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

Flow Cytometry

Flow cytometry, often referred to as, and synonymous with fluorescent-activated cell sorting (FACS; a trademark name of BD Biosciences), is a semiquantitative technique that predominantly measures the fluorescent, although also the optical (i.e., scattering), characteristics of single cells.[41] Based upon the principles of fluorescence, the technique of flow cytometry is built upon the ability for cells to be streamlined into a fluid laminar flow (known as 'hydrodynamic focusing') which is sequentially excited by a specified laser light. The emission of the activated fluorochrome on the NP by the specific laser used is captured and enables a computational read-out of the fluorescent intensity (e.g., via a histogram) of the sample of interest.[42]

Flow cytometry can be used to rapidly (in some cases, with fast flow cytometers [dependent upon specific internal settings] it can take ≥1 s to record ≤10,000 events in one sample) investigate the cellular characteristics, such as size, granularity, surface and intracellular proteins, as well as determine such intracellular components as pH, calcium and DNA (cell cycle) within large cell populations.[43] Through such an application, clinicians are able to characterize specific and multiple cell populations, at any one time, of a patient as to their specific disease state (e.g., immunotyping of cell populations).[42,44] Furthermore, this highly sensitive characterization of specific cell populations is used as an advantageous tool within biotechnology-based research strategies.[43]

It is important to note however, that apart from the need for detectable fluorescence, one of the major limitations attributed to flow cytometry is that no distinction between internalized and membrane attached (also referred to as 'cellular associated') NPs can be made. In this regard, flow cytometry can only be used to support that of microscopy-based research strategies in order to achieve a rapid semiquantification (i.e., an approximation) of the amount of NPs associated with cells. An example of this is in the study by Clift et al.,[45] in which a series of different surface charged quantum dots were assessed via flow cytometry. One-colour (i.e., one fluorophore) flow cytometry was performed using the J774A.1 murine macrophage cell line, which showed an increase in the fluorescent intensity of the cell types over time (2 h) (Figure 1), as observed also qualitatively with the confocal microscope. Such a comparison does not consider a specific number of NPs, but is based upon relative fluorescence. In a recent study by Gottstein et al.,[46] a correlative approach was undertaken in order to specifically determine the number of internalized particles versus total number of associated particles, and how normalization to a standard could provide a direct comparison with other methods used to quantify the NP–cell interaction. Nonetheless, a limitation of both these studies is that two separate techniques were used to correlate against different sample preparations. It is possible however that these analyses could be performed on the same sample, at the same time by using imaging flow cytometry, as this technique combines the effectiveness of fluorescence microscopy with the power of flow cytometry.[47] Possible limitations of this technique, however, may be the limited optical resolution due to fast acquisition and processing of ultra large and complex data loads, respectively. Yet, despite the advances made with this technology, there is still a severe lack of research in this area, and therefore further, in-depth investigation is necessary.

Figure 1.

Assessment of the cellular associated carboxylated surface functionalized quantum dots with J774A.1 mouse macrophage cells by flow cytometry over a 2-h period. (A) A histogram of the collected cellular associated fluorescent events over a period of 120 min (purple = negative control, i.e., no fluorescent signal, peak denoted within the first logarithmic decade; green = 30 min; red = 60 min; blue = 120 min). (B) Geometric mean fluorescent intensity (MFI) data showing a semiquantitative (i.e., approximation) of the cellular associated fluorescence over the 2-h analytical period.
(A) Adapted with permission from [45] © Elsevier.

Besides fluorescence, flow cytometry can also assess the optical properties of cells and can use these characteristics to determine the NP–cell interaction. Specifically, this is achieved by studying the change in size of the cells by analyzing the side-scatter (SSC) function and/or forward scatter (FSC) function of the technique. This was recently shown by Zucker et al.,[48] who reported the size of human-derived retinal pigment epithelial cells (ARPE-19) to increase following exposure to TiO2 NPs. Despite this, however, the SSC function cannot solve the matter of whether or not NPs are internalized or associated with cells.

