Dark Field Transmission Electron Microscopy as a Tool for Identifying Inorganic Nanoparticles in Biological Matrices

Abstract

Dark field transmission electron microscopy has been applied herein to visualize the interactions of inorganic nanomaterials with biological systems. This new application of a known technique addresses a deficiency in status quo visualization techniques. High resolution and low noise images can be acquired to locate and identify crystalline nanoparticles in complex biological matrices. Moreover, through the composition of multiple images taken at different angular beam tilts, it is possible to image a majority of nanoparticles present at a site in dark field mode. This facilitates clarity regarding the internalization of nanomaterials in cellular systems. In addition, comparing dark field images recorded at different angular tilts yields insight into the character of nanoparticle faceting.

Impacts of gold nanoparticle charge and ligand type on surface binding and toxicity to Gram-negative and Gram-positive bacteria

“…Biological TEM samples were prepared by a typical process of fixation, dehydration, and embedding in a resin matrix. 29 , 30 Briefly, bacterial suspensions in HEPES at OD 0.2 were pelleted and washed three times in 0.1 M sodium cacodylate buffer, then fixed in a 2.5% gluteraldehyde in 0.1 M sodium cacodylate buffer solution for 1 hour. The pellet was flipped halfway through fixation to improve gluteraldehyde penetration.…”

“…Dark field TEM images were collected in dark field mode with a variety of objective aperture sizes depending on the instrument magnification. 30 …”

Methodologies and approaches for the analysis of cell–nanoparticle interactions

“…A variation on this approach was performed by Klein et al, who used dark‐field microscopy combined with EM. In their study, dark field images were obtained in a transmission EM set‐up by tilting the beam radially and collecting only scattered electrons.…”

Cell-Promoted Nanoparticle Aggregation Decreases Nanoparticle-Induced Hyperthermia under an Alternating Magnetic Field Independently of Nanoparticle Coating, Core Size, and Subcellular Localization

“…In order to determine whether nanoparticles orientation during cellular aggregation could be the cause of this different magnetic behavior observed, MNP aggregates were isolated from lysosomes and orientation analyzed using bright and dark field transmission electron microscopy. This technique has been used to locate and identify crystalline nanoparticles in complex biological matrices 75 . When APS-NMP aggregates from Pan02 lysosomes were analyzed in bright field mode, inorganic nanoparticles with high mass and crystallinity appear dark (Figure 9).…”