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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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Aggregation of HIV-1 observed at high virion concentrations. (a) Size histogram for single fluorescent particles from chamber oscillation along y-axis and its double-Gaussian fit (dashed line) (N=128). Concentration of the HIV-1 inside the chamber is 1.2 × 108 virions/ml. (b) Size histogram for single fluorescent particles from chamber oscillation along y-axis (N=103). Concentration of the HIV-1 inside the chamber is 4.0 × 108 virions/ml. (c) Distribution of EGFP-Vpr TPF intensity from trapped virions that are confirmed to be single based on diameter measurement in (a) (N=92).
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Figure 4: Aggregation of HIV-1 observed at high virion concentrations. (a) Size histogram for single fluorescent particles from chamber oscillation along y-axis and its double-Gaussian fit (dashed line) (N=128). Concentration of the HIV-1 inside the chamber is 1.2 × 108 virions/ml. (b) Size histogram for single fluorescent particles from chamber oscillation along y-axis (N=103). Concentration of the HIV-1 inside the chamber is 4.0 × 108 virions/ml. (c) Distribution of EGFP-Vpr TPF intensity from trapped virions that are confirmed to be single based on diameter measurement in (a) (N=92).

Mentions: Freely-diffusing particles in the complete media may undergo concentration-dependent aggregation. To examine the aggregation status of HIV-1 virions under native conditions in the complete media, we conducted diameter measurement for single particles trapped at various concentrations in the microfluidic chamber. Fig. 4a shows the histogram of ϕBFP determined for 128 apparent single particles at 1.2 × 108 virions/ml in the complete media (grey bars), which displays two distinct populations as indicated by the double-Gaussian fit (dashed lines). The primary peak, with a mean of 156 nm and a standard deviation of 30 nm, accounts for 72% of the particles. This number is very close to the average diameter of 154 nm for single HIV virions measured at 4.0 × 107 virions/ml, suggesting that these particles indeed correspond to single virions. The secondary peak in this histogram, with a mean of 305 nm and a standard deviation of 44 nm, is about twofold larger than the size of a typical HIV-1 virion. These particles were rarely observed when trapping at a concentration of 4.0 × 107 virions/ml, suggesting that they resulted from a concentration-dependent aggregation. This conclusion is further supported by the same experiment done at even higher concentration of virion particles. Fig. 4b shows the histogram of ϕBFP determined for 103 apparent single particles at 4.0 × 108 virions/ml in the complete media. No clear peaks were present. Particle diameters ranged from 90 to 440 nm, with an average diameter of 232 ± 78 nm (median 219 nm). This feature is reproducible from an independent repeat of the same experiment, suggesting a concentration-dependent aggregation of HIV-1 particles. Fig. 4c shows the distribution of EGFP TPF intensity from single HIV-1 virions upon their initial trapping. This distribution was broad and asymmetric, ranging from 1,000 to 40,000 a.u., with a major peak around 30,000. This variation of TPF intensity exclusively resulted from EGFP-Vpr molecules packaged into virions because control virions without EGFP showed no detectable TPF under these conditions. This substantial variation of the initial TPF amplitudes suggests that one cannot simply use TPF intensity to confirm a single virus. Rather, size or trap stiffness measurement is required. Moreover, virion diameter is a better parameter than Dvolt in resolving subpopulations within a virus pool (Supplementary Fig. 5).


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)

Aggregation of HIV-1 observed at high virion concentrations. (a) Size histogram for single fluorescent particles from chamber oscillation along y-axis and its double-Gaussian fit (dashed line) (N=128). Concentration of the HIV-1 inside the chamber is 1.2 × 108 virions/ml. (b) Size histogram for single fluorescent particles from chamber oscillation along y-axis (N=103). Concentration of the HIV-1 inside the chamber is 4.0 × 108 virions/ml. (c) Distribution of EGFP-Vpr TPF intensity from trapped virions that are confirmed to be single based on diameter measurement in (a) (N=92).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Aggregation of HIV-1 observed at high virion concentrations. (a) Size histogram for single fluorescent particles from chamber oscillation along y-axis and its double-Gaussian fit (dashed line) (N=128). Concentration of the HIV-1 inside the chamber is 1.2 × 108 virions/ml. (b) Size histogram for single fluorescent particles from chamber oscillation along y-axis (N=103). Concentration of the HIV-1 inside the chamber is 4.0 × 108 virions/ml. (c) Distribution of EGFP-Vpr TPF intensity from trapped virions that are confirmed to be single based on diameter measurement in (a) (N=92).
Mentions: Freely-diffusing particles in the complete media may undergo concentration-dependent aggregation. To examine the aggregation status of HIV-1 virions under native conditions in the complete media, we conducted diameter measurement for single particles trapped at various concentrations in the microfluidic chamber. Fig. 4a shows the histogram of ϕBFP determined for 128 apparent single particles at 1.2 × 108 virions/ml in the complete media (grey bars), which displays two distinct populations as indicated by the double-Gaussian fit (dashed lines). The primary peak, with a mean of 156 nm and a standard deviation of 30 nm, accounts for 72% of the particles. This number is very close to the average diameter of 154 nm for single HIV virions measured at 4.0 × 107 virions/ml, suggesting that these particles indeed correspond to single virions. The secondary peak in this histogram, with a mean of 305 nm and a standard deviation of 44 nm, is about twofold larger than the size of a typical HIV-1 virion. These particles were rarely observed when trapping at a concentration of 4.0 × 107 virions/ml, suggesting that they resulted from a concentration-dependent aggregation. This conclusion is further supported by the same experiment done at even higher concentration of virion particles. Fig. 4b shows the histogram of ϕBFP determined for 103 apparent single particles at 4.0 × 108 virions/ml in the complete media. No clear peaks were present. Particle diameters ranged from 90 to 440 nm, with an average diameter of 232 ± 78 nm (median 219 nm). This feature is reproducible from an independent repeat of the same experiment, suggesting a concentration-dependent aggregation of HIV-1 particles. Fig. 4c shows the distribution of EGFP TPF intensity from single HIV-1 virions upon their initial trapping. This distribution was broad and asymmetric, ranging from 1,000 to 40,000 a.u., with a major peak around 30,000. This variation of TPF intensity exclusively resulted from EGFP-Vpr molecules packaged into virions because control virions without EGFP showed no detectable TPF under these conditions. This substantial variation of the initial TPF amplitudes suggests that one cannot simply use TPF intensity to confirm a single virus. Rather, size or trap stiffness measurement is required. Moreover, virion diameter is a better parameter than Dvolt in resolving subpopulations within a virus pool (Supplementary Fig. 5).

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