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 Microscopy

Fluorescence microscopy provides detection and localization of NPs along with biochemical information by biomarkers of choice, such as F-actin distribution or position of the nucleus. The main drawback of traditional wide-field fluorescence microscopy is the limited axial resolution (resolution in the z-dimension) due to out-of-focus light, especially in thick specimens (thicker than the focal plane). This makes it challenging to pinpoint the precise localization of small objects, such as intracellular NPs. Confocal microscopy applies a pinhole system that can eliminate most out-of-focus light and therefore allows for optical sectioning: a valuable tool to obtain high axial resolution and 3D reconstructions of fluorescently labeled structures.[52] Two types of confocal microscopes are generally in use: the confocal laser scanning microscopes (LSM) provide the best z-resolution and signal-to-noise ratio but require longer acquisition times; and confocal spinning-disc systems are dedicated to fast dynamic processes but suffer from poorer z-resolution and lower signal-to-noise ratio compared with LSM.

The spatial resolution of light microscopy is limited by wavelength of the light and the numerical aperture of the lens, practically: about 200 nm in the lateral dimensions and 500–900 nm in the axial dimensions, however, deconvolution algorithms may increase the resolution up to 2–3 fold.[53] From the definition of NPs, it follows that single NP events cannot be individually resolved and prevents distinguishing between single particles and small NP agglomerates. Instead, particle events can be counted, where one particle event corresponds to an individual vesicular structure containing NPs (Figure 2). Thus, relative differences in the amount of incorporated particles per cell can be quantified and for instance the differences in NP uptake into different cell types can be assessed.[54,55] In a recent study we have used well-defined fluorescently labeled polymer-coated Au NPs with an electron dense Au core and directly compared two different quantitative microscopy methods: the fluorophore was analyzed by LSM combined with digital imaging analysis and the Au core by means of stereology on TEM. It could be shown that by light microscopy the number of particle events is underestimated by a factor of ten when compared with the total NP number received by TEM.[36]

Figure 2.

Visualization of fluorescently labeled iron oxide NPs inside dendritic cells in vitro by laser scanning microscopy (LSM). LSM pictures of monocyte-derived dendritic cells treated with Oregon green 488-labelled iron oxide NPs during 4 h. Afterwards the cells were fixed and stained for the cell nuclei (orange) and for F-actin (light yellow), which facilitated detection of intracellular (arrowheads) localization of iron oxide NPs. xy-projections (top panel) and xz-projections (lower panel) were obtained from multiple consecutive optical sections, yellow arrowheads mark the positions of the respective projections.

Much attention has been put on further improvement in spatial resolution of the confocal microscopy methods. By interference of incident or emitted light from above and below the sample the axial resolution could be further improved and isotropic voxels (lateral resolution = axial resolution, ~200 nm) can now be achieved by 4π microscopy. Progress in the lateral resolution is evident by the many super-resolution light microscopy methods that have been developed in the last years, such as stimulated emission depletion microscopy (STED), photoactivated localization microscopy (PALM) or stochiastic optical reconstruction microscopy (STORM). PALM and STORM allow the visualization of photoswitchable fluorescence probes at the nanometer resolution,[56,57] and STED aims to reduce the size of the excitation spot.

Spinning-disc confocal microscopy finds its purpose in applications involving fast processes in living cells. Combined with tracking routines the kinetics and dynamics of fluorescently labeled NPs can be studied.[58] Clever use of the scanning module of the LSM can also provide information on fast dynamic processes. Fluorescence correlation spectroscopy (FCS) tracks the fluorescent signal in one pixel and allows for measuring the diffusion constant, and derived from this, the size of the tagged object.[59] FCS can provide information on NP concentration and dynamics at high temporal resolution (microseconds) within a very small region of the cell. Since only one point is scanned, FCS is limited to processes occurring in a single location. Using 2D fast-Fourier transform algorithms to calculate the spatial autocorrelation function of an image is the idea behind image correlation spectroscopy (ICS).[60] ICS is an image-processing method and thus applicable to any kind of microscopic (or nonmicroscopic image) image or stack of images. Spatial power spectrum analysis provides the number and size of aggregates in the image and in cells the degree of aggregation and the average number of fluorescently labeled protein aggregates for an entire image can be determined. In contrast with FCS, ICS can characterize the mode of motion of and quantify transport parameters on different length scales from single-cell to subcellular level.[61,62] Raster image correlation spectroscopy (RICS) successfully bridges the timescales of FCS and ICS and provides spatially resolved dynamic information in the microsecond-to-second time range.[63]

The possibility to capture the process of uptake of (fluorescently labelled) NPs by living cells or to perform colocalization studies with fluorescently labeled organelles in 3D objects (fixed or living cells)[54] makes confocal microscopy an excellent tool to gain new insights into NP–cell interactions, NP uptake mechanisms and NP intracellular fate.[36,54,64]