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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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Measurement of diameter for single HIV-1. (a) The power spectrum of a trapped virion when the chamber was oscillated at 10 Hz with amplitude of 176 nm along y-axis of the sample plane (Methods). The black curve is fitting of the thermal noise background to aliased Lorentzian with Dvolt=9.37×10−5 V2/s and fc= 233 Hz. (b) Size histogram for single fluorescent particles from chamber oscillation along y-axis (N=137). Concentration of the HIV-1 inside the chamber is 4.0 × 107 virions/ml. (c) Distribution of the optical trap stiffness for each virion trapped (N=137).
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Figure 3: Measurement of diameter for single HIV-1. (a) The power spectrum of a trapped virion when the chamber was oscillated at 10 Hz with amplitude of 176 nm along y-axis of the sample plane (Methods). The black curve is fitting of the thermal noise background to aliased Lorentzian with Dvolt=9.37×10−5 V2/s and fc= 233 Hz. (b) Size histogram for single fluorescent particles from chamber oscillation along y-axis (N=137). Concentration of the HIV-1 inside the chamber is 4.0 × 107 virions/ml. (c) Distribution of the optical trap stiffness for each virion trapped (N=137).

Mentions: Single HIV-1 particles are heterogeneous in diameter37. To help distinguish individual viral particles of different diameters, we set out to use the sensitive laser deflection signal to directly measure the diameter for each trapped virion in the culture fluid (Methods). This method works by calibration of the laser deflection signal using a distance standard38 and the Dvolt can be converted to the diffusion coefficient of the particle in space (D, in the unit of µm2/s)38. The diameter of the particle, ϕBFP, can then be calculated using the Stokes-Einstein equation (Supplementary Methods). We first tested this method by trapping polystyrene beads of various sizes in water. These results were compared with the diameters of the beads determined from transmission electron microscopy (TEM) images (ϕTEM, Supplementary Fig. 2). Over the range of bead sizes we tested, these two methods yielded good agreement, with ϕBFP slightly larger than ϕTEM by 5.5% on average, which reflected either the error in laser deflection measurement or hydration of particles in water. Fig. 3a shows the power spectrum of a trapped virion obtained from this calibration procedure in complete media, with voltage converted to distance as shown on the right axis, which yields a diameter of 165 nm for this particular virion. Fig. 3b shows the histogram of ϕBFP determined for 137 single HIV-1 particles in complete media at a concentration of 4.0 × 107 virion/ml, which displayed a population with a mean of 154 nm, and a median of 148 nm. The reduced chi-squared statistic from this analysis varied from 1.10 to 1.16 (Supplementary Fig. 3), indicating a good quality of power spectrum fitting. This virion size distribution was quantitatively similar to what was reported previously for authentic, mature HIV-1 virions by cryoelectron microscopy (cEM)37. Our mean diameter was 6.2% larger than 145 ± 25 nm for single HIV virions measured by cEM. This positive deviation was consistent with the trend seen for reference beads, suggesting that these particles indeed corresponded to single virions in complete media. This positive deviation can result from either hydration of the particles in culture media or potential shrinkage of the feature size under EM (Supplementary Note 2). From calibrated power spectra for single HIV-1 virions, we also obtained stiffness of the optical trap (Supplementary Methods), which is shown in Fig. 3c with a mean value of 3.2 fN/nm. This stiffness is about 30-fold lower than typically reported values for bigger particles3 and explains the difficulty in trapping virions of this size. The variation in trap stiffness as compared to variations in particle diameter also suggests that HIV-1 virions deviate from true Rayleigh scatterers. To examine how well the measured particle diameter and trap stiffness can differentiate particles of different size, we conducted sequential trapping of one, two and three particles as illustrated in Supplementary Fig. 1, and measured ϕBFP and trap stiffness for the apparent single, double and triple particles, respectively. The distributions of these values are plotted in Supplementary Fig. 4a and b. As shown, both ϕBFP and trap stiffness conform to statistically different distributions for single versus double or triple particles (Supplementary Table 2), suggesting that these parameters are sensitive enough to distinguish a single particle from particle aggregates.


