The uptake of nanoparticles into cells has conventionally been studied with transmission electron microscopy of thin sections. However, the preparation into thin sections leads to a loss of nanoparticles, and also prevents the analysis of the whole three-dimensional cellular volume. Furthermore, conventional electron microscopy is not capable of studying live cells. D.B. Peckys and N. de Jonge from Vanderbilt University Medical Center have now improved upon a liquid scanning transmission electron microscopy (STEM) method that allows for quantitative assessment of nanoparticle uptake in living cells. The researchers used a microfluidic chamber composed of microchips where fibroblast cells were imaged with minimal radiation damage to reveal the distribution of gold-nanoparticles (Au-NP) within the cells.
In the March 16th issue of Nano Letters (DOI: 10.1021/nl200285r; p. 1733), the research team described the application of a novel microfluidic chamber device for live-cell examination. The device was made from two parallel silicon microchips. Each microchip contained a central silicon nitride (SiN) membrane of 50 nm thickness that was transparent to the electron beam as well as light. One microchip was coated with poly-l-lysine (PLL) for increased cell adherence; the other chip had an attached 6 μm spacer to form a reservoir for the cells and buffer. Live green monkey kidney fibroblast cells (COS-7) were incubated for 2 hours in the presence of 30 nm Au-NPs. The cells were then washed and incubated in a medium. Twenty-four hours later, the cells were enclosed in the chamber and imaged live with a STEM under a continuous flow of buffer. Experiments demonstrated that COS-7 cells remained viable in the microfluidic chamber.
STEM analysis was carried out shortly after the cells were loaded. The cells were viewed at 16,000x magnification using an accelerating voltage of 200 kV and a probe current of 0.16 nA. The researchers were seeking to identify several quantitative values pertaining to the number and placement of the Au-NPs inside the cell vesicle. The STEM images showed high concentrations of Au-NPs as dark spots clustered inside cell vesicles, which were 200–300 nm in diameter. However, there was a degree of image distortion due to certain factors including beam-specimen interaction, the liquid medium, distance to focal plane, and the degree of pixelation.
Despite these conditions, the researchers were still able to resolve separate nanoparticles by enhancing the image using image processing software, Image J. They conducted quantitative measurements of several factors such as the total number of Au-NPs per vesicle cluster and the average density of Au-NPs on the surface of the vesicles.
The researchers concluded that their research showed this method of liquid STEM is suitable for further study of nanoparticles uptake into living cells.