While not specific to determining the precise localization of NPs, rather confocal and transmission electron microscopy are pertinent, flow cytometry can be incorporated into studies in order to give a semiquantification of the NP–cell interaction. Taking this issue into consideration, a number of different methods have been applied to gain information as to the intracellular and extracellular NP content, respectively. A common technique used is the application of Trypan blue as a quencher of extracellular fluorescence.[49] Such a process could be advantageous, however, if the fluorescence of the NPs is located on the surface/outer layer of the NP, although if the fluorescence is intrinsic of the NP (i.e., polystyrene nanoparticles), Trypan blue will have no significant effect on diminishing the extracellular fluorescent signal. Thus, in such cases additional washing steps have been suggested as a possible solution to detaching cell membrane associated NPs. This approach, however, is useful if the NPs are only loosely bound to the surface, as strongly bound (i.e., via attached proteins) NPs may not be able to be removed so easily. Further to these outlooks, additional approaches to deciphering intracellular NP content to those attached to the cell membrane include the use of chemical agents and temperature. For example, cytochalasin B and D have previously been used to inhibit the active uptake of NPs by cells, as has placing cells at 4°C for a specific period of time (i.e., 30 min). It is then common practice to correlate the findings of these approaches to samples that have only been exposed at 37°C (i.e., negative control). However, such experimental approaches can cause significant difficulties in distinguishing the biologically relevant changes in fluorescence between samples.

Predominantly flow cytometry is used in the determination of changes in cellular phenotype, such as after NP exposure, via specific, fluorescent antibodies, as recently reported by Blank et al..[50] Advantageously, flow cytometry is able to analyze numerous cell types simultaneously, using a combination of different specific fluorescent biomarkers (which do not cause any spectral overlap). Therefore, when using multiple fluorophores (i.e., ≥2 colour work), it is imperative that the fluorescent wavelengths are not overlapping or are correlated against one another, a process which is known as compensation. With the most recently produced flow cytometers, compensation is an automatic process, although with older models it is a manual process and can incite human error to the sample. However, also automatic compensation may produce abnormalities. In order to execute proper compensation, the instrument has to know which population of events has to be assumed positive and which negative. Therefore, both automatic and manual compensation requires profound experience by the user in both knowledge of the positive and basic knowledge about manual compensation per se. Compensation error is the most commonly attributed limitation to flow cytometry. It is important therefore that consistent methods and outlooks are utilized at these points and that the same person undertakes such analytics to maintain the level of error.

Additional concepts that must be taken into consideration when using flow cytometry to assess the NP–cell interaction are: that cell samples are prepared according to their specific culture conditions, as well as that in order to clarify the cell population of interest it is best to identify the specific cell type using not a single but rather a set of specific surface protein markers; the stability of the fluorophore on the NP under biological pH, including endocytotic vesicle pH (4–5); and the strength of the fluorophore under intense laser light over time. The latter two aspects are of increasing importance when investigating NP types of different sizes, shapes, surface charge or fluorescent signals against one another and concomitantly. Finally, proper analysis of cell–NP interactions by flow cytometry requires a number of control samples, consisting of untreated and unstained cells in order to measure the level of autofluorescence, isotype controls matching the respective specific antibodies, vitality stainings (e.g., annexin V and propidium iodide staining for detection of apoptosis and necrosis, respectively), or cells treated with specific substances in order to impede specific particle uptake mechanisms (e.g., cytochalasin-D to prevent actin-polymerization required for phagocytosis). In addition to flow cytometry analyzers (i.e., instruments that only analyze the sample of interest), it is possible to use a flow cytometer that has the capability to sort cells according to their characteristics. This technique has all the attributes of an analyzer (as discussed above), yet it is then possible to obtain subsamples of interest for further analysis. Such a technique is of extreme interest when considering multicellular cultures, since it can provide vital information as to the precise location of the NPs within the multicellular structure, the load of NPs per cell type (i.e., 'semiquantification' based on NP fluorescence), as well as understanding further the NP–cell interaction in a highly specific, sophisticated alternative system compared with animal models.

In summary, flow cytometry is a straightforward technique that is easy to use and allows for rapid, high-volume analysis providing semiquantitative analysis as to the NP–cell interaction. Limitations of flow cytometry (e.g., extra-/intra-cellular NP localization) may be easily compensated in a powerful combination with microscopic techniques. Just recently a paper has been published showing the quantification of fluorescently labeled carbon nanotubes by multispectral imaging flow cytometery where imaging of single cells in the flow were produced,[51] however, further research is needed to show if this method is useful for additional fluorescently labeled NPs.