Active-Feedback 3D Single-Molecule Tracking Using a Fast-Responding Galvo Scanning Mirror

Tan, Xiaochen; Hou, Shangguo; Niver, Anastasia; Zhang, Chen; Johnson, Alexis; Welsher, Kevin

doi:10.1021/acs.jpca.3c02090

Cited by 3 publications

(1 citation statement)

References 45 publications

(67 reference statements)

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“…This sampling rate can be improved by improving the response time of the feedback element. By replacing the XY piezoelectric stage with a galvoscanning mirror, the sampling rate could reach approximately 5 kHz . This sampling rate could theoretically be improved further.…”

Section: Resultsmentioning

confidence: 99%

Hour-Long, Kilohertz Sampling Rate Three-Dimensional Single-Virus Tracking in Live Cells Enabled by StayGold Fluorescent Protein Fusions

Lin,

Exell,

Lin

et al. 2024

J. Phys. Chem. B

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A viral infection process covers a large range of spatiotemporal scales. Tracking the viral infection process with fluorescent labels over long durations while maintaining a fast sampling rate requires bright and highly photostable labels. StayGold is a recently identified green fluorescent protein that has a greater photostability and higher signal intensity under identical illumination conditions compared to existing fluorescence protein variants. Here, StayGold protein fusions were used to generate virus-like particles (StayGold-VLPs) to achieve hour-long 3D single-virus tracking (SVT) with 1000 localizations per second (kHz sampling rate) in live cells. The expanded photon budget from StayGold protein fusions prolonged the tracking duration, facilitating a comprehensive study of viral trafficking dynamics with high temporal resolution over long time scales. The development of StayGold-VLPs presents a simple and general VLP labeling strategy for better performance in SVT, enabling exponentially more information to be collected from single trajectories and allowing for the future possibility of observing the entire life cycle of a single virus.

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Section: Resultsmentioning

confidence: 99%

Hour-Long, Kilohertz Sampling Rate Three-Dimensional Single-Virus Tracking in Live Cells Enabled by StayGold Fluorescent Protein Fusions

Lin,

Exell,

Lin

et al. 2024

J. Phys. Chem. B

Self Cite

View full text Add to dashboard Cite

show abstract

Hour-long, Kilohertz Sampling Rate 3D Single-virus Tracking in Live Cells Enabled by StayGold Fluorescent Protein Fusions

Lin,

Exell,

Lin

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

The viral infection process covers a large range of spatiotemporal scales. Tracking the viral infection process with fluorescent labels over long durations while maintaining a fast sampling rate requires bright and highly photostable labels. StayGold is a recently identified green fluorescent protein that has a greater photostability and higher signal intensity under identical illumination conditions as compared to existing fluorescence protein variants. Here, StayGold protein fusions were used to generate virus-like particles (StayGold-VLPs) to achieve hour-long 3D single-virus tracking (SVT) with one thousand localizations per second (kHz sampling rate) in live cells. The expanded photon budget from StayGold protein fusions prolonged the tracking duration, facilitating a comprehensive study of viral trafficking dynamics with high temporal resolution over long timescales. The development of StayGold-VLPs presents a simple and general VLP labeling strategy for better performance in SVT, enabling exponentially more information to be collected from single trajectories and allowing for the future possibility of observing the whole life cycle of a single virus.

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Single-nanoparticle electrophoretic mobility determination and trapping using active-feedback 3D tracking

Johnson,

Welsher

2024

Preprint

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Nanoparticles (NP) are versatile materials with widespread applications across medicine and engineering. Despite rapid incorporation into drug delivery, therapeutics, and many more areas of research and development, there is a lack of robust characterization methods. Light scattering techniques such as dynamic light scattering (DLS) and electrophoretic light scattering (ELS) use an ensemble-averaged approach to the characterization of nanoparticle size and electrophoretic mobility (EM), leading to inaccuracies when applied to polydisperse or heterogeneous populations. To address this lack of single-nanoparticle characterization, this work applies 3D Single-Molecule Active Real-time Tracking (3D-SMART) to simultaneously determine NP size and EM on a per-particle basis. Single-nanoparticle EM is determined by using active feedback to “lock on” to a single particle and apply an oscillating electric field along one axis. A maximum likelihood approach is applied to extract the single-particle EM from the oscillating nanoparticle position along the field-actuated axis, while mean squared displacement is used along the non-actuated axes to determine size. Unfunctionalized and carboxyl-functionalized polystyrene NPs are found to have unique EM based on their individual size and surface characteristics, and it is demonstrated that single-nanoparticle EM is a more precise tool for distinguishing unique NP preparations than diffusion alone, able to determine the charge number of individual NPs to an uncertainty of less than 30. This method also explored individual nanoparticle EM in various ionic strengths (0.25-5 mM) and found decreased EM as a function of increasing ionic strength, in agreement with results determined via bulk characterization methods. Finally, it is demonstrated that the electric field can be manipulated in real time in response to particle position, resulting in one-dimensional electrokinetic trapping. Critically, this new single-nanoparticle EM determination and trapping method does not require microfluidics, opening the possibility for the exploration of single-nanoparticle EM in close proximity to live tissue and more comprehensive characterization of nanoparticles in biologically relevant environments.

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Active-Feedback 3D Single-Molecule Tracking Using a Fast-Responding Galvo Scanning Mirror

Cited by 3 publications

References 45 publications

Hour-Long, Kilohertz Sampling Rate Three-Dimensional Single-Virus Tracking in Live Cells Enabled by StayGold Fluorescent Protein Fusions

Hour-Long, Kilohertz Sampling Rate Three-Dimensional Single-Virus Tracking in Live Cells Enabled by StayGold Fluorescent Protein Fusions

Hour-long, Kilohertz Sampling Rate 3D Single-virus Tracking in Live Cells Enabled by StayGold Fluorescent Protein Fusions

Single-nanoparticle electrophoretic mobility determination and trapping using active-feedback 3D tracking

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