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)

Measurement of diameter for single HIV-1. (a) The power spectrum of a trapped virion when the chamber was oscillated at 10 Hz with amplitude of 176 nm along y-axis of the sample plane (Methods). The black curve is fitting of the thermal noise background to aliased Lorentzian with Dvolt=9.37×10−5 V2/s and fc= 233 Hz. (b) Size histogram for single fluorescent particles from chamber oscillation along y-axis (N=137). Concentration of the HIV-1 inside the chamber is 4.0 × 107 virions/ml. (c) Distribution of the optical trap stiffness for each virion trapped (N=137).
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Related In: Results  -  Collection

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Figure 3: Measurement of diameter for single HIV-1. (a) The power spectrum of a trapped virion when the chamber was oscillated at 10 Hz with amplitude of 176 nm along y-axis of the sample plane (Methods). The black curve is fitting of the thermal noise background to aliased Lorentzian with Dvolt=9.37×10−5 V2/s and fc= 233 Hz. (b) Size histogram for single fluorescent particles from chamber oscillation along y-axis (N=137). Concentration of the HIV-1 inside the chamber is 4.0 × 107 virions/ml. (c) Distribution of the optical trap stiffness for each virion trapped (N=137).
Mentions: Single HIV-1 particles are heterogeneous in diameter37. To help distinguish individual viral particles of different diameters, we set out to use the sensitive laser deflection signal to directly measure the diameter for each trapped virion in the culture fluid (Methods). This method works by calibration of the laser deflection signal using a distance standard38 and the Dvolt can be converted to the diffusion coefficient of the particle in space (D, in the unit of µm2/s)38. The diameter of the particle, ϕBFP, can then be calculated using the Stokes-Einstein equation (Supplementary Methods). We first tested this method by trapping polystyrene beads of various sizes in water. These results were compared with the diameters of the beads determined from transmission electron microscopy (TEM) images (ϕTEM, Supplementary Fig. 2). Over the range of bead sizes we tested, these two methods yielded good agreement, with ϕBFP slightly larger than ϕTEM by 5.5% on average, which reflected either the error in laser deflection measurement or hydration of particles in water. Fig. 3a shows the power spectrum of a trapped virion obtained from this calibration procedure in complete media, with voltage converted to distance as shown on the right axis, which yields a diameter of 165 nm for this particular virion. Fig. 3b shows the histogram of ϕBFP determined for 137 single HIV-1 particles in complete media at a concentration of 4.0 × 107 virion/ml, which displayed a population with a mean of 154 nm, and a median of 148 nm. The reduced chi-squared statistic from this analysis varied from 1.10 to 1.16 (Supplementary Fig. 3), indicating a good quality of power spectrum fitting. This virion size distribution was quantitatively similar to what was reported previously for authentic, mature HIV-1 virions by cryoelectron microscopy (cEM)37. Our mean diameter was 6.2% larger than 145 ± 25 nm for single HIV virions measured by cEM. This positive deviation was consistent with the trend seen for reference beads, suggesting that these particles indeed corresponded to single virions in complete media. This positive deviation can result from either hydration of the particles in culture media or potential shrinkage of the feature size under EM (Supplementary Note 2). From calibrated power spectra for single HIV-1 virions, we also obtained stiffness of the optical trap (Supplementary Methods), which is shown in Fig. 3c with a mean value of 3.2 fN/nm. This stiffness is about 30-fold lower than typically reported values for bigger particles3 and explains the difficulty in trapping virions of this size. The variation in trap stiffness as compared to variations in particle diameter also suggests that HIV-1 virions deviate from true Rayleigh scatterers. To examine how well the measured particle diameter and trap stiffness can differentiate particles of different size, we conducted sequential trapping of one, two and three particles as illustrated in Supplementary Fig. 1, and measured ϕBFP and trap stiffness for the apparent single, double and triple particles, respectively. The distributions of these values are plotted in Supplementary Fig. 4a and b. As shown, both ϕBFP and trap stiffness conform to statistically different distributions for single versus double or triple particles (Supplementary Table 2), suggesting that these parameters are sensitive enough to distinguish a single particle from particle aggregates.

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