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

Abstract and Introduction


With the increasing production and use of engineered nanoparticles it is crucial that their interaction with biological systems is understood. Due to the small size of nanoparticles, their identification and localization within single cells is extremely challenging. Therefore, various cutting-edge techniques are required to detect and to quantify metals, metal oxides, magnetic, fluorescent, as well as electron-dense nanoparticles. Several techniques will be discussed in detail, such as inductively coupled plasma atomic emission spectroscopy, flow cytometry, laser scanning microscopy combined with digital image restoration, as well as quantitative analysis by means of stereology on transmission electron microscopy images. An overview will be given regarding the advantages of those visualization/quantification systems, including a thorough discussion about limitations and pitfalls.


"Dreams apart, numerical precision is the very soul of science, and its attainment affords the best, perhaps the only criterion of the truth of theories and the correctness of experiments" is written in the most famous work by D'Arcy Wentworth Thompson almost hundred years ago.[1] Reliable numerical precision is more than ever relevant, including nanosized particle (NPs; 1–100 nm[2]) research, whose immense potential for diagnostic and therapeutic applications stands in sharp contrast to a growing number of critical reports regarding their potential toxicity.[3] In order to understand how NPs interact with cellular systems, potentially causing adverse effects, their detection, localization and quantification within cells is of central importance to understand how physicochemical parameters might influence the possible interaction with a specific cell type. Once intracellular NPs are identified, their distribution in different cellular compartments, such as endosomes, lysosomes, mitochondria, the nucleus or cytosol may also provide some indication as to their potential biological impact, as well as how to specifically design nanocarriers for cell targeting and drug delivery.

The method of choice for the intracellular detection of NPs depends on the characteristics of the particles (e.g., chemical composition, fluorescence, size, and structure or electron density), as well as on the cellular structure of interest.

Elemental analysis techniques and a variety of microscopic techniques are commonly used for NP detection and/or quantification as they are relatively easy to apply and provide a rapid understanding of the NP–cell interaction. Inductively coupled plasma (ICP) techniques including ICP-optical emission spectroscopy (OES) and ICP-mass spectrometry (MS),[4,5] are powerful tools for detecting and analyzing trace and ultra-trace elements. They yield elemental information on the liquid sample or the dissolved solid sample after dissolution and consequently allow the quantification of NPs in colloidal suspensions and in a cell culture experiment without any need for additional labeling. However, these methods do not provide any information about NP distribution within the cell population or location within the cells. Flow cytometry is a high-throughput method that provides information on the presence and distribution of fluorescently labeled NPs in a population of cells, but not their localization within the cells. For the localization of NPs within a cell and in specific compartments, microscopic techniques such as light and transmission electron microscopy must be applied.[6] Using either living cells or fixed, light microscopy provides visualization of fluorescently tagged NPs in a cellular landscape but with its resolution limited to approximately 200–500 nm (but is dependent on the microscopy and analysis method used, for a review see[7]) cannot resolve the NP and distinguish between a single particle or agglomerates. Transmission electron microscopy (TEM) with a resolution range from Ångstrom to nanometer is the method of choice for resolving electron dense NPs, but requires a complex and long sample section preparation, including fixation of the cells. Stereological approaches are required to understand the 3D distribution of NPs within a defined reference volume.[8] In recent years many new methods have been established to quantify the uptake of NPs by cells by microscopically and analytical techniques[8,9] since it has been shown to be relevant that the applied and intracellular dose/NP number can be related to any observed cell response.[10]

The present review aims to provide an overview of the most commonly used methods in the community to identify and quantify cellular-associated NPs in in vitro assays, discussing the specific advantages and disadvantages of each method. Other techniques are relevant for in vivo imaging, such as MRI, computed tomography or optical imaging (for reviews see[11,12]), but are outside the scope of this review.