Limits...
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.

Show MeSH

Related in: MedlinePlus

BFP interferometry to distinguish single HIV-1 particle from aggregates in complete media. (a) The laser deflection signal measured in real time using BFP interferometry. (b) Power spectra calculated from the data shown in (a) and fit to aliased Lorentzian with Dvolt=8.53×10−5 V2/s, fc= 223 Hz (blue) and Dvolt=0.0046 V2/s, fc= 982 Hz (red). Histograms for Dvolt (c) and fc (d) derived from Lorentzian fitting parameters for apparent one (blue, N=110), two (hashed, N=41) and three (red, N=21) particles trapped, with cartoons representing one, two and three particles above each histogram.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4125448&req=5

Figure 2: BFP interferometry to distinguish single HIV-1 particle from aggregates in complete media. (a) The laser deflection signal measured in real time using BFP interferometry. (b) Power spectra calculated from the data shown in (a) and fit to aliased Lorentzian with Dvolt=8.53×10−5 V2/s, fc= 223 Hz (blue) and Dvolt=0.0046 V2/s, fc= 982 Hz (red). Histograms for Dvolt (c) and fc (d) derived from Lorentzian fitting parameters for apparent one (blue, N=110), two (hashed, N=41) and three (red, N=21) particles trapped, with cartoons representing one, two and three particles above each histogram.

Mentions: Freely-diffusing particles in culture fluid may undergo concentration-dependent aggregation. How do we know that the trapped particle correspond to a single HIV-1 virion? Independent of EGFP fluorescence, we could also detect the trapping of the virus from changes in laser deflection at the objective’s back focal plane (BFP) (Fig. 2a, blue). The occasional trapping of an apparent viral aggregate produced quantitatively different laser deflection signals29,36 (Fig. 2a, red), suggesting that one could potentially use this signal to distinguish viral particles of different size. We analyzed the time courses of the laser deflection by conversion to power spectra in the frequency domain (Fig. 2b). For both the single particle (blue) and virion aggregate (red), the power spectra could be well described with Lorentzian up to 10 kHz (green curves, Supplementary Methods), consistent with the Brownian motion of a particle in a harmonic photonic field3. Furthermore, this fitting yielded very different parameters for the single particle and virion aggregate: Dvolt, the diffusion coefficient (in the unit of V2/s) and fc, the corner frequency (in Hz). To test the sensitivity of these parameters in differentiating particles of different size, we delivered diluted virions through a microfluidic channel into the complete media followed by trapping of the virion in the vicinity of the channel opening (Supplementary Fig. 1). This scheme allowed us to trap one, two and three particles sequentially using a single laser beam (Supplementary Fig. 1), and measure Dvolt and fc for the apparent single, double and triple particles, respectively. The distributions of these values are plotted in Fig. 2c and d. As shown, both Dvolt and fc conform to statistically different distributions for single versus double or triple particles (Supplementary Table 1), suggesting that the laser deflection is 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)

BFP interferometry to distinguish single HIV-1 particle from aggregates in complete media. (a) The laser deflection signal measured in real time using BFP interferometry. (b) Power spectra calculated from the data shown in (a) and fit to aliased Lorentzian with Dvolt=8.53×10−5 V2/s, fc= 223 Hz (blue) and Dvolt=0.0046 V2/s, fc= 982 Hz (red). Histograms for Dvolt (c) and fc (d) derived from Lorentzian fitting parameters for apparent one (blue, N=110), two (hashed, N=41) and three (red, N=21) particles trapped, with cartoons representing one, two and three particles above each histogram.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: BFP interferometry to distinguish single HIV-1 particle from aggregates in complete media. (a) The laser deflection signal measured in real time using BFP interferometry. (b) Power spectra calculated from the data shown in (a) and fit to aliased Lorentzian with Dvolt=8.53×10−5 V2/s, fc= 223 Hz (blue) and Dvolt=0.0046 V2/s, fc= 982 Hz (red). Histograms for Dvolt (c) and fc (d) derived from Lorentzian fitting parameters for apparent one (blue, N=110), two (hashed, N=41) and three (red, N=21) particles trapped, with cartoons representing one, two and three particles above each histogram.
Mentions: Freely-diffusing particles in culture fluid may undergo concentration-dependent aggregation. How do we know that the trapped particle correspond to a single HIV-1 virion? Independent of EGFP fluorescence, we could also detect the trapping of the virus from changes in laser deflection at the objective’s back focal plane (BFP) (Fig. 2a, blue). The occasional trapping of an apparent viral aggregate produced quantitatively different laser deflection signals29,36 (Fig. 2a, red), suggesting that one could potentially use this signal to distinguish viral particles of different size. We analyzed the time courses of the laser deflection by conversion to power spectra in the frequency domain (Fig. 2b). For both the single particle (blue) and virion aggregate (red), the power spectra could be well described with Lorentzian up to 10 kHz (green curves, Supplementary Methods), consistent with the Brownian motion of a particle in a harmonic photonic field3. Furthermore, this fitting yielded very different parameters for the single particle and virion aggregate: Dvolt, the diffusion coefficient (in the unit of V2/s) and fc, the corner frequency (in Hz). To test the sensitivity of these parameters in differentiating particles of different size, we delivered diluted virions through a microfluidic channel into the complete media followed by trapping of the virion in the vicinity of the channel opening (Supplementary Fig. 1). This scheme allowed us to trap one, two and three particles sequentially using a single laser beam (Supplementary Fig. 1), and measure Dvolt and fc for the apparent single, double and triple particles, respectively. The distributions of these values are plotted in Fig. 2c and d. As shown, both Dvolt and fc conform to statistically different distributions for single versus double or triple particles (Supplementary Table 1), suggesting that the laser deflection is 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