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Optical trapping of individual human immunodeficiency viruses in culture fluid reveals heterogeneity with single-molecule resolution.

Pang Y, Song H, Kim JH, Hou X, Cheng W - Nat Nanotechnol (2014)

Bottom Line: Here, using optical tweezers that can simultaneously resolve two-photon fluorescence at the single-molecule level, we show that individual HIV-1 viruses can be optically trapped and manipulated, allowing multi-parameter analysis of single virions in culture fluid under native conditions.We show that individual HIV-1 differs in the numbers of envelope glycoproteins by more than one order of magnitude, which implies substantial heterogeneity of these virions in transmission and infection at the single-particle level.Analogous to flow cytometry for cells, this fluid-based technique may allow ultrasensitive detection, multi-parameter analysis and sorting of viruses and other nanoparticles in biological fluid with single-molecule resolution.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109, USA.

ABSTRACT
Optical tweezers use the momentum of photons to trap and manipulate microscopic objects, contact-free, in three dimensions. Although this technique has been widely used in biology and nanotechnology to study molecular motors, biopolymers and nanostructures, its application to study viruses has been very limited, largely due to their small size. Here, using optical tweezers that can simultaneously resolve two-photon fluorescence at the single-molecule level, we show that individual HIV-1 viruses can be optically trapped and manipulated, allowing multi-parameter analysis of single virions in culture fluid under native conditions. We show that individual HIV-1 differs in the numbers of envelope glycoproteins by more than one order of magnitude, which implies substantial heterogeneity of these virions in transmission and infection at the single-particle level. Analogous to flow cytometry for cells, this fluid-based technique may allow ultrasensitive detection, multi-parameter analysis and sorting of viruses and other nanoparticles in biological fluid with single-molecule resolution.

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Optical trapping of HIV virions in culture fluid. (a) HIV-1 virions were delivered into a microfluidic chamber and trapped by the IR laser focused at the center of the chamber. The xyz dimensions are shown as indicated, with y perpendicular to the figure plane. (b) TPF image of a trapped HIV virion. The scale bar is 10 µm. (c) Representative TPF time courses from individually-trapped HIV virions. All traces were fit with single exponential decay (red), with time constants for each trace as follows: 60.7 (square), 49.5 (cross), 51.5 (circle) and 54.1 s (triangle). Time zero started with the onset of TPF collection.
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Figure 1: Optical trapping of HIV virions in culture fluid. (a) HIV-1 virions were delivered into a microfluidic chamber and trapped by the IR laser focused at the center of the chamber. The xyz dimensions are shown as indicated, with y perpendicular to the figure plane. (b) TPF image of a trapped HIV virion. The scale bar is 10 µm. (c) Representative TPF time courses from individually-trapped HIV virions. All traces were fit with single exponential decay (red), with time constants for each trace as follows: 60.7 (square), 49.5 (cross), 51.5 (circle) and 54.1 s (triangle). Time zero started with the onset of TPF collection.

Mentions: To demonstrate the optical trapping of animal viruses in biological fluid, we have recently prepared the HIV-1 virions derived from the X4-tropic NL4-3 provirus clone30. These viruses were tagged internally with EGFP fused to Vpr31,32, an accessory protein of HIV that physically associates with the virion core33 and thus serves as a fluorescent marker31 for virion manipulation. The infectivity of this virus was 0.1% in TZM-bl cells30, a value that was comparable to wild-type viruses. Control experiments showed that EGFP-Vpr also distinguished virions from microvesicles (Supplementary Note 1). Without any fixation procedure, we diluted the live virus stock in the complete media and injected them into a microfluidic chamber (Fig. 1a). Upon trapping of a virion by the 830 nm infrared laser29 focused at the center of the chamber, the virion was immediately ‘visible’ in the dark background due to simultaneous two-photon excitation of EGFP by the trapping laser29,34 (Fig. 1b). Such events were not observed when complete media only, viruses without EGFP-Vpr labels, or culture supernatants transfected with EGFP-Vpr were used as controls, suggesting that the fluorescent particles resulted from virions tagged with EGFP. Under these conditions, a single fluorescent particle could be trapped in culture fluid and manipulated in various patterns by steering the laser beam without interference from other virions (Supplementary Video). Typical two-photon fluorescence (TPF) time courses from individually-trapped virions are shown in Fig. 1c, where the initial bursts of fluorescence well above background indicated the arrival of HIV-1 virions at the optical trap. The subsequent decay of TPF with time could all be fit by single exponentials with an average time constant of 53.7±8.0 s (N=19), very close to the rate of photobleaching that we measured previously for EGFP either on a surface (50.8±4.2 s)34 or inside a cell (52.7±5.2 s)35. This comparison confirmed the presence of EGFP in these particles.


