Encyclopedia of Experiments
Biological Techniques
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Encyclopedia of Experiments Biological Techniques
Darkfield Microscopy to Visualize Diffusional Dynamics of Nanorods on Cell Membrane

Darkfield Microscopy to Visualize Diffusional Dynamics of Nanorods on Cell Membrane

Transcription

To investigate gold nanorod diffusional dynamics on a live cell membrane using darkfield microscopy, DFM, combined with single-particle tracking, begin with a sterilized coverslip in a culture dish containing suitable medium. Add the cell suspension onto the coverslip. The medium facilitates cell growth.

Incubate with positively-charged gold nanorods that adsorb onto the negatively-charged cell membrane via electrostatic interactions. Pipette the medium from the dish to the groove of a glass slide. Place the coverslip cell-side-down on the groove, and seal.

Place the slide on the darkfield microscope stage. The microscope illuminates the sample with a high-angle oblique light passing through a specialized darkfield condenser.

Upon the illumination of the nanorods, the free electrons on the nanorod surface oscillate at a frequency resonant with the incident light. The oscillating electrons scatter light at a specific wavelength captured by the objective lens, enabling nanorod visualization.

Similarly, the cell membrane — composed of lipids and proteins — scatters the incident light at a higher refractive index than the surrounding medium, creating a bright image against a dark background. Capture time series DFM images.

Using a suitable software, perform single-particle tracking analysis on the DFM images to visualize the diffusion of the nanorods through the cell membrane with high spatiotemporal resolution.

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