Optical manipulation of a single human virus for study of viral-cell interactions

Abstract

Although Ashkin and Dziedzic first demonstrated optical trapping of individual tobacco mosaic viruses in suspension as early as 1987, this pioneering work has not been followed up only until recently. Using human immunodeficiency virus type 1 (HIV-1) as a model virus, we have recently demonstrated that a single HIV-1 virion can be stabled trapped, manipulated and measured in physiological media with high precision. The capability to optically trap a single virion in suspension not only allows us to determine, for the first time, the refractive index of a single virus with high precision, but also quantitate the heterogeneity among individual virions with single-molecule resolution, the results of which shed light on the molecular mechanisms of virion infectivity. Here we report the further development of a set of microscopic techniques to physically deliver a single HIV-1 virion to a single host cell in solution. Combined with simultaneous epifluorescence imaging, the attachment and dissociation events of individual manipulated virions on host cell surface can be measured and the results help us understand the role of diffusion in mediating viral attachment to host cells. The establishment of these techniques opens up new ways for investigation of a wide range of virion-cell interactions, and should be applicable for study of B cell interactions with particulate antigens such as viruses.

Keywords: HIV-1; Optical trapping; manipulation; micropipette; single cell; single virus; virus-cell interactions.

Figure 1

(A) Design of the microfluidic chamber for single cell immobilization, single virion delivery, imaging and single cell collection. The chamber contains 3 channels: upper and bottom channels are used to flow in components such as cells or viruses; the center channel is used for experiments. Capillary tubings are placed between the center channel and the other two channels to deliver cells or beads into the center channel. A micropipette (4–5 μm diameter at the end of the opening) is installed to immobilize a single cell by the hydrostatic pressure established with a movable syringe filled with buffers. A collection tube (~40 μm diameter) is installed on the side of the micropipette to collect cells similarly using hydrostatic pressure driven by a second movable syringe. (B) Experimental setup for optical tweezers. The optical trap is focused at the center of the microfluidic chamber. (C) Test of the hydrostatic pressure at the micropipette opening using a polystyrene bead (2.8 μm diameter). The bead is trapped by optical tweezers and placed over the pipette opening. We systematically changed the height of the syringe connected with the micropipette and measured the corresponding Stokes force acting on the bead using optical tweezers. The scale bar is 10 μm. (D) The Stokes force acting on the bead varied linearly with the height of the syringe, confirming the establishment of a hydrostatic pressure.

Figure 2

Manipulation of a single T cell by a micropipette. (A) A SUP-T1 cell is trapped by optical tweezers and transported to the opening of a micropipette. (B) Upon release of the optical tweezers and application of a negative pressure below a threshold at the micropipette opening, the cell can be immobilized stably atop the pipette. (C) At the threshold negative pressure, the single cell is deformed to form a hemisphere inside the pipette. The diameter of the hemisphere approximately equals the diameter of the pipette opening. For both (B) and (C), the cell can be immobilized atop the pipette stably without damage. (D and E) when the negative pressure is further increased above the threshold, the single cell is continuously deformed and stretched into the pipette. (F) When a positive pressure is generated by moving the syringe, the process shown in D and E can be reversed and the single cell finally recovers its original morphology and can be released from the pipette. The scale bars are 10 μm each.

Figure 3

A single cell was trapped by optical tweezers, and delivered to a collection tube (A). We switched off the optical trap, and the cell went into the collection tube due to the applied negative pressure (B–C). Finally, the cell was recovered and placed on a coverslip under microscope (D). The scale bars are 10 μm each.

Figure 4

TPF images of individual 293T cells atop a micropipette. These cells were transiently transfected with EGFP-Vpr fusion protein as described in Materials and Methods. (A–D) show four individual cells with varied expression levels of the fusion protein. The scale bars are 5 μm each. (E–H) Bright field images of a single cell during the scanning of the micropipette. Panels E to H show four different locations of the micropipette relative to the laser focus, which was indicated by the red stars in each panel. No movement of the cell relative to the micropipette was detected during the scanning process, as confirmed by overlaying these bright field images using the contours of the micropipette as a reference. The scale bars are 10 μm each.