Optical trapping of individual human immunodeficiency viruses in culture fluid reveals heterogeneity with single-molecule resolution.

Pang Y, Song H, Kim JH, Hou X, Cheng W - Nat Nanotechnol (2014)

Optical trapping of HIV virions in culture fluid. (a) HIV-1 virions were delivered into a microfluidic chamber and trapped by the IR laser focused at the center of the chamber. The xyz dimensions are shown as indicated, with y perpendicular to the figure plane. (b) TPF image of a trapped HIV virion. The scale bar is 10 µm. (c) Representative TPF time courses from individually-trapped HIV virions. All traces were fit with single exponential decay (red), with time constants for each trace as follows: 60.7 (square), 49.5 (cross), 51.5 (circle) and 54.1 s (triangle). Time zero started with the onset of TPF collection.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125448&req=5

Figure 1: Optical trapping of HIV virions in culture fluid. (a) HIV-1 virions were delivered into a microfluidic chamber and trapped by the IR laser focused at the center of the chamber. The xyz dimensions are shown as indicated, with y perpendicular to the figure plane. (b) TPF image of a trapped HIV virion. The scale bar is 10 µm. (c) Representative TPF time courses from individually-trapped HIV virions. All traces were fit with single exponential decay (red), with time constants for each trace as follows: 60.7 (square), 49.5 (cross), 51.5 (circle) and 54.1 s (triangle). Time zero started with the onset of TPF collection.
Mentions: To demonstrate the optical trapping of animal viruses in biological fluid, we have recently prepared the HIV-1 virions derived from the X4-tropic NL4-3 provirus clone30. These viruses were tagged internally with EGFP fused to Vpr31,32, an accessory protein of HIV that physically associates with the virion core33 and thus serves as a fluorescent marker31 for virion manipulation. The infectivity of this virus was 0.1% in TZM-bl cells30, a value that was comparable to wild-type viruses. Control experiments showed that EGFP-Vpr also distinguished virions from microvesicles (Supplementary Note 1). Without any fixation procedure, we diluted the live virus stock in the complete media and injected them into a microfluidic chamber (Fig. 1a). Upon trapping of a virion by the 830 nm infrared laser29 focused at the center of the chamber, the virion was immediately ‘visible’ in the dark background due to simultaneous two-photon excitation of EGFP by the trapping laser29,34 (Fig. 1b). Such events were not observed when complete media only, viruses without EGFP-Vpr labels, or culture supernatants transfected with EGFP-Vpr were used as controls, suggesting that the fluorescent particles resulted from virions tagged with EGFP. Under these conditions, a single fluorescent particle could be trapped in culture fluid and manipulated in various patterns by steering the laser beam without interference from other virions (Supplementary Video). Typical two-photon fluorescence (TPF) time courses from individually-trapped virions are shown in Fig. 1c, where the initial bursts of fluorescence well above background indicated the arrival of HIV-1 virions at the optical trap. The subsequent decay of TPF with time could all be fit by single exponentials with an average time constant of 53.7±8.0 s (N=19), very close to the rate of photobleaching that we measured previously for EGFP either on a surface (50.8±4.2 s)34 or inside a cell (52.7±5.2 s)35. This comparison confirmed the presence of EGFP in these particles.

Bottom Line: Here, using optical tweezers that can simultaneously resolve two-photon fluorescence at the single-molecule level, we show that individual HIV-1 viruses can be optically trapped and manipulated, allowing multi-parameter analysis of single virions in culture fluid under native conditions.We show that individual HIV-1 differs in the numbers of envelope glycoproteins by more than one order of magnitude, which implies substantial heterogeneity of these virions in transmission and infection at the single-particle level.Analogous to flow cytometry for cells, this fluid-based technique may allow ultrasensitive detection, multi-parameter analysis and sorting of viruses and other nanoparticles in biological fluid with single-molecule resolution.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109, USA.

ABSTRACT
Optical tweezers use the momentum of photons to trap and manipulate microscopic objects, contact-free, in three dimensions. Although this technique has been widely used in biology and nanotechnology to study molecular motors, biopolymers and nanostructures, its application to study viruses has been very limited, largely due to their small size. Here, using optical tweezers that can simultaneously resolve two-photon fluorescence at the single-molecule level, we show that individual HIV-1 viruses can be optically trapped and manipulated, allowing multi-parameter analysis of single virions in culture fluid under native conditions. We show that individual HIV-1 differs in the numbers of envelope glycoproteins by more than one order of magnitude, which implies substantial heterogeneity of these virions in transmission and infection at the single-particle level. Analogous to flow cytometry for cells, this fluid-based technique may allow ultrasensitive detection, multi-parameter analysis and sorting of viruses and other nanoparticles in biological fluid with single-molecule resolution.

Show MeSH
Related in: MedlinePlus