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Super Resolution and Single Particle Fluorescence Microscopy

Super-Resolution Structured Illumination Microscopy (SR-SIM) provides resolutions down to 120 nm, allowing visualization of individual herpesvirus capsids (HSV-1) attached to the nucleus (HSV-1 C-capsid diameter is 125 nm).

 

Representative 2D Super-Resolution Microscopy image of a nucleus (blue) with attached capsids (green). A histogram of a capsid cross-section profile for a capsid GFP signal and a 3D reconstruction of the nucleus and capsids.

Figure: Imaging of reconstituted capsid-nuclei system confirms specific capsid binding to the NPCs at the nuclear membrane. Representative super-resolution SIM image showing GFP-HSV-1 C-capsids (green) bound to isolated reconstituted rat liver nuclei (blue DAPI stain). A histogram of a capsid cross-section profile for a capsid GFP signal along the white line shows that individual C-capsids are resolved (HSV-1 C-capsid diameter ≈ 125 nm).

 

In parallel, using spinning disk confocal fluorescent microscopy (FM), single-molecule measurements, we investigate the average ensemble kinetics of the number of viruses which ejected their DNA versus time during an infection. This allows investigation of virus population dynamics.

 

Graphical representation of single molecule fluorescence experimental setup.

Figure: Single-molecule fluorescence measurements of the ensemble kinetics for DNA ejection from WT phage λ over time in 10 mM MgCl2 Tris-buffer at 22 and 37°C. YOYO dye shows particles with ejected DNA stretched in the flow. The ejection is triggered by LamB receptor addition at time 0 and flown continuously with YOYO in the flow chamber.