Pang Daiwen's research group of Nankai University: Quantum dot single virus tracing technology cracks the dynamic infection mechanism of viruses

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Written by Jevin

Single virus tracking (SVT) provides the opportunity to monitor the journey of a single virus in real time and explore the interactions between viruses and cellular structures in living cells, which helps characterize complex infection processes and reveal relevant dynamic mechanisms
.
However, the low brightness and poor photostability of traditional fluorescent labels greatly limit the development of SVT technology, and multi-color SVT over a long period of time still has challenges
.

On November 30, 2022, Professor Pang Daiwen's team of Nankai University used the virus they had previously studied systematically as an example to provide a detailed operational procedure
for QSVT implementation.
Specific procedures for performing QSVT experiments in living cells, including virus preparation, QD labeling strategies, imaging methods, image processing, and data analysis
, are described.

style="box-sizing: border-box;" _msthash="251139" _msttexthash="580814">Virus tracking technology

 01 

Single-virus tracking (QSVT) technology based on quantum dots (QD) is an imaging method that uses quantum dots as fluorescent labels to label different viral components, and fluorescence microscopy studies the infection process of individual viruses and the dynamic relationship
between viruses and cellular components.
In QSVT experiments, the behavior of individual viruses or viral components labeled with quantum dots can be monitored in real time for long periods of
time within host cells.
After reconstructing the trajectory of a single virus, relevant dynamic information can be extracted in detail to reveal the infection mechanism
of the virus.
The extremely high brightness and excellent photostability of quantum dots facilitate high-contrast and long-span imaging of individual viruses from milliseconds to hours, and allow positioning of individual viruses with nanoscale precision, and color-adjustable emission with narrow full width and half maximum, making quantum dots an excellent marker for simultaneous multi-component labeling of viruses with single-virus sensitivity
.

As a result, quantum dots greatly facilitate the development of single-virus tracking (SVT) technology, especially for applications such as viral journeys that require long-term and multicolor imaging and single-virus level studies of virus-cell
interactions.

"Six-stage" operation process

 02

Over the past decade, the Pondaiwen team has been conducting research related to QSVT and has done a lot of work to advance the technology and systematically explore the mechanisms
of viral infections.
In recent years, QSVT technology has made many methodological improvements, especially in image
processing and imaging algorithms.
QSVT technology has become a well-established and commonly used method
of virus tracking.
The research team used a set of operational procedures to illustrate how QSVT can easily be used to decipher the virus's complex infection process and reveal the underlying mechanisms, divided into six stages
.

Phase 1: Virus amplification and purification

Viral stocks should first be amplified and purified to obtain samples that meet the requirements for effective labeling of viruses to avoid interference with non-viral structures during imaging
.

Phase 2: Cell labeling and drug inhibition

Cells need to be seeded in a glass-bottom dish for confocal imaging
.
Since the dynamic interaction between cells and viral components is a continuous process throughout the life cycle of the virus, labeling cellular structures with FPs or organic dyes can be used to identify specific locations of the virus in living cells, and inhibition of cellular function using common inhibitors can be used to identify viral infection cellular uptake and transport pathways
.
Therefore, cell labeling and drug inhibition are optional steps to study different stages of viral infection, and cell samples
can be treated individually or simultaneously prior to imaging depending on the purpose of the experiment.

Phase 3: Flag the virus with QD

In the viral process of virus infection of living cells, as the infection process advances, the different components of the virus are dynamically decomposed, so the internal and external components of the virus should be labeled with QD alone or simultaneously to fully understand the mechanism
of virus infection.
Once the labeled virus is incubated with the cells cultured in the dish, infection begins and imaging can be performed
.

Stage 4: Image acquisition

The infection behavior of a single virus in living cells can be monitored
with fluorescence microscopy equipped with a detector with sufficient sensitivity and rapid acquisition capabilities.
Two-dimensional (2D), three-dimensional (3D), and multicolor QSVT technologies can accurately track the movement behavior
of individual viral particles by using spinning disk confocal microscopy (SDCM).

Stage 5: Image processing

After acquiring a microscope image containing a large amount of information, the trajectory of viral infection behavior, including noise reduction, particle localization, and trajectory reconstruction
, needs to be obtained through an image processing step.

Phase 6: Data analysis

Finally, by analyzing the viral trajectory, including transport characteristics, fluorescence intensity analysis and multicolor image analysis, the dynamic parameters related to viral infection were extracted to reveal the potential mechanism of viral infection
.

Application value and significance