aspirate mixture into a reaction tube.
10. Centrifuge at 1000 x g for 3 minutes.
11. Aspirate media, careful not to disturb the pellet.
12. Resuspend in 10 mL of media.
13. Add 400 μL of media to each chamber of the chamber slide.
14. Add 5 μL of cell suspension to each chamber.
15. Grow overnight in incubator at 37°C with 5% CO₂.

6. Addition of pathogens to sample

1. Pipet 5-10 μL of labeled pathogens of interest (from steps 1-3) into chamber.
2. Mix thoroughly by pipeting up and down, careful not to touch the bottom of the chamber to disturb the adhered macrophages.

7. Loading sample onto spinning disk confocal microscope (in video, scan through components)

1. Turn on all components to spinning disk confocal microscope.
2. Align microscope for DIC imaging.
3. Remove chamber slide from incubator.
4. Insert chamber slide into specialized stage.
5. Remove top of chamber slide (necessary for DIC imaging).

8. Preparation for optical trapping (in video, scan through components and how it's integrated into microscope)

1. Turn on shutter for optical trap.
2. Turn on IR laser.
3. Open shutter (on the laser) to optical trap.
4. Confirm shutter in front of IR laser is closed by checking with IR card.

9. Selection and manipulation of pathogen with optical trap

1. Focus on macrophages on adhered slide.
2. Find pathogens freely fluctuating in solution adjacent to macrophages.
3. Move stage such that the pathogen is in the vicinity of the trap.
4. Open shutter and engage the trap.
5. Move sample to bring macrophages into contact with the stationary trapped pathogen.
6. Image with spinning disk confocal microscope, either in DIC, fluorescence, or combination of both. Typically, trapping is done in DIC, and real-time imaging is done with fluorescence.

10. Representative Results:

To exert full spatial and temporal control of pathogens and beads in an in vivo and in vitro environment, we designed a custom-built trapping apparatus integrated onto a spinning disk confocal microscope (schematic shown in Fig. 1). At full strength, the laser provided 350 mW of power and after coupling the light with the various optical components in the optical trap apparatus, ˜80 mW of power at the TIRF objective, as measured with a power meter, was used to form the trap in the chamber.

In order to position an object in the chamber relative to the trapped object, the stage was moved while holding the trapped object stationary with the trapping laser. The stage was moved at slow enough velocities, so that the drag force on the trapped particle did not exceed the maximum trapping force.

Separate populations of C. albicans (typical size - ˜5 μm) were labeled with each of three colors (AF488, AF568, and AF647, corresponding to green, blue, and red in the figure, respectively) to illustrate imaging with multiple fluorescence channels while simultaneously optically trapping the pathogen. A single C. albicans was trapped and moved in a square pattern through a cluster of other yeast, as indicated by gray arrows, demonstrating the ability to capture and manipulate the specific location of a single pathogen chosen by the operator even in a crowded environment (Fig. 2A).

To illustrate further the flexibility of this system to trap the different shape morphologies exhibited by pathogenic organisms, the optical tweezer was also able to hold and situate a C. albicans particle with a pseudohyphae. The C. albicans labeled with AF647 (red) was moved along a trajectory as outlined by the white arrow and placed next to fluorescent GFP-LC3-RAW cells (Fig. 2B). The yeast portion of the C. albicans was trapped as the pseudohyphae trailed along.

We also positioned Aspergillus fumigatus next to a RAW mouse macrophage cell in order to analyze the absolute time frame of phagocytosis with this particular cell line and pathogen (Fig. 3A). Once the pathogen makes contact with the cell, the trap is turned off, and time-lapse imaging is employed to observe the subsequent cellular events (Fig. 3B). The optical trap was also used to capture primary T-cells isolated from blood and directed adjacent to beads coated with anti-CD3 antibody in order for the T-cell to form an immunological synapse with the bead (Fig. 4), show the additional versatility to directly trap and manipulate immune cells.

Figure 1. Overview and schematic of combined optical trap and spinning disk confocal microscope setup. Instrument layout showing the trapping beam (purple), illumination path for brightfield imaging (orange), fluorescence excitation beam (red), fluorescence emission (green), charge-coupled device (CCD) camera, electron-multiplying charge coupled device (EM-CCD) camera, dichroic mirrors (D1 and D2), mirrors (M1 and M2), and lenses (L1, L2, L3, L4). All other components of the trap-confocal microscope system are labeled in the figure.

Figure 2. (A) Fluorescence images of trapped and manipulated C. albicans. A trapped fluorescently-labeled (Alexa Fluor 488 (AF488), blue) organism in a field of other fluorescently-labeled C. albicans (AF568, green and AF647, red). The stage is moved around the trapped, blue CA particle as indicated by the gray arrows. (B) Trapped pseudo-hyphal form of C. albicans next to GFP-LC3 RAW cell. Fluorescently labeled psuedohyphal form of CA (red, AF647) optically trapped and moved adjacent to GFP-LC3 expressed RAW macrophages. The CA organism is moved along the trajectory as indicated by the white arrow.

Figure 3. Trapping and positioning of A. fumigatus next to a phagocytic RAW cell. (A) Brightfield images of a trapped A. fumigatus, as indicated by the white arrow, moved and positioned along the path as indicated by the red arrow. The trapped pathogen is slightly out of focus due to the trap pushing the organism slightly above the focal plane. A. fumigatus is moved until it is placed adjacent to the desired RAW cell. (B) Fluorescence imaging of phagocytosis of A. fumigatus by RAW cell. After the trapped pathogen is placed next to a RAW cell, the phagocytosis process is activated. At 30 s, the membrane of the RAW cell starts to change and form a cup around the particle. At 60 s, the cup is fully formed. From 90s to 150s, the A. fumigatus is engulfed, and by 180 s, the particle is fully internalized

Figure 4. Trapping primary T-cell to form synapse with anti-CD3 coated beads. DIC images of a primary T-cell moved by the optical trap to a position next to a bead coated with anti-CD3 antibody. The cell then forms an immunological synapse, which cannot be easily discerned in the images.

Dyskusje

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...

Materiały

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