Diana Peckys and Niels de Jonge have developed a new approach for the study of nanoparticle (NP) and cell interactions. The traditional method for EM imaging of NPs involves dehydrating, embedding and sectioning the sample into multiple thin sections (<100 nm). Such processing can lead to extraction or loss of NPs from the sample, particularly in membrane regions. In addition, thin sections may cut through three-dimensional structures, such as vesicles that contain high densities of NPs. In this study the Poseidon™ liquid TEM holder and E-Chips™ were utilized to eliminate these disadvantages.

The microfluidic chamber was comprised of two Poseidon™ E-Chips™, both with a 50 nm thin silicon nitride membrane in their center to provide a transparent window for photons and electrons. The E-Chips™ are used in combination to enclose the liquid and seal the cells from the EM vacuum. One E-Chip™ had a flat surface and served as the substrate on which adherent cells were cultured. The second E-Chip™ contained two, 6mm thick, integrated spacers that formed a flow channel. These spacers prevent compression of the cells when the E-Chips™ are assembled into the microfluidic chamber, and allow the introduction of buffer to maintain the cell’s hydrated state during imaging.

A resolution of 3 nm was obtained on fully hydrated, intact COS7 fibroblast cells that were living at the onset of imaging. EM images were collected in STEM mode using a Philips CM200 microscope. An average electron dose of 3 e^-/Ų, a factor 10 below cryo-TEM dose limits, was used to produce the initial image. Because the STEM image contains signals from the full three-dimensional space of the cell, this technique can be used for quantitative studies of NP uptake and spatial distribution without sectioning sample. Citing the absence of signs of radiation damage in the recorded images, and the low electron dose, Peckys and de Jonge hypothesize that the first image recorded on a cell can be used to assess the distribution of NPs inside a live cell.

Serum-protein coated gold-NPs were observed sequestered intracellular vesicles, likely lysozomes. Analysis of these vesicles indicated that the NPs were primarily localized on the vesicle membranes, rather than filling the entire intravesicular space.  The distributions of these NPs were analyzed further, and found to occupy 67±11% of the available membrane area. Thus, Liquid STEM is an efficient and robust technique for whole cell analysis of uptake and distribution NPs

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