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

Fluorescence-based Quantification Techniques

The utilization of so-called 'encoded' NPs for the detection or targeting of specific cells or intracellular compartments is a highly researched area and considered one of the most powerful tools for the medical application of NPs such as the codification of particles through highly active fluorescence reporters.[35] Fluorescence generates very intense signals that allow detection in very short data acquisition times with straightforward analysis. In fact, fluorescence-encoded particles can be rapidly processed by methods that detect fluorescence in single cells or tissues such as confocal laser scanning microscopy (LSM), and/or flow cytometry.[15,36] A wide variety of fluorochromes are available with emitting wavelengths ranging from the blue–violet end of the visible spectrum to the infrared. Individual fluorochromes are characterized by their wavelengths at which they maximally absorb excitatory and emit fluorescent light.[37] During the last decade many new NPs with fluorescence specificity have been introduced, which can be located either within the core, the shell (core-shell NPs with fluorophores embedded in their shell[15,38]), attached to the surface or be an intrinsic characteristic (i.e., semiconductor nanocrystals – quantum dots – that are robust and bright light emitters[39]). However, pitfalls such as intracellular signal quenching or uncoupling of fluorescent dyes remain and become relevant when comparing NPs, for example, between particle type, size, charge or fluorescent signal.

It is important to mention that quantification of fluorescence has always to be taken in relation to a control since stability of the fluorophore, which is paramount for quantification purposes, can only partially be controlled. In addition, interferences with the optical systems have to be avoided.[40]