Quantification of Nanoparticles at the Single-cell Level: An Overview About State-of-the-art Techniques and Their Limitations

Dimitri Vanhecke; Laura Rodriguez-Lorenzo; Martin JD Clift; Fabian Blank; Alke Petri-Fink; Barbara Rothen-Rutishauser


Nanomedicine. 2014;9(12):1885-1900. 

In This Article

Emerging Techniques in Microscopy/Spectroscopy

Many recent new techniques have been described recently which are promising in furthering NP quantification in cells and only some of them will be mentioned here.

High-angle annular dark-field scanning transmission electron microscopy (HAADF STEM) is a scanning electron microscope that can facilitate the acquisition of high contrast images of the ultrastructure of cells without poststaining with heavy metals and allows the distinguishing of NPs of different composition at first sight by exploiting atomic number (Z) contrast. This can be an advantage for, for example, the analysis of complex NP mixtures or NP composites envisaged for biological application.[96] Moreover, the potential of low-energy HAADF STEM with respect to quantification of the NP size in 3D has been demonstrated by calculating the HAADF STEM intensity on the basis of Monte Carlo simulations.[97] In the same direction, STEM in combination with analytical techniques such as energy dispersive x-ray spectroscopy (EDS) is a promising method to quantify NPs in the cell, as well as to distinguish small NPs from metal precipitates from stains that can appear as nanosized objects.[98] However, these approaches still require mechanical sectioning to render eukaryotic cells suitable for analysis despite the risk of compression artifacts distorting interpretation of recorded images. It is worth noting that advances in rapid scanning x-ray fluorescence microscopy (XFM) and ion beam imaging techniques have allowed high resolution images of internalized metal oxide NPs to be acquired from intact cells.[99] The correlative photon and ion beam imaging techniques, for example, XFM FIB-SEM can also provide both high-resolution and statistically powerful information of the 3D distribution and composition of metal oxide NPs at the single-cell level.[99]

Surface enhanced Raman scattering (SERS) spectroscopy is gradually emerging as an alternative analytical technique to quantify and visualize the NPs at the single-cell level. SERS spectroscopy is a linear Raman technique carried out on nanostructured metal nanoparticles.[100] SERS has received considerable attention due to its high sensitivity, fingerprinting ability, resistance to photobleaching, minimal sample preparation, and ease of operation. Recently, it has been demonstrated that it is possible to accurately correlate the SERS signal with the concentration of NPs labeled with Raman reporters.[101] Thus, SERS was successfully applied to the determination of the number and distribution of Au NPs taken-up by macrophage cells and was in good correlation with the results of ICP-MS[102] In addition, SERS flow cytometry can improve significantly the ability of multiplex detection since the vibrational bands are narrower than the fluorescence, allowing the quantification of different type of NPs taken up into the single cell.[103]

For fluorescence quantification methods high content analysis (HCA) platforms have evolved over the last years that integrate and automatize quantitative fluorescence microscopy and image analyses.[104] The uptake of siRNA NPs into Calu-3 cells was analyzed by HCA and validated by confocal microscopy techniques that showed significant difference in uptake when the siRNA NPs where specifically modified.[105]

All these mentioned methods are promising, however, their accuracy, reproducibility and ease to use has to be shown in future studies.