Figure 1.
Overview and schematic of combined optical trap and spinning disk confocal microscope setup.
Instrument layout (a) 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. (b) Detailed schematic of a trapped pathogen (green) is shown. The laser light (purple) refracts through the pathogen, creating a momentum change (red, labeled Fa and Fb), pushing the pathogen up and into the focused trapping beam. As the pathogen is trapped, it can then be positioned next to a phagocytic cell (blue) where the cell surface proteins on the pathogen can engage in receptors on the phagocytic cell surface.
Figure 2.
Trapping and positioning of polystyrene bead next to a fluorescent J774 cell.
(a–f) Combined fluorescent and brightfield images extracted from a real-time movie of a fluorescent polystyrene bead (yellow) positioned next to a fluorescent J774 cell expressing YFP-actin (green) (see Supporting Information for Video S1). The bead is trapped in a field of other fluorescent beads (orange) and another fluorescent J774 cell (also labeled green) as the stage is moved to position the cell next to the trapped bead. The red arrows indicate the deflection of the stage to direct the bead adjacent to the J774 cell, and the starting position of the trapped bead is indicated by a star (*).
Figure 3.
Fluorescence images of trapped and manipulated C. albicans.
(a) 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, and follows the sequence in frames i–x. The starting position of the CA is indicated by a star (*), and briefly moves out of the field of view in frames v–vii. Full length movie of trapped C. albicans can be seen in Supporting Information, Video S2. (b) Trapped C. albicans with attached, budding daughter cell (labeled with AF647, red, and highlighted in the gray circle) in a highly dense field of AF647-labeled and AF488-labeled (green) organisms. The stage is maneuvered around the red C. albicans, as indicated by the gray arrows. Frames i-viii shows the sequence of movements to position C. albicans in a different area of the stage. The starting position of the mother-daughter pair is indicated by a star (*).
Figure 4.
Trapping and positioning of A. fumigatus next to a phagocytic RAW cell.
(a–g) 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. The trap is fine enough to select the desired particle even as another microbe moves close to the trapped organism (c). A. fumigatus is moved until it is placed adjacent to the desired RAW cell (h). Movie of positioned A. fumigatus can be seen in Video S4.
Figure 5.
Fluorescence imaging of phagocytosis of A. fumigatus by RAW cell.
(a) 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 90 s to 150 s, the A. fumigatus is engulfed, and by 180 s, the particle is fully internalized. Movie of complete phagocytosis of A. fumigatus can be seen in Video S5. (b) 3-D rendering of RAW cell with ingested A. fumigatus. Close-up of RAW cell to verify that the pathogen is fully phagocytosed. The cell is rotated in each successive frame, and the axis in the lower left corner of each frame shows the degree of rotation (readers are able to virtually rotate the cell in Video S6). Please note that Video S6 is a QTVR movie, which is actually an animation that is based on the user's mouse movements.
Table 1.
Average velocity of stage moved around trapped particles.
Figure 6.
Synapse formation of Jurkat cell to trapped polystyrene bead coated with anti-CD3.
(a–e) Brightfield images of Jurkat cell shows that the cell has already formed a synapse with the trapped anti-CD3 bead at 0 s. The Jurkat's protrusion actually pushes the bead out of the trap (at 30 s to 90 s) and starts to bring the bead back to the cell (at 120 s). Movie of synapse formation can be seen in Video S7.