Comparison of Confocal and Super-Resolution Reflectance Imaging of Metal Oxide Nanoparticles

Abstract

The potential for human exposure to manufactured nanoparticles (NPs) has increased in recent years, in part through the incorporation of engineered particles into a wide range of commercial goods and medical applications. NP are ideal candidates for use as therapeutic and diagnostic tools within biomedicine, however concern exists regarding their efficacy and safety. Thus, developing techniques for the investigation of NP uptake into cells is critically important. Current intracellular NP investigations rely on the use of either Transmission Electron Microscopy (TEM), which provides ultrahigh resolution, but involves cumbersome sample preparation rendering the technique incompatible with live cell imaging, or fluorescent labelling, which suffers from photobleaching, poor bioconjugation and, often, alteration of NP surface properties. Reflected light imaging provides an alternative non-destructive label free technique well suited, but not limited to, the visualisation of NP uptake within model systems, such as cells. Confocal reflectance microscopy provides optical sectioning and live imaging capabilities, with little sample preparation. However confocal microscopy is diffraction limited, thus the X-Y resolution is restricted to ~250 nm, substantially larger than the <100 nm size of NPs. Techniques such as super-resolution light microscopy overcome this fundamental limitation, providing increased X-Y resolution. The use of Reflectance SIM (R-SIM) for NP imaging has previously only been demonstrated on custom built microscopes, restricting the widespread use and limiting NP investigations. This paper demonstrates the use of a commercial SIM microscope for the acquisition of super-resolution reflectance data with X-Y resolution of 115 nm, a greater than two-fold increase compared to that attainable with RCM. This increase in resolution is advantageous for visualising small closely spaced structures, such as NP clusters, previously unresolvable by RCM. This is advantageous when investigating the subcellular trafficking of NP within fluorescently labelled cellular compartments. NP signal can be observed using RCM, R-SIM and TEM and a direct comparison is presented. Each of these techniques has its own benefits and limitations; RCM and R-SIM provide novel complementary information while the combination of modalities provides a unique opportunity to gain additional information regarding NP uptake. The use of multiple imaging methods therefore greatly enhances the range of NPs that can be studied under label-free conditions.

Super-resolution microscopy as a powerful tool to study complex synthetic materials

“…This technique was recently used to follow the internalization of iron and cerium oxide nanoparticles with 100 nm resolution 114 .…”

Mechanisms for cellular uptake of nanosized clinical MRI contrast agents

“…Cellular TEM processing was carried out using standard TEM processes of fixation, dehydration, infiltration, trimming and sectioning as detailed previously (Guggenheim et al 2016). Cellular TEM imaging was carried out on an FEI Tecnai G2 Spirit operating at 80 keV (FEI, Center for Advanced Microscopy, Northwestern University, Chicago, IL).…”

“…Microscopy remains one of the most utilized techniques to study NPs, particularly for in vitro investigation. This is due to the resolution achievable and the ability to directly visualize signals in context with other cellular components when coupled with labelling or staining (B€ ose et al 2014;Guggenheim et al 2016;Karreman et al 2016). Traditionally, Transmission Electron Microscopy (TEM) or confocal fluorescence imaging are used, however TEM is time consuming, laborious and low throughput, while the use of fluorescent tags has numerous limitations, including low sensitivity, difficult bio-conjugation, low quantum efficiency and photo-bleaching effects, in addition to potential alteration of NP surface chemistry if the label is surface-attached, a factor known to influence biological activity (Sugden 2004;Sigmund et al 2008;Patskovsky et al 2014;Bartczak et al 2015;Quinn et al 2015).…”

Cold Atmospheric Plasma induces silver nanoparticle uptake, oxidative dissolution and enhanced cytotoxicity in Glioblastoma multiforme cells

“…Nanoparticle detection in cells and tissues are often achieved by employing electron microscopy techniques and confocal microscopy to investigate translocation of NPs in cells [72][73][74]. While these techniques have extensively accomplished identification of NPs, they lack the potential to validate NPs presence in cells or tissue via spectral mapping.